Studies on the effect of thymoquinone on oxidative stress
and cell wall integrity of Candida glabrata
A thesis submitted in fulfilment of the requirements for the degree of Doctor of
Philosophy
Hala Saied Almshawit
BSc (Hons), MSc.
School of Applied Sciences
College of Science Engineering and Health
RMIT University
November 2015
Preface
DECLARATION
I certify that except where due acknowledgement has been made, the work is that of the author
alone; the work has not been submitted previously, in whole or in part, to qualify for any other
academic award; the content of the thesis/project is the result of work which has been carried out
since the official commencement date of the approved research program; any editorial work, paid
or unpaid, carried out by a third party is acknowledged; and, ethics procedures and guidelines
have been followed.
Hala Saied Almshawit
3/12/2015
ii
Preface
ORIGINALITY STATEMENT
I hereby declare that this submission is my own work and to the best of my knowledge it
contains no materials written by another person, or substantial proportion of materials which
have been accepted for the award of any other qualifications at RMIT or any other educational
institution, except where due acknowledgement is made in the thesis. Any contribution made to
the research by others, with whom I have worked at RMIT, is explicitly acknowledged in the
thesis.
I also, declare that the intellectual content of this thesis is the product of my own work, except to
the extent that assistance from others in the project’s design and conception or in style,
presentation and linguistic expression acknowledged.
Signed …………………………
Hala Saied Almshawit
03/12/2015
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Preface
COPYRIGHT STATEMENT
I hereby grant the Royal Melbourne Institute of Technology University or its agents the right to
archive and to make available my thesis in whole or part in the University libraries in all forms
of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain
all proprietary rights, such as patent right. I also retain the right to use in future works (such as
articles or books) all or part of this thesis or dissertation.
I also authorise University Microfilms to use the 488 words abstract of my thesis in Dissertation
Abstract International. I have either used no substantial portions of copyright materials in my
thesis or I have obtained permission to use copyright materials; where permission has not been
granted I have applied/ will apply for a partial restriction of the digital copy of my thesis or
dissertation.
Signed …………………………
Hala Saied Almshawit
03/12/2015
iv
Preface
AUTHENTICITY STATEMENT
I certify that the Library deposit digital copy is a direct equivalent of the final officially approved
version of my thesis. No emendation of content has occurred and if there are any variations in
formatting, they are the result of the conversion to digital format.
Signed …………………………
Hala Saied Almshawit
03/12/2015
v
Preface
ABSTRACT
Thymoquinone is a major active ingredient of black seeds which are produced by the plant,
Nigella sativa. Thymoquinone exhibits various chemotherapeutic and chemo preventative
activities including immunostimulation, antimicrobial, anticancer and chemoprotection activities
(reviewed by (Darakhshan et al., 2015). Despite available data on thymoquinone’s anticancer
mechanism of action, there is no description of its antifungal mode of action. This thesis
presents data on thymoquinone’s effect on the redox status of C. glabrata and reveals a binding
site of TQ on the surface of these cells.
The data presented in chapter 2 demonstrates for the first time the growth inhibitory effect of TQ
on the species C. krusei and C. glabrata beside other Candida species. This chapter also shows
that in C. glabrata, thymoquinone exhibited concentration-dependent fungicidal activity on
planktonic cells and caused necrotic death. In addition, the antifungal effect of thymoquinone
against C. glabrata biofilms was examined. The biofilms were stabilised using a poly propylene
biofilm reactor designed to mimic Calgary biofilm device (Almshawit et al., 2014b). The
advantages of this novel reactor are described.
Thymoquinone may act as
anti-oxidant or pro-oxidant depending on the milieu (Mansour et
al., 2002). Chapter 3 demonstrated that thymoquinone causes oxidative stress in C. glabrata as a
result of increased reactive oxygen species generation. This chapter also reports depletion of
reduced glutathione levels and reduced mitochondrial membrane potential. In addition, this
chapter provides experimental characterisation of the ALO1 gene and evidence of production of
D-erythroascorbic acid by C. glabrata.
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Chapter 4 shows that activation of CWI-MAPK in C. glabrata results following treatment with
thymoquinone, hydrogen peroxide, doxycycline or hypo-osmotic treatment. However, CWIMAPK mutants were neither sensitive nor resistant to thymoquinone. In contrast the downstream
kinase mutant (C. glabrata Δslt2) was sensitive to H2O2 (Chapter 5). Activation of CWI-MAPK
by hypo-osmolarity was exploited to develop a hypothesis that this mechanism could be used to
screen potential CWI-MAPK inhibitors. Survival of cells in water was used as a model to screen
such inhibitor. The results give insights into important facts that should be considered when
using water suspensions of C. glabrata cells.
Finally chapter 5 describes for the first time an interaction between thymoquinone and the fungal
cell wall. This study demonstrates that thymoquinone induced stress promotes chitin synthesis
after induction of CWI-MAPK. Chitin was shown to bind thymoquinone and facilitate its
transport through the cell wall inside the cell. This was supported by the observation that
inhibition of chitin synthesis led to a decrease in thymoquinone antifungal efficacy. This finding
is important in a reconsideration of the use of TQ together with other cell wall active antifungal
compounds such as nikkomycin Z.
vii
Preface
PUBLICATIONS AND CONFERENCE PROCEEDINGS
Almshawit, H., Macreadie, I. & Grando, D. 2014a. A simple and inexpensive device for biofilm
analysis. Journal of Microbiological Methods, 98, 59-63.
Almshawit, H., Pouniotis, D. & Macreadie, I. 2014b. Cell density impacts on Candida glabrata
survival in hypo-osmotic stress. FEMS Yeast Research, 3, 508-516.
Manuscripts in preparation:
Almshawit H, Anand S. & Macreadie I., Fungicidal effect of thymoquinone involves generation
of oxidative stress.
Almshawit H and Macreadie I. Elevated cell wall chitin in Candida glabrata increases
thymoquinone antifungal efficacy.
Conference proceedings:
Almshawit, H., Macreadie, I., and Grando, D., A simple and inexpensive device for biofilm
analysis that mimics Calgary device. Lorne infection and immunity conference, 2014, Mantra
Lorne, Australia.
Almshawit, H., Macreadie, I., and Grando, D., A simple and inexpensive device for biofilm
analysis that mimics Calgary device. AusBiotech, 2014, Gold Coast Convention and Exhibition
Centre, Australia.
Almshawit, H., Anand, S., and Macreadie, I., The effect of thymoquinone on oxidative stress
and the cell wall in C. glabrata, 19th congress of International Society for Human and Animal
Mycology 2015. Melbourne Convention and Exhibition Centre, Australia.
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ACKNOWLEDGMENTS
I am using this opportunity to express my gratitude to everyone who supported me throughout
this journey. First, I appreciate deeply the opportunity of awarding me PhD scholarship from the
Libyan Ministry of Higher Education and Scientific Research to achieve this study.
I would like to express my sincerest thanks to Assoc. Prof. Ian Macreadie for the guidance and
kindness.
Thank you Ian for allowing me to think freely and thanks for your excellent
communication. I would also like to express my deepest appreciation to Assoc. Prof. Danilla
Grando for her help to coordinate my study. I appreciate your encouragement and your advice
regarding my writing. I would keenly like to thank Dr. Anna Walduck and Dr. Gregory Nugent
for their guidance, valuably constructive criticism and friendly advice which helped in
development of this work.
Special gratitude I give to my close friends in the laboratory. I am thankful for your inspiring
and technical support. Thank you to my entire lab. mates at RMIT who made my PhD study
period an unforgettable experience.
I like to dedicate this work to my dear husband Mohammed in appreciation of his unlimited
support and encouragement during whole my post graduate study. I would also like to give
special gratitude and thanks to my parents Zohra and Saied who paved my scientific way since the
childhood and have given me the force to start and continue my scientific life.
Last but not least a big thanks to my lovely children Weam, Abdul-wakil, Aos and Judie for your
patient and incredible happiness that you add to my life.
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Preface
Table of contents
DECLARATION…………………………………………………………………………...
ORIGINALITY STATEMENT…………………………………………………………….
COPYRIGHT STATEMENT……………………………………………………………….
AUTHENTICITY STATEMENT…………………………………………………………..
ABSTRACT…………………………………………………………………………………
PUBLICATIONS AND CONFERENCE PROCEEDINGS………………………………..
ACKNOWLEDGMENTS…………………………………………………………………..
LIST OF FIGURES…………………………………………………………………………
LIST OF TABLES………………………………………………………………………….
LIST OF ABBREVIATIONS………………………………………………………………
CHAPTER 1: Introduction………………………………………………………………….
ii
iii
iv
v
vi
viii
ix
xv
xviii
xix
1
1.1 Candida glabrata…………………………………..…………………………………………….
2
1.1.1 General characteristics of Candida glabrata………………………...……………….. 2
1.1.2 Candida glabrata virulence factors……………………………………………….
3
1.1.2.1 Adherence and biofilm formation …………………………………………
4
1.1.2.2 Pigment production ……………………………………………………….
6
1.1.2.3 Morphology and Phenotypic switching……………………………………
7
1.1.2.4 Enzyme production………………………………………………………..
8
1.1.3 Host interaction …………………………………………………………………..
8
1.1.4 Cell wall structure and cell wall integrity MAPK………………………………...
11
1.2 Thymoquinone
1.2.1 Thymoquinone in Nigella sativa………………………………………………………..
15
15
1.2.2 Chemical properties of thymoquinone……………………………………………. 17
1.2.3 Thymoquinone toxicity…………………………………………………………… 18
1.2.4 The therapeutic potential of thymoquinone………………………………………
19
1.3 Rational and scope of this study………………………………………………………...
26
CHAPTER 2: Antifungal effect of thymoquinone against Candida spp. planktonic cells
and C. glabrata biofilms……………………………………………………………………
28
2.1 Introduction…………………………………………………………………………….
28
2.2 Materials and methods………………………………………………………………….
34
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2.2.1 Yeast strains and media…………………………………………………………...
34
2.2.2 Determination of TQ and fluconazole minimum inhibitory concentrations……… 34
2.2.3 Testing of TQ fungicidal activity…………………………………………………. 36
2.2.4 Biofilm establishment……………………………………………………………..
37
2.2.4.1 Microwell plates…………………………………………………………
37
2.2.4.2 Polypropylene biofilm reactor…………………………………………..
38
2.2.4.2.1 Reactor assembly…………………………………………………
38
2.2.4.2.2 Biofilm establishment…………………………………………….
40
2.2.4.3 Enumeration of CFU in biofilms and testing PP reactor reproducibility…. 41
2.2.4.4 Viewing attached cells by environmental scanning electron microscope
41
2.2.5 Testing TQ toxicity against C. glabrata biofilm………………………………….
41
2.2.6 Susceptibility of C. glabrata to protein-polymer TQ nanoparticles………………
44
2.2.7 In vivo staining of caspase activity and membrane integrity…………………….
44
2.2.8 Cell cycle analysis………………………………………………………………... 45
2.2.9 Data analysis……………………………………………………………………...
45
2.3 Results
2.3.1 Thymoquinone inhibits C. glabrata growth……………………………………...
46
2.3.2 Thymoquinone time–kill curve study…………………………………………….
46
2.3.3 Biofilm formation and reproducibility…………………………………………… 51
2.3.4 Candida glabrata biofilm observed by scanning electron microscopy…………..
58
2.3.5 Thymoquinone fungicidal activity against C. glabrata biofilms………………...
58
2.3.6 Susceptibility of C. glabrata to TQ nanoparticles………………………………..
58
2.3.7 Thymoquinone induces FITC-VAD-FMK activation in dead cells……………..
61
2.3.8 Thymoquinone treated cells did not exhibit DNA degradation………………….
63
2.4 Discussion
2.4.1 Thymoquinone anti-candida effect……………………………………………….
65
2.4.2 Polypropylene biofilm reactor affordability and reproducibility…………...........
67
2.4.3 Thymoquinone causes necrotic death in C. glabrata………………………….......... 71
2.5 Conclusion………………………………………………………………………………
73
CHAPTER 3: Thymoquinone’s effect on the oxidative status of C. glabrata……………….. 74
3.1 Introduction……………………………………………………………………………..
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74
Preface
3.2 Materials and methods
3.2.1 Chemicals…………………………………………………………………………. 76
3.2.2 Yeast strains and media…………………………………………………………..
76
3.2.3 Effect of antioxidants…………………………………………………………….
78
3.2.4 Detecting reactive oxygen species (ROS)………………………………………..
78
3.2.5 ROS and cell membrane integrity………………………………………………… 79
3.2.6 Estimation of intracellular reduced glutathione content……………………… ….
80
3.2.7 Measuring of mitochondrial membrane potential……………………………........ 80
3.2.8 Amplification of ALO1 gene by polymerase chain reaction……………………..
81
3.2.9 Agarose gel electrophoresis………………………………………………….........
82
3.2.10 Vector digestion and ALO1 ligation……………………………………………..
83
3.2.11 Overexpression of ALO1 in C. glabrata………………………………………………
86
3.2.12 Measurement of intracellular D-erythroascorbic acid…………………………
86
3.2.13 Assay for resistance to oxidative stress………………………………………….
87
3.2.14 Data analysis……………………………………………………………………..
88
3.3 Results
3.3.1 Antioxidants protect against the toxicity of TQ…………………………………..
89
3.3.2 Thymoquinone stimulates generation of reactive oxygen species (ROS) ……….
89
3.3.3 Induction of ROS generation causes cell death…………………………………...
92
3.3.4 Thymoquinone depletes cellular glutathione……………………………………...
94
3.3.5 Thymoquinone causes depolarization of mitochondria…………………………...
96
3.3.6 Characterization of the ALO1 in C. glabrata………………………………………….. 98
3.3.7 Candida glabrata transformants produce higher amounts of EASC……………..
104
3.3.8 Effect of EASC over production on resistance to oxidative stress………………..
105
3.4 Discussion
3.4.1 Thymoquinone induces oxidative stress in C. glabrata……………………………… 107
3.4.2 Reduced glutathione protects from TQ oxidation………………………………..
108
3.4.3 Candida glabrata can produce intracellular D-erythroascorbic acid…………….
109
3.5 Conclusion………………………………………………………………………………
110
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4. Chapter 4: A model of hypo-osmotic stress to screen cell wall MAPK inhibitors in C.
glabrata.
4.1 Introduction……………………………………………………………………………..
111
4.2 Materials and methods
4.2.1 Strains and media………………………………………………………………….
114
4.2.2 Detection of Slt2 phosphorylation………………………………………………..
114
4.2.3 Viability assays……………………………………………………………………
116
4.2.4 Propidium iodide staining…………………………………………………………
116
4.2.5 Bio-protection assays……………………………………………………………...
117
4.2.6 Testing for apoptosis………………………………………………………………
118
4.2.7 Toxicity of compounds on cell’s survival in water……………………………….
119
4.2.8 Statistical analysis…………………………………………………………………
119
4.3 Results
4.3.1 Thymoquinone, hydrogen peroxide and hypo-osmolarity induces SLT2
phosphorylation in C. glabrata………………………………………………………………….
120
4.3.2 Cell viability and cell density…………………………………………………….
122
4.3.3 Bio-protection…………………………………………………………………….
125
4.3.4 Apoptotic cell death………………………………………………………………
130
4.3.5 Effect of Pkc1 inhibitor on C. glabrata in water…………………………………
134
4.3.6 Toxicity of XMD8-92, sorafenib and doxycycline in water……………………..
136
4.3.7 Doxycycline induces Slt2 phosphorylation……………………………………….
137
4.3.8 Toxicity of TQ on cells in water………………………………………………….
140
4.4 Discussion
4.4.1 The hypothesis of inhibiting CWI-MAPK to improve TQ efficacy………………
142
4.4.2 Candida glabrata survival in hypo-osmolarity…………………………………… 142
4.4.3 Apoptotic cells death in water…………………………………………………….
143
4.4.4 Cells in water as a model for screen CWI-MAPK inhibitors……………………..
145
4.4.4.1 CWI-MAPK is necessary for maintaining viability in water……………..
145
4.4.4.2 Screening of CWI MAPK inhibitor………………………………………..
146
4.5 Conclusion………………………………………………………………………………
148
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CHAPTER 5: Effect of thymoquinone on the cell wall…………………………………….
149
5.1 Introduction…………………………………………………………………………….
149
5.2 Materials and methods
5.2.1 Yeast strains and media…………………………………………………………...
152
5.2.2 Testing sensitivity of CWI-MAPK mutants to thymoquinone and hydrogen
peroxide………………………………………………………………………………….
153
5.2.3 Observation of TQ’s fluorescence and PI staining……………………………….
153
5.2.4 Qualitative analysis of chitin levels………………………………………………
154
5.2.5 Testing the interaction between thymoquinone and chitin inhibitor……………..
154
5.3 Results
5.3.1 Activity of SLT2 is required for hydrogen peroxide resistance but not
thymoquinone……………………………………………………………………………
155
5.3.2 Thymoquinone generates a blue fluorescence on C. glabrata cell wall…………..
157
5.3.3 Thymoquinone stimulates chitin production………………………………………
160
5.3.4 Chitin mutants are more resistant to thymoquinone but not hydrogen peroxide….
161
5.3.5 Chitinase inhibitor antagonise with thymoquinone………………………………..
162
5.4 Discussion
5.4.1 Cell wall integrity MAPK and TQ antifungal effect……………………………...
166
5.4.2 Thymoquinone targets chitin……………………………………………………...
167
5.4.3 Chitin, CWI-MAPK and TQ sensitivity…………………………………………...
171
5.5 Conclusion………………………………………………………………………………
172
CHAPTER 6: Final conclusions…………………………………………………………….
172
References…………………………………………………………………………………..
177
Appendix A…………………………………………………………………………………. 204
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LIST OF FIGURES
Figure 1.1
The cell wall structure in Candida spp…………………………………..........
12
Figure 1.2
Cell wall integrity (CWI) signalling pathway in Candida glabrata………….
14
Figure 1.3
Thymoquinone chemical structure, Nigella sativa seeds (black seeds) and
Nigella sativa flowers………………………………………………………..
16
Figure 1.4
Oxido-reduction cycling of TQ……………………………………………...
23
Figure 2.1
Calgary Biofilm Device (CBD)……………………………………………...
29
Figure 2.2
Polypropylene biofilm reactor mimicking CBD consists of a 200 µl pipette
tips box and 0.4 ml PP microcentrifuge tubes……………………………….
Figure 2.3
37
Diagram summarising steps of C. glabrata biofilm establishment in PP
reactor and treating them with TQ. ……………………………………….…
41
Figure 2.4
In vitro kinetics of fungicidal activity of thymoquinone against C. glabrata.
47
Figure 2.5
Population densities versus concentrations of TQ…………………………...
48
Figure 2.6
Adhesion of C. glabrata isolates and C. albicans ATCC14053 to
polysterene…………………………………………………………………………..
50
Figure 2.7
Effect of washing process on C. glabrata A2a2 biofilms architecture……...
51
Figure 2.8
Establishment of biofilm by the isolates on the surface of PP microcentrifuge
tubes after shaking for 2 h in media………………………..
Figure 2.9
Comparisons between biofilm formation of A2a2 strain at different sites in
the PP reactor………………………………………………………………...
Figure 2.10
55
Scanning electron micrograph of Candida glabrata clinical isolate (A2a2)
biofilm formed on the biofilm reactor……………………………………….
Figure 2.12
54
Biofilms of C. glabrata ATCC2001 and the clinical isolate C. glabrata
A2a2…………………………………………………………………………………..
Figure 2.11
53
57
Activities of different concentrations of thymoquinone (TQ) against C.
glabrata A2a2 biofilms……………………………………………………...
58
Figure 2.13
Monitoring of metacaspase1 activation and cell membrane integrity……….
61
Figure 2.14
Monitoring the cell cycle of TQ treated cells by using DAPI………………..
62
Figure 3.1
Schematic diagrams of ALO1 plasmid constructs developed in this study…..
83
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Figure 3.2
Elimination of TQ toxicity using 10 mM of reduced glutathione and ascorbic
acid…………………………………………………………………………….. 88
Figure 3.3
Thymoquinone induces ROS production in C. glabrata ATCC2001…………
89
Figure 3.4
Pro-oxidant effect of thymoquinone leads to cell death……………………….
91
Figure 3.5
Thymoquinone caused a depletion in reduced glutathione in C. glabrata
ATCC90030…………………………………………………………………… 93
Figure 3.6
Thymoquinone causes a decrease in mitochondrial membrane potential…..
Figure 3.7
Digestion of HHT2-ALO1 vector with XhoI and XmaI enzymes resulted in a
band of approximately 1.6 kb…………………………………………….........
Figure 3.8
95
96
Alignment of CAGL0H04125g ORF published in the CGD with the
sequenced insert (16a) in this study…………………………………………… 97
Figure 3.9
ALO1 protein sequence which resulted from translation of the sequenced Cg
ALO1 gene in this study………………………………………………………. 100
Figure 3.10
Diagram represents FAD binding domain (interval 25-161) and ALO1
(interval 188-510) domain, which is specific to D-arabinono-1,4-lactone
oxidase, in the submitted C. glabrata ALO1 sequence…………………….....
Figure 3.11
100
Multiple ALO1 protein sequence alignments in C. glabrata ATCC2001
(CAGL0H04125g) with S. cerevisiae (YML086C) and C. albicans
(orf19.7551)………………………………………………………….
Figure 3.12
The Extracted Ion Chromatogram (EIC) for protonated EASC and ESI-MS
spectra…………………………………………………………………………
Figure 3.13
101
103
Sensitivity of C. glabrata 16a to H2O2 and TQ comparing with the wild type
C. glabrata ATCC2001(WT) and the control strain (C. glabrata
HHT2)…………………………………………………………………………. 104
Figure 4.1
Induction of SLT2 phosphorylation by TQ, H2O2 and water treatment………
119
Figure 4.2
The relationship between cell density and cell survival in water. …………….
121
Figure 4.3
Analysis of cell viability by flow cytometry…………………………………..
122
Figure 4.4
UV-CD spectra of high cell density filtrates…………………………………..
124
Figure 4.5
Bio-protection of low cell density by filtrates of high cell density……………
125
Figure 4.6
Protection of cells against hypo osmolarity by sorbitol or by diluted yeast
minimal media (YMM)………………………………………………………..
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Preface
Figure 4.7
Effect of dialysis on survival of high cell density in hypo-osmolarity………..
Figure 4.8
FITC-annexin V staining on high density cells dialysed for 3 days to examine
127
externalization of phosphatidylserine…………………………………………. 130
Figure 4.9
Fluorescence microscopy and flow cytometry analysis of DAPI staining to
cells growing in rich medium and cells dialysed in water…………………….
Figure 4.10
Low concentrations of staurosporine (Sts) kills C. glabrata ATCC2001 in
water but not in RPMI1640 media…………………………………………….
Figure 4.11
131
133
Susceptibility of C. glabrataATCC90030 to doxycycline as tested with MIC
assay or in water……………………………………………………………….
135
Figure 4.12
Doxycycline induces SLT2 phosphorylation………………………………….
136
Figure 4.13
Effect of thymoquinone on cells survival in water……………………………
138
Figure 5.1
Susceptibility of C. glabrata Δbck1, Δmkk2, Δslt2 and their parental strains
HTL and TG11 to TQ (The two upper panels) and sensitivity of C. glabrata
Δslt2 (Cg Δslt2) to hydrogen peroxide (The panel at the bottom).…………… 153
Figure 5.2
Candida glabrata ATCC2001 treated with TQ emits blue fluorescence……..
Figure 5.3
Epi-fluorescence microscopy images demonstrate the binding of
thymoquinone to C. glabrata cell wall which leads to cell death…………….
155
156
Figure 5.4
Thymoquinone induces chitin production in C. glabrata……………………... 158
Figure 5.5
Sensitivity of C. glabrata CHS3 mutants to TQ and H2O2……………………
Figure 5.6
Microscopic examination of C. glabrata Δchs3A demonstrate failure in bud
Figure 5.7
160
scar septum formation…………………………………………………………
161
Nikkomycin Z rescues C. glabrata from TQ adverse effect………………….
162
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LIST OF TABLES
Table 1.1
Summary of the most relevant properties of thymoquinone……………
17
Table 1.2
List of selected studies demonstrating data on thymoquinone toxicity…
21
Table 2.1
Strains used in this study………………………………………….
34
Table 2.2
Minimum inhibitory concentration of thymoquinone and fluconazole…
46
Table 2.3
The variation of colonization capability on PP tubes surface by
different C. glabrata strains after 24 h………………………………….
55
Table 3.1
Strains used in this study………………………………………………..
76
Table 3.2
Primers used in this study……………………………………………….
80
Table 5.1
Strains used in this study………………………………………………..
149
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LIST OF ABBREVIATIONS
ANOVA
analysis of variance
ATCC
American Type Culture Collection
BCK1
Bypass of C Kinase
bp
base pairs
ºC
degree Celsius
CBD
Calgary Biofilm Device
CFU
colony forming unit
CDS
coding sequences
CLSI
Clinical and Laboratory Standards Institute
CGD
Candida genome database
CWI
cell wall integrity
d
day
DAPI
4,6-diaminido-2-phenylindole dihydrochloride
DNA
deoxyribonucleic acid
DMSO
dimethyl sulfoxide
DOX
doxycycline
EDTA
Ethylenediaminetetraacetic acid
EASC
D-erythroascorbic acid
ESEM
environmental scanning electron microscope
ERK
extracellular signal-regulated kinase
g
gram
h
hour
H2DCF-DA
2′,7′ dichlorodihydrofluorescein diacetate
IC50
The half maximal inhibitory concentration
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kb
kilobase
kDa
kilodaltons
L
litre
LD50
median lethal dose
M
molar
MAPK
mitogen activated protein kinase
MBEC
Minimum Biofilm Eradication Concentration
mg
milligram
MIC
minimum inhibitory concentration
min
minute
MilliQH2O
double-distilled water
MKK
Mitogen-activated Kinase Kinase
ml
millilitre
mM
millimolar
MW
molecular weight
MOPS
3-[N-morpholino] propanesulfonic acid
MTD
maximum tolerated dose
NEB
New England Biolabs, Inc.
OD600
optical density at 600 nm wavelength
ORF
open reading frame
PBS
phosphate buffered saline
PCR
polymerase chain reaction
PI
propidium iodide
PP
polypropylene
Raf
Rapidly accelerated fibrosarcoma
RLM1
Resistance to Lethality of MKK1P386 overexpression
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ROS
reactive oxygen species
rpm
revolutions per minute
RT
room temperature
RTK
receptor tyrosine kinase
SEM
standard error of the mean
Swi
Switching deficient
Slt2
Suppressor of the Lytic phenotype
sts
staurosporine
TOR
target of rapamycin
TQ
thymoquinone
t
time
Ura
uracil
UV
ultraviolet
W/V
weight/volume
WT
wild-type
xg
acceleration
µg
microgram
µl
microliter
µm
micrometer
µM
micromolar
xxi
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CHAPTER 1:
Introduction
The kingdom fungi contains yeast organisms responsible for human infections. The genus
Candida contains species responsible for the commonest forms of these infections. Over the past
three decades the incidence of Candida infections has considerably increased. This is attributed
to various factors including the increased numbers of immunocompromised patients (e.g. AIDS
and cancer patients), a growing aged population, the increased use of indwelling medical devices
and antibiotic misuse. Candida albicans is considered the main cause of candidiasis. However,
non-albicans Candida species such as Candida glabrata are important due to their increased
incidence of infection. In the 1960s Candida glabrata was known as a non-pathogenic fungus
(Fidel et al., 1999), however by the 1990s C. glabrata was reported as the 4th most common
cause of Candida blood stream infections, and in the 2000s it became one of the most common
cause of blood stream infections in the USA (Li et al., 2007). Additionally, C. glabrata infection
is of concern because of the organism’s ability to develop resistance towards common
antifungals (Silva et al., 2012), which necessitates a continued search for new antifungal agents.
Plant-derived antifungal agents (Phytochemicals) have been historically used to treat fungal
infections (Halberstein). Thymoquinone is one of these phytochemicals which showed several
pharmacological effects and can be extracted from Nigella sativa seeds (reviewed by
(Forouzanfar et al., 2014). This study investigates thymoquinone’s antifungal action particularly
towards C. glabrata.
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1.1 Candida glabrata
1.1.1 General characteristics of Candida glabrata
C. glabrata is a yeast that forms blastoconidia with cell sizes ranging from 1 to 4 µm in diameter
(Bialkova et al., 2006). It is a haploid yeast, petite-positive (can grow in the absence of oxygen
as a reduced colony size mutant) and genetically is considered to be the Candida species most
closely related to Saccharomyces cerevisiae (Barns et al., 1991, Marcet-Houben et al., 2009). C.
glabrata is an asexual yeast despite the presence of genes potentially responsible for conjugation
and mating switching (Bialkova et al., 2006). It has a genome of 12.3 Mb with 38.8% G+C
content, which is close to that of S. cerevisiae (38.3%). Its genome comprises 13 chromosomes
(sizes of
.5-1.4 Mb) and encodes 5283 coding sequences (CDS), with an average CDS size of
493 codons. Although the vast majority of S. cerevisiae genes have obvious C. glabrata
orthologs, C. glabrata has a simplified metabolic capacity when compared to S. cerevisiae,
probably because of its association with and adaptation to the mammalian host. For instance, the
C. glabrata genome has lost genes involved in galactose and sucrose assimilation, phosphate,
nitrogen and sulfur metabolism, as well as thiamine, pyridoxine and nicotinic acid biosynthesis
(Kaur et al., 2005). C. glabrata has been considered a non-dimorphic yeast. Thus C. glabrata
was classified historically under the genus Torulopsis. However, in 1978, it was demonstrated
that production of pseudohyphae was not a reliable factor to distinguish members of the Candida
genus. Consequently, T. glabrata was classified into the genus Candida (Fidel et al., 1999). In a
subsequent study to this reclassification, C. glabrata was found to be able to form pseudohyphae
in response to nitrogen source starvation which is commonly thought to arise from its search for
nutrients when they are limiting (Roetzer et al., 2011a).
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C. glabrata is a common cause of superficial and systematic infections including oral, vaginal
and urinary tract candidiasis and candidemia. Moreover its incidence has increased worldwide.
The International Fungal Surveillance Participant Group reported in 2004 that C. glabrata
comprises 7.5% to 18.3% of bloodstream fungal isolates in the study period 1992 to 2001
(Pfaller et al., 2004a). A recent Australian candidemia study reported that 32% of Candida
isolates were identified as C. glabrata (from 2004 to 2007 it was 15%) and this species was the
most resistant to antifungal drugs amongst other candida isolates tested (Chen et al., 2015).
The increased incidence of C. glabrata infections is of concern because of the organism’s ability
to rapidly develop resistance to antifungal compounds such as fluconazole, itraconazole,
voriconazole and amphotericin B (Foster et al., 2007, Diekema et al., 2002, Borst et al., 2005).
Resistance has also been found to develop after prolonged periods of azole prophylaxis such as
that used in hematopoietic stem cell transplant recipients (Bennett et al., 2004). Moreover,
following exposure to fluconazole C. glabrata can develop high-level resistance to other azoles
such as voriconazole: this is called cross-resistance (Panackal et al., 2006, Hitchcock et al.,
1993). Echinocandins (e.g. caspofungin) have generally retained excellent activity against C.
glabrata (Kauffman et al., 2014). However isolated cases of resistance have been also reported
against this antifungal group. Overall, the broad emergence of co-resistance over time to both
azoles and echinocandins in clinical isolates of C. glabrata has been reported (Pfaller et al.,
2012), creating a need for continued development of new antifungal drugs.
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1.1.2 Candida glabrata virulence factors
Virulence determinants are a subset of genes that have evolved and are primarily expressed in
vivo to promote survival, allow replication of the fungus and facilitate disease progression in the
mammalian host (Haynes, 2001). Compared with Candida albicans there is a limited number of
studies examining the virulence determinants of C. glabrata (Kaur et al., 2005). The
pathogenicity of C. glabrata has mainly been attributed to its ability to adhere, production of
tissue damaging hydrolytic enzymes and formation of biofilms on host tissue and medical
devices (Rodrigues et al., 2014).
1.1.2.1 Adherence and biofilm formation
Adherence of Candida glabrata cells to components of mammalian tissues has been
hypothesized to be important in virulence (Calderone et al., 2001). Its epithelial adhesion (EPA)
protein family is a specific family of glycosylphosphatidylinositol (GPI)-anchored cell wall
proteins which are involved in mediating adhesion to human epithelial cells in vitro. The
genomes of CBS138 and the BG2 strains contain at least 17 and 23 EPA-related genes,
respectively. However, only three of them (EPA1, EPA6 and EPA7) have been shown to be
involved in adhesion (Kaur et al., 2005, Maestre-Reyna et al., 2012, Caudle, 2010). EPA1 is a
lectin that binds to host-encoded N-acetyl lactosamine-containing glycol conjugates in human
epithelial cells (HEp2) (Cormack et al., 1999). The other two adhesins EPA6 and EPA7 are more
involved in C. glabrata biofilm formation (Iraqui et al., 2005). Deletion of EPA1 reduces
adherence in vitro and is associated with a reduction of expression of other EPA genes. The EPA
genes are localized to the sub-telomeric regions of chromosomes and thus their expression is
controlled by SIR and RIF genes (termed as sub-telomeric silencing). Therefore, C. glabrata
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sir3∆ and rif1∆ strains showed increased colonization of the kidney in a murine model after
increased expression of EPA6 and EPA7 (Castano et al., 2005). More recently it has been found
that Cst6p also regulates expression of EPA6 and consequently biofilm formation (Riera et al.,
2012).
Another four adhesion-like proteins, Awp1/2/3/4 have been identified by de Groot’s group
(2008). These proteins were also implicated in C. glabrata adherence to human epithelia and
biofilm formation. The presence of these proteins in the cell wall depended on the growth phase
and on the genetic background that was used, and that in turn affected the adhesion capacity and
cell surface hydrophobicity (de Groot et al., 2008). This study demonstrated that Awp2, Awp3,
Awp4, and Epa6 were present in C. glabrata ATCC2001 but not in ATCC90876. In contrast,
Awp1 was found only in C. glabrata ATCC90876. In addition, Awp3 was identified only in logphase cells while Awp4 and Epa6 were identified only in stationary-phase cells (de Groot et al.
2008). YPS genes encoding extracellular glycosylphosphatidylinositol-linked aspartyl proteases
were also found to be important for adherence to mammalian cells, cell wall integrity and
survival in macrophages (Kaur et al., 2007). In addition, the chromatin remodelling Swi/Snf
complex has been shown to regulate positively biofilm formation in C. glabrata (Riera et al.,
2012). All these facts suggest a contribution of these adhesion (-like) genes of C. glabrata to its
adaptability and virulence in vivo.
Since C. glabrata cells are able to adhere and surround themselves by an external matrix, it has
been widely accepted that the pathogenesis of C. glabrata infections is related also to their
ability to form biofilms. This property is a strain-dependent ability which is mediated by the
expression of a large number of adhesins (Almshawit et al., 2014, de Groot et al., 2013, Riera et
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al., 2012). Candida biofilms may adhere to abiotic medical device materials and subsequently
invade human tissues and the immune system mechanism. Furthermore, biofilms are believed to
cause elevation in antifungal treatment resistance (Seneviratne et al., 2008a) and help them
withstand the competitive pressure from other microorganisms in comparison to their planktonic
cells counterpart (Mohammadi et al., 2012, Nett et al., 2007b, Toulet et al., 2012). Moreover,
adhesion and dispersion of cells from biofilms are an important part of the biofilm development
cycle which is associated with disseminated invasive diseases such as candidemia (Braga et al.,
2008, Uppuluri et al., 2010a). Therefore, antibiotic treatment of biofilm-related catheter infection
is difficult and usually requires implant replacement which entails an additional cost and
discomfort for the patient (Kucharikova et al., 2015). In talking about C. glabrata biofilm
virulence, the role of mixed organism biofilm pathogenicity should not be ignored. For instance,
for mixed C. albicans and C. glabrata biofilm it is thought that C. glabrata takes advantage of
the C. albicans hyphae extension that penetrates and invades into the host tissue (Coco et al.,
2008). In contrast, mixed bacterial biofilms (e.g. Lactobacilli sp. or Pseudomonas aeruginosa)
inhibited C. glabrata biofilm formation (Bandara et al., 2010, Chew et al., 2015).
1.1.2.2 Pigment production
Pigment production is thought to protect fungal cells from the host defence system (Wang et al.,
1995). Indole-derived pigment was described for C. glabrata by Mayser and colleagues (2007).
They described this pigment as similar to that produced by the skin pathogen Malassezia furfur.
It is a by-product of tryptophan catabolism via the Ehrlich pathway when tryptophan is provided
as a sole nitrogen source (Mayser et al., 2007). Indeed there is no direct evidence of this
protection in vivo where systemic C. glabrata infections may originate from niches where
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tryptophan and other nitrogen sources are readily available (Brunke et al., 2010). Nevertheless
compared to non-pigmented cultures, in vitro pigmented C. glabrata cultures exhibited decreased
killing by hydrogen peroxide, enhancement of protection against oxidative burst inside the
neutrophil phagosomes and caused increased damage of human epithelial cells in a monolayer
infection model (Brunke et al., 2010). This protection is similar to that provided by melanin in
fungi such as Cryptococcus neoformans or Aspergillus fumigatus (Jacobson, 2000, Wang et al.,
1995).
1.1.2.3 Morphology and Phenotypic switching
C. glabrata clinical isolates have been shown to have a limited capacity to form pseudohyphae,
indicating that the ability of this species to form true hyphae is not essential for its pathogenesis
(Gow et al., 2002, Grubb et al., 2008). However Roetzer and colleagues (2011a) showed that
colonies with ability to form pseudohyphae exhibited an advantage in colonizing the liver and
spleen in a mouse model. These colonies were isolated on CuSO4-containing agar as dark brown
C. glabrata colonies.
Defects in cell separation may also enhance the virulence of C. glabrata. In a study that
investigated the deletion of ACE2 (part of the RAM network regulation of Ace2 activity and
cellular morphogenesis) on budding patterns, the mutant showed a significant increase in
virulence in a murine model of disseminated candidiasis (Kamran et al., 2004). This was thought
to be due to a defect in cell separation and this resulted in the growth of cells in large clumps.
Although the aggregates did not cause vascular occlusion, the virulence was related in part to an
increase in pro-inflammatory cytokine levels including alpha tumor necrosis factor and
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interleukin-6. However, the specific downstream targets of C. glabrata ACE2 that account for
the proinflammatory phenotype of the ace2 mutant are not known yet (Kamran et al., 2004).
1.1.2.4 Enzyme production
Candida glabrata is not major producer of secreted enzymes in vivo, but recent studies have
described production of hemolysins and phospholipases. The genetic expression of hemolytic
activity in C. glabrata is however poorly understood. Candida glabrata has no known receptors
for haem (Nevitt et al., 2011), but like many other Candida species C. glabrata can obtain
elemental iron from host cells by degrading haemoglobin using haemolysins. Interestingly, in
vitro production of hemolysins is strain-dependent and this might be a reason for variation in
virulence between C. glabrata strains (Luo et al., 2004). Candida glabrata also produces
phospholipases which hydrolyze phospholipids into fatty acids (Mohan das et al., 2008). Their
production destroys the host mucosa and aids effective invasion of the involved tissues
(reviewed by Rodrigues et al., 2014).
According to Polakova et al., (2009) C. glabrata isolates can also alter their chromosome
number by duplication of chromosome segments carrying a centromere and subsequently adding
novel telomeric ends. This plasticity is associated with antifungal drug resistance and it seems to
be an advantage in colonizing the human body where there is much fluctuation in the
environmental conditions (Poláková et al., 2009).
1.1.3 Host interaction
Infection of mucosal surfaces with Candida glabrata has been reported in the mouth,
oesophagus, intestines, and vagina. Since different Candida species can be commensals of
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human mucosae, the origin of candidiasis is considered to be mainly endogenous. The
establishment of Candida glabrata infection in vivo requires four essential steps: adherence,
invasion, immune interaction and efficient nutrient acquisition (Brunke et al., 2014, Seider et al.,
2014).
As described in a previous section, C. glabrata has a large family of genes called EPA which are
essential for expressing different adhesions to different epithelial human cells. Based on in vitro
studies it seems to be that the host environment signalling controls the expression of EPA genes.
For example, Sir complex-mediated transcriptional silencing requires NAD+ as a cofactor, thus
there is an indirect influence on the silencing activity by external nicotinic acid concentrations.
This acid is present in low concentrations in the human urinary tract. Deficiency of nicotinic acid
decreases SIR2 activity resulting in derepression of EPA6. This leads to an increased adherence
of C. glabrata to host tissues and thus C. glabrata infection thrives in this niche (reviewed in
(Brunke et al., 2013, Domergue et al., 2005). In addition, exposure to preservatives such as
sorbic acid and parabens induces EPA6 expression via activation of the transcription factors Flo8
and Mss11 which control weak organic acid stress. These preservatives are used often in food
and health products including over-the-counter vaginal products. Candida glabrata cells also
have better chance to adhere to vaginal epithelium because of the low pH which leads to
activation of Flo8 and Mss11 transcription factors and consequently an induction of EPA6
expression (Mundy et al., 2009, Tscherner et al., 2011).
Little is known about the C. glabrata invasion mechanism. It is likely to occur accidently or
iatrogenically in susceptible individuals. Infection can be transmitted from contaminated
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catheters or surgically related inoculation. C. glabrata can also penetrate across the epithelial
surfaces as an association with the absorption of other microbes, mainly C. albicans (Coco et al.,
2008, Silva et al., 2011). According to Coco et al. 2008 it is likely that C. albicans hyphae help
to maintain the structural integrity of denture-associated biofilm, and form a sanctuary for the
smaller C. glabrata cells. Silva and colleagues (2010) have also shown by fluorescence
microscopy how C. glabrata is transported to deeper tissue layers through adherence to C.
albicans.
Invasion of naked epithelial surfaces via trauma or/and endocytosis is another proposed way of
invasion (reviewed in Brunke & Hube, 2013). Once C. glabrata enters the bloodstream, it starts
to employ immune evasion strategies (Seider et al., 2014). While strong neutrophil infiltration is
characteristic for C. albicans infections, C. glabrata infection is associated with stimulation of
mononuclear cells rather than neutrophil attraction (reviewed in (Brunke et al., 2013, Jacobsen et
al., 2010, Westwater et al., 2007). This observation is attributed to the fact that C. glabrata
induces more GM-CSF than C. albicans and less of the other inflammatory cytokines like IL-1a
or Il-8 (reviewed by (Brunke et al., 2013). According to Keppler-Ross et al., (2010), C. glabrata
is recognized and phagocytosed by macrophages more frequently than C. albicans. The yeast
form is more susceptible to uptake by macrophages compared to hyphae. This selectivity seems
to be mannan dependent, but independent of glucan or chitin (Keppler-Ross et al., 2010).
Candida glabrata exploits the macrophage’s phagosomes as niches for replication until the
phagosome bursts and releases the pathogen. To be able to survive in the harsh environment of
phagosomes, intracellular survival strategies have evolved. One of these strategies is to repress
and detoxify reactive oxygen species that can be produced by primary human macrophages. In
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addition, intracellular persistence is an important strategy to hide from other branches of the
immune system such as neutrophils and humoral immune components. Candida glabrata has
been shown to modify the phagosome into a non-acidified environment and alter phagosomal
biogenesis via arresting of phagosomal maturation in a late endosomal stage to prevent fusion
with lysosomes. In this manner, C. glabrata can multiply until the host cell finally lyses and
releases the fungi (Kasper et al., 2014, Seider et al., 2011).
Efficient nutrient acquisition is another crucial factor for survival inside the phagosomes. In
contrast with C. albicans which escapes from the nutrient-poor environment in the phagosome,
C. glabrata depends on autophagy to survive inside the phagosome. It remodels its chromatin to
facilitate reprogramming of cellular energy metabolism which also provides protection against
DNA damage (Rai et al., 2012, Seider et al., 2014). C. glabrata might obtain nitrogen from
amino acids which are present in free form in low millimolar concentrations at different host
sites such as urine or blood in the same way that C. albicans liberates these amino acids from
proteins and peptides through the action of hydrolases and uptake them using permeases (Brunke
et al., 2014). However, it is not clear yet how this microorganism can obtain enough other
nutrients such as a carbon source, minerals and vitamins (Brunke et al., 2013). Therefore, more
C. glabrata infection studies are required to detail events inside the cell, organ and/or organism
(Rodrigues et al., 2014).
1.1.4 Cell wall structure and cell wall integrity MAPK
The cell wall determines the shape of the fungal cell. It is essential for the integrity,
environmental stress response and recognition by host cells. Common to many other fungi the
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major constituents of the cell wall in C. glabrata are mannoproteins and polysaccharides (Ueno
et al., 2011). The polysaccharides include β-1,3-glucan and β-1,6-glucan linked to lower levels of
chitin (1.4–7.1% of the entire cell wall mass (Aguilar-Uscanga et al., 2003)) via β-1,4-glucan
(Fig. 1.1). Chitin forms the innermost part close to the cell membrane and β-(1,6)-glucan
linkages/structure are displayed outwards to link with the outer cell wall mannoproteins. Some of
the chitin and glucan chains may extend throughout the entire depth of the cell wall structure and
chitin is concentrated mostly in the septa ( > 90%) (Cabib et al., 2013, Molano et al., 1980). The
cell wall forms about 20% of the cell's dry weight in C. glabrata and it appears to have 50%
more mannoprotein and a thinner glucan layer than baker's yeast and C. albicans. Indeed, βglucans in the cell wall of C. glabrata may be more effectively masked from host immune
recognition by the receptor dectin-1 than in C. albicans (Cabib et al., 2013, de Groot et al., 2008,
Santos et al., 2000, West et al., 2013). Nevertheless, as previously mentioned C. glabrata is more
recognizable to macrophages than C. albicans and this might be attributable to their cell wall’s
higher content of mannan (Keppler-Ross et al., 2010).
Mannoproteins
β-1,6 glucan (linkers)
β-1,3 glucan
Chitin (skeleton)
Figure 1.1 The cell wall structure in Candida spp.
Adapted from Prof. Neil Gow’s profile < http://www.abdn.ac.uk/ims/profiles/n.gow> , viewed 17th August 2015
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Taking S. cerevisiae as model yeast, cell wall stress recognition is mediated through the response
of a specific cell wall integrity (CWI) MAPK pathway which is known also as Pkc1-MAPK. As
reviewed by Levin (2011) this signal transduction pathway activates an adaptive transcriptional
response. Environmental stress is detected mainly via Wsc1 and Mid2 sensors. Then Rom1/2, the
guanine nucleotide exchange factors, will be recruited and consequently trigger the GTPase
Rho1. Rho1 activates Pkc1which triggers the MAPK cascade. The linear pathway consisting of
the MAPKKKK Pkc1, MAPKKK Bck1, MAPKKs Mkk1/2 and finally the mitogen activated
kinase, Slt2, which activates the transcription factors Rlm1 and Swi4/Swi6 (Fig. 1.2). Following
the activation of transcription factors the expression of cell wall biosynthesis genes will be
regulated to produce compounds that are involved in remodelling of the yeast cellular envelope
(reviewed in Levin et al., 2011). Such genes are CHS1, MNN10 and CWH41 (Schwarzmuller et
al., 2014). A search through the Candida Genome Data Base (CGD) reveals that all of the S.
cerevisiae genes encoding CWI MAPK proteins have homologs in C. glabrata except for the
Mkk1 homolog.
Activation of CWI MAPK has been demonstrated to be inducible under conditions that stress the
cell surface, mainly under hypo-osmotic shock. However, due to the conversation between
MAPK pathways. CWI can be activated under other stresses such as growth at high
temperatures, hypo-osmotic shock, polarized growth, actin perturbation, oxidative stress or the
presence of compounds or mutations that interfere with cell wall biosynthesis (Krasley et al.,
2006a, Soriano-Carot et al., 2012, Krasley et al., 2006b, Martin et al., 2015, Fuchs et al., 2009).
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Wsc1 Wsc2 Wsc3 Mid2 Mtl1
(stress sensors)
Rom1/2
Rho1
PKC pathway
Pkc1
MAP4K
Bck1
MAP3K
Mkk2
Slt2
MAP2K
MAPK
Nucleus
Rlm1
SBF (Swi4/Swi6)
Skn7
Figure 1.2 Cell wall integrity (CWI) signalling pathway in Candida glabrata. Signals are
initiated at the stress sensors which are located at the plasma membrane. The extracellular
domains of these proteins recruits the Rom1/2 GEFs to the plasma membrane, then the sensors
stimulate nucleotide exchange on Rho1. The Rho1 activates the Pkc1-MAPK cascade. The
transcription factors Rlm1 and SBF (Swi4/Swi6) are activated by the pathway. Skn7 may also
contribute to the CWI transcriptional program (dashed line). The summary of the core elements
of Pkc1-MAPK pathway was adapted from (Levin, 2011) except Mkk1 protein which was
excluded from C. glabrata in this study.
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1.2
Thymoquinone
1.2.1 Thymoquinone in Nigella sativa
The Nigella sativa seed (also called black cumin, or black seed) is a member of Ranunculaceae
family (Fig. 1.3). The seed produces an annual flowering plant native to Mediterranean
countries, Pakistan and India. The black seed has an interesting cultural history that traces back
to the Egyptian Dynasty, having been discovered in Pharaoh Tutankhamen’s tomb (Botnick et
al., 2 12). In Hebrew the black seed is called “Ketzah” (Bible, in the book of Isaiah 28:25-27)
and sprinkling the seeds over bread, cake and flavouring dishes is a postbiblical custom (Botnick
et al., 2012). In Islamic culture it is known as “The blessed seed” and there is an ideological
belief that this seed can cure different diseases. This belief is based on the words of Mohamad
"There is healing in black seed for all diseases except death" * (Buḫārī et al., 1997, Duke, 2
7).
The biological activity of Nigella sativa seeds is mainly attributed to components of its essential
oil including p-cymene, carvacrol, thymo-hydroquinone, dithymoquinone (nigellone), thymol,
nigellicine, nigellidine, α-hedrin and thymoquinone (TQ) (Botnick et al., 2012). Thymoquinone
is considered the major active ingredient of black seeds and most of the known biological actions
of this seed have been attributed to TQ (Darakhshan et al., 2015). There are monoterpene
composition changes upon seed maturation, in a pattern that reflects the proposed biosynthetic
pathway to thymoquinone (Botnick et al., 2012). Different studies have shown varying
concentrations of TQ in N. sativa essential oil. For instance, it has been found to constitute 27.8
% – 57 % (Ali et al., 2003) or 0.2 % w/v of the essential oil (Houghton et al., 1995). In another
study it was shown to constitute 7.9-13.8 % w/w of the seed (Botnick et al., 2012). This
* In other version “The black seed is healing for ..etc”
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difference might relate to the different sources of the seed and different extraction methods that
have been used.
(a)
(b)
(c)
Figure 1.3 (a) Thymoquinone chemical structure , (b) Nigella sativa seeds (black seeds) , (c)
Nigella sativa flowers.
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1.2.2 Chemical properties of thymoquinone
Thymoquinone (2-isopropyl-5-methylbenzo-1, 4-quinone) is present in the fixed oil of N. sativa
and is the most abundant constituent of its volatile oil. The most relevant properties of TQ are
summarized in table 1.1. The UV–visible spectra of TQ has one sharp peak (max) at 254–257
nm. Thymoquinone exists in tautomeric forms mostly as the keto form (∼90%) which is
responsible for the pharmacological properties of this compound. It is a hydrophobic molecule
and its solubility depends on time. Thymoquinone is light sensitive and unstable in aqueous
solutions, particularly at an alkaline pH (reviewed by(Darakhshan et al., 2015, Salmani et al.,
2014). As TQ is soluble to about 5
μg/ml after 24 h, the aqueous solubility is not a major
hindrance for drug formulations. This solubility might be sufficient to exert a pharmacologic
effect if administered via the parenteral route (Salmani et al., 2014).
Table 1.1 Summary of the most relevant properties of thymoquinone. Adapted from (Darakhshan et al.,
2015).
IUPAC name
2-Isopropyl-5-methylbenzo-1,4-quinone
Molecular formula
C10H12O2
Composition
C (73.15%), H (7.37%), O (19.49%)
Molar mass
164.2011 g/mol
Appearance
Crystalline and dark yellow
CAS number
490-91-5, 73940-92-8
PubChem CID
10281
Melting point
44-47 ºC
Boiling point
230-232 ºC
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1.2.3 Thymoquinone toxicity
There have been a number of reports investigating in vitro and in vivo measurements of
thymoquinone toxicity. Reported data regarding the maximum tolerated dose (MTD) for TQ in
cell lines and animals is presented in table 1.2. As an example, 32.8 µg/ml of TQ was not toxic to
a normal human lung fibroblasts cell line (80% viability) at the highest concentration used in that
study (Gurung et al., 2010). Another study showed erythrocyte death following treatment with ≥
1.6 µg/ml (Qadri et al., 2009). Normal human cervical squamous cells were sensitive to about 18
µg/ml after 72h (IC50) of treatment with thymoquinone. In the same study, thymoquinone was
less toxic to normal cells than cisplatin (IC50 = 2.8µg/ml) which is a known treatment for human
cervical squamous carcinoma. Thymoquinone showed higher efficiency in killing these cancer
cells as well (Ng et al., 2011). Animal models differ in their toxicity patterns depending on the
animal type and mode of administration. In mice, the lethal dose (LD50) of TQ after
intraperitoneal injection was 104.7 mg/kg and after oral ingestion 870.9 mg/kg. The LD50 in rats
after intraperitoneal injection was determined to be 57.5 mg/kg (Al-Ali et al., 2008). Another
study on TQ toxicity in rats suggested lower maximum tolerated dose (MTD) values for
intraperitoneal injections. Values obtained were 22.5 mg/kg in male rats and 15 mg/kg in
females, whereas for oral ingestion it was 250 mg/kg in both male and female Wistar rats
(Abukhader, 2012). More recently, in mice, TQ cream was used safely for vaginal candidiasis at
concentrations of 10% (Abdel Azeiz et al., 2013).
A suitable dose for the whole seed extract in humans can be extrapolated from animal studies
based on the human equivalent dose of no observed adverse effect level (NOAEL). The human
dose was estimated to be approximately 0.6 mg/kg/day oral TQ or . 5 ml/kg/day oral of N.
sativa extract reported in a study of postmenopausal women and this study reported no side
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effects (Shuid et al., 2012). A chronic toxicity study has been performed using N. sativa as an
adjunct to therapy for Helicobacter pylori. In humans it was found that 3 g per day for 4 weeks
was considered safe (Salem et al., 2010). Alkharfy et. al., 2014 used an equation of allometric
relationship in animals that can be used to predict a starting dose in man:
Doseman=Doseanimal*(Wtman/Wtanimal)0.75
(Wt = weight)
Based on the effective dose obtained from mouse model in their study and according to the body
weight ratio of man to mouse, the expected effective dose in humans was between 0.05 and 0.25
mg/kg. In conclusion these studies suggest a wide margin of therapeutic safety for thymoquinone
doses.
1.2.4 The therapeutic potential of thymoquinone
Advances in technology have allowed the identification of the active components in Nigella
sativa extracts. Despite lack of scientific verification of their effectiveness and safety, they were
used as treatment of different diseases (Al-Rowais, 2002).
Since the first extraction of thymoquinone by El-Dakhakhany in 1963 many studies have tested
this compound for its therapeutic efficacy (El–Dakhakhny, 1963, Woo et al., 2012a). The antioxidant and anti-inflammatory effects of thymoquinone have been reported in various disease
models, including diabetes, asthma and encephalomyelitis (reviewed by Woo et al., (2012). It has
been reported to act as a free radical and superoxide radical scavenger, as well as maintaining the
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Chapter1
activity of various anti-oxidant enzymes such as glutathione peroxidase, catalase and
glutathione-S-transferase (Badary et al., 2003, Nagi et al., 2009). On the other hand, as a quinone
TQ can undergo enzymatic or non-enzymatic redox cycling with their corresponding
semiquinone radicals to generate superoxide anion radicals (Fig. 1.4) (Bolton et al., 2000). This
induction of reactive oxygen species (ROS) generation has been reported in various studies (ElNajjar et al., 2010, Dergarabetian et al., 2013). There is reported consensus that the antioxidant
or pro-oxidant properties of TQ are dependent on the milieu of the interaction (El-Najjar et al.,
2010, Mansour et al., 2002, Staniek et al., 2010).
Thymoquinone has been found to inhibit different proinflammatory cytokines and chemokines
including, monocyte chemoattractant protein-1, TNF-α, IL-1β, COX-2 and iNOS. It inhibits the
activation of transcription factor NF-кB, ERK and Akt signaling pathways and its effect was
more dramatic than the specific histone deacetylases (HDAC) inhibitor-Trichostatin A (Chehl et
al., 2009, Woo et al., 2012a, Darakhshan et al., 2015). In addition the studyn of Alkharfy et al.,
(2011) has demonstrated the protective effect of TQ against sepsis induced by E. coli and LPS in
a mouse model.
Alleviation of oxidative stress and inflammation is an important strategy that could be affected
by TQ. This might help to prevent various chronic diseases such as bone and joint complications,
respiratory diseases such as asthma and dyspnoea, gastric dysfunction and stomach ulcer,
nephrotoxicity, Aβ-induced neurotoxicity and other diseases (reviewed by Darakhshan et al.,
2015).
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Chapter1
Table 1.2 List of selected studies demonstrating data on thymoquinone toxicity.
In vitro studies
Tested Model
Toxic dose of TQ
Reference
Normal human lung
32.8 µg/ml resulted in 80% viability
(Gurung et al., 2010)
Erythrocyte cells
≥ 1.6 µg/ml (suicidal)
(Qadri et al., 2009)
Normal human cervical
18 µg/ml (IC50 after 72h)
fibroblasts cell line
squamous cells
Normal human osteoblasts
~ 6.5 µg/ml (IC50 after 24h)
(Roepke et al., 2007)
Human pancreatic ductal
8.2 µg/ml resulted in 80% viability after 72h.
(Banerjee et al., 2009)
˃ 16 µg/ml
(Vance et al., 2008)
epithelial cells
Monkey kidney epithelial
cells
In vivo studies
Mode of dministration
Toxic dose
Reference
Mice
Intraperitoneal injection
104.7 mg/kg (LD50)
(Al-Ali et al., 2008)
Mice
Intraperitoneal injection
5 mg/kg were safe
(Khan et al., 2015)
Mice
Oral ingestion
870.9 mg/kg (LD50)
(Al-Ali et al., 2008)
Mice
Oral ingestion
2.4 g/kg (LD50)
(Badary et al., 1998)
Mice
Cream
˃ 10%
(Ahmed
Z.
Abdel
Azeiz, 2013)
Rat
Intraperitoneal injection
57.5 mg/kg (LD50)
(Al-Ali et al., 2008)
Rat
Intraperitoneal injection
22.5 mg/kg (male)
(Abukhader, 2012)
15 mg/kg (Female)
Oral ingestion
250 mg/kg (male & female)
Rat
Oral ingestion
Human
Oral
Human
Skin
Human
Oral
10 mg/kg/day was safe
(Kara et al., 2012)
.6 mg/kg/day (safe)
(Shuid et al., 2012)
1%
in
96%
ethanol
(53% (Coenraads
irrational)
1975)
400 mg/day (not toxic)
(Al-Amri
2009)
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et
et
al.,
al.,
Chapter1
Accumulation of superoxide may contribute to the pro-oxidant effect of TQ particularly in the
absence of detoxifying enzymes, which is common in numerous cancers. The anti-tumor effect
of thymoquinone has been investigated in tumor mice models for colon, fore stomach, prostate,
fibrosarcoma, Ehrlich ascites carcinoma, pancreatic, lung cancer and its toxicity towards breast
cancer cell lines has been frequently reported (reviewed by Banerjee et al., 2009, Motaghed et
al., 2013, Ng et al., 2011, Woo et al., 2012).
The combination of thymoquinone with conventional chemotherapeutic drugs could reduce the
toxicity of the latter and produces better therapeutic effects. Thymoquinone also increases the
radiosensitizing effect on human breast carcinoma cells. More importantly, TQ has showed some
selectivity to cancer cells compared to normal cells (reviewed in Rahmani et al., 2014).
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Chapter1
Figure 1.4 Oxido-reduction cycling of TQ. Thymoquinone can be converted to thymohydroquinone by a
two-step one-electron reduction, or by a one-step two-electron reduction enzymatic reaction. In one
electron reduction of TQ, microsomal NADPH cytochrome P450 reductase, microsomal NADH
cytochrome-b5 reductase, and mitochondrial NADH ubiquinone oxido-reductase, catalyse the conversion
of TQ to semiquinone. Semiquinone which is a pro-oxidant can be converted into thymohydroquinone.
Otherwise, a one-step reaction may cause direct formation of thymohydroquinone. A non-enzymatic
reaction also may take place and reduce TQ through interaction with GSH to generate glutathionyldihydro-TQ. The reduced products of TQ, glutathionyl-dihydro-TQ and thymohydroquinone are known
for their anti-oxidant activity. Superoxide anion generated through oxidation of reduced TQ can be
detoxified by SOD and CAT; Adapted from (Darakhshan et al., 2015).
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Chapter1
Banerjee et al., (2009) have reviewed the anticancer effect(s) of thymoquinone and reported that
these effects are mediated through different modes of action, including antiproliferation,
apoptosis induction, cell cycle arrest, ROS generation and anti-metastasis/anti-angiogenesis. This
quinone was found to exhibit anticancer activity through the modulation of multiple molecular
targets, including p53, p73, p16, PTEN, STAT3 and PPAR-g. It interferes with essential steps of
neovascularization from proangiogenic signalling to endothelial cell migration and tube
formation. Thymoquinone down-regulates the nuclear factor-κB (NF-κB), Bcl-2 family, and NFκB-dependent
anti-apoptotic genes
(X-linked inhibitors
of apoptosis,
survivin,
and
cyclooxygenase-2) which result in chemosensitization. The review by Rahmani et al., (2014) has
reported that TQ has a vital role in preventing cancer via modulation of phase I (cytochrome
p450) and phase II detoxifying enzymes (glutathione transferase and N-acetyl transferase).
Despite all of these promising data from in vitro and animal models studies, there has been no
reported phase I pilot studies to evaluate TQ’s promising anticancer effects in humans. One
study that has been reported by Al-Amri and Bamosa (2009) examined the effectiveness of oral
TQ as an anticancer drug in advanced malignant breast cancer patients. The authors reported
neither toxicity nor therapeutic response when patients were treated with 400 mg/day for 2
weeks. (AbuKhader, 2013) suggested that the low effectiveness could be due to the oral
administration. This might lead to TQ biotransformation due to induction of phase II enzymes in
the liver such as DT-diaphorase which catalyses two electron reduction of TQ into a
hydroquinone. Another reason could be that binding of TQ to serum proteins reduces its activity
in vivo. Thymoquinone-loaded poly (lactide-co-glycolide) nanoparticles and TQ-loaded
- 24 -
Chapter1
liposomes particles might overcome these challenges and these alterations can be applied
clinically to optimize the desired result to treat cancer patients (AbuKhader, 2013).
Thymoquinone has shown antimicrobial properties against bacteria including Staphylococcus
spp., Streptococcus spp., Salmonella spp., Pseudomonas aeroginosa, Escherichia coli, Shigella
flexneri, Klebsiella pneumonia, Mycobacterium phlei (an acid-fast bacterium), Vibrio
parahaemolyticus, Bacillus cereus, Listeria monocytogenes and shows weak antibacterial
activity against Enterococcus faecalis, Enterococcus faecium and
Streptococcus salivarius
(Chaieb et al., 2011, Halawani, 2009, Jrah Harzallah et al., 2011, Kouidhi et al., 2011, Shohayeb
et al., 2012). In addition, thymoquinone’s anti-parasitic activity against Plasmodium spp.,
Trypanosoma spp. and Leishmania spp. has been reported (reviewed in (Bero et al., 2014). In
another study a reduction in the cytogenetic damage caused by schistosomiasis infection has
been attributed to thymoquinone (Aboul-Ela, 2002). The anti-viral ability of N. sativa extracts
have been tested more frequently than purified thymoquinone. The whole seed antiviral property
was demonstrated against murine cytomegalovirus and hepatitis C virus in human and this
property was attributed mainly to its immune modulation ability (reviewed in (Forouzanfar et al.,
2014). In vitro, purified thymoquinone could efficiently inhibit the survival of Epstein-Barr
virus (EBV) infected B cells and altered EBV gene expression (Zihlif et al., 2013).
Thymoquinone’s antifungal property has been reported for dermatophyte molds (Aljabre et al.,
2005), Aspergillus niger (Al-Qurashi et al., 2007) and Candida albicans (Abdel Azeiz et al.,
2013). Aqueous and oil extracts of Nigella sativa have been found to inhibit the growth of C.
albicans (Bhuvan et al., 2010, Bita et al., 2012), C. parapsilosis, C. tropicalis, Aspergillus spp.,
Cryptococcus spp. and Issatchenkia orientalis (Bhuvan et al., 2010). Thymoquinone was found
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Chapter1
to be effective also against dairy spoilage yeast which has been suggested as useful for the dairy
industry as a chemical preservative of natural origin (Halamova et al., 2010). Other yeast
including C. glabrata and C. krusei have been not tested for their susceptibility to TQ.
In contrast to the amount of data which has been reported for thymoquinone antitumor mode of
action, there are few reports on the antimicrobial mode of action of TQ. An investigation into
thymoquinone’s mode of action as an antifungal compound will be a major focus of the current
study.
1.3 Rational and scope of this study
Candida glabrata causes life threatening disseminated candidiasis mainly in immunodeficient
individuals. Given thymoquinone’s broad pharmaceutical profile, it is likely that thymoquinone
can inhibit Candida glabrata growth and acts on one or more fundamental metabolic functions.
As thymoquinone may act as immunostimulator, have anticancer activities and be a
chemoprotective, it would be useful if it also simultaneously prevents Candida infection in those
more susceptible to fungal infections. In addition, therapeutic options available to treat fungal
infections are limited compared with antibacterials. Some antifungal treatments can trigger
adverse side-effects because of the close biological similarity between fungal and mammalian
cells. Furthermore, the emerging resistance of C. glabrata to antifungals further limits the
available options (Román et al., 2007). Chapter 2 investigates the antifungal properties of
thymoquinone towards different Candida species with more focus on C. glabrata planktonic
- 26 -
Chapter1
cells and biofilms. Chapter 2 also aims to optimise an in vitro model to stabilise C. glabrata
biofilms and test their sensitivity to thymoquinone.
Studying thymoquinone molecular targets in the context of drug-pathogen interactions may assist
with anti-fungal therapy and drug development. Yet relatively little is known about TQ effects
on the cellular level of fungi. Chapter 3 aims to report the effects that occur at the cellular level
of C. glabrata with regards to oxidative status.
Previous studies on Saccharomyces cerevisiae have demonstrated regulation of oxidative stress
response through Slt2p activation (a component of cell wall integrity (CWI) MAPK) (Krasley et
al., 2006a, Vilella et al., 2005). In C. glabrata activation of Slt2p is proposed in Chapter 4 as a
response to oxidative stress and a hypothesis is investigated. An important question to address in
this thesis is if TQ causes activation of CWI MAPK, does chemical inhibition of this signalling
pathway increase the efficacy of TQ?
Finally Chapter 5 further investigates this hypothesis by utilizing mutant strains of C. glabrata to
highlight interactions between the C. glabrata cell wall and thymoquinone.
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CHAPTER 2:
Antifungal effect of thymoquinone against Candida spp. planktonic cells and
C. glabrata biofilms
2.1 Introduction
Candida spp. are the third leading cause of infection resulting in catheter-related mortality after
Staphylococcus
aureus
and
coagulase-negative staphylococci
(Bouza et
al.,
2014).
Thymoquinone (TQ) has been shown to exert antifungal properties (Abdel Azeiz et al., 2013, AlQurashi et al., 2007, Bhuvan et al., 2010, Bita et al., 2012); however C. glabrata and other
Candida species such as C. krusei have not been tested for their sensitivity to TQ. Antifungals
such as azoles and echinocandins have been used safely against many Candida infections,
unfortunately C. glabrata more rapidly develops resistance to these antifungal agents than other
Candida species (Alexander et al., 2013, Chen et al., 2015, Pfaller et al., 2012, Posteraro et al.,
2006).
Therefore, development of new effective, affordable and safe antifungal agents is
required. Thymoquinone might be an option to develop such an antifungal drug.
Candidiasis which is caused most frequently by C. albicans, and to a lesser extent by C.
glabrata, C. tropicalis, or C. parapsilosis is often associated with the formation of a biofilm
extracellular matrix. Candida glabrata cells are able to adhere, surround themselves by an
external matrix and establish biofilms on surfaces of medical devices such as central venous and
urinary catheters (Nett et al., 2007a, Toulet et al., 2012, Mohammadi et al., 2012). The major
clinical relevance of Candida biofilms is their elevated antifungal resistance compared to their
planktonic counterparts (Seneviratne et al., 2008b). In addition, adhesion and dispersion of cells
- 28 -
Chapter2
from biofilms are an important part of the biofilm developmental cycle which is associated with
disseminated invasive diseases such as candidemia (Uppuluri et al., 2010a, Braga et al., 2008).
In vitro antimicrobial susceptibility broth dilution standard assays such as CLSI (Clinical and
Laboratory Standards Institute) and EUCAST (The European Committee on Antimicrobial
Susceptibility Testing) are methods developed for testing the susceptibility of planktonic cells
rather than the biofilm form.
In published studies, different groups showed significantly
different results for biofilms antifungal susceptibility. Despite using the same strain, the variation
was present because of different biofilm formation models and different viability measuring
assays (Maiolo et al., 2014, Ramage et al., 2001). Therefore there is effort by the researchers to
standardise models to test the susceptibility of biofilms to antimicrobial agents.
Several in vitro models have demonstrated the impact of different nutrients and surface materials
(in flow or static conditions) on adhesion and biofilm properties of several Candida species.
These models include a 96-well polystyrene plate, as well as discs of catheters made from
different substrates in 6- or 24-well plates and the Calgary Biofilm Device (CBD) (Tournu et al.,
2012, Ceri et al., 2001b). Many of them have been assembled from readily available laboratory
materials and showed good reproducibility (Uppuluri et al., 2010b). The CBD is one of the most
widely used methods for testing the susceptibility of microbial biofilm to antibiotics and toxic
compounds (Coenye et al., 2011). It consists of two parts: a plastic lid with 96 polystyrene pegs,
which sits in the wells of a standard 96-well plate (MBEC Assay System, Innovotech, Canada
(Harrison et al., 2006)). The CBD, shown in Figure 2.1, facilitates the production of 96 biofilms
grown on pegs. These biofilms are used in standard experiments for biofilm susceptibility and
minimum biofilm eradication concentration (MBEC) assays after exposure to toxic chemicals
(Ceri et al., 2001a).
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Chapter2
Figure 2.1 Calgary Biofilm Device (CBD)
Innovotech website <http://www.innovotech.ca/products_mbec.php>, viewed on 17th August
2015
Following formation of the biofilm, quantification is the critical step to obtain data on the effect
of different treatments on the biofilms. These are based on the quantitation of matrix and both
living and dead cells. Qualitative data can be obtained by using crystal violet staining which
binds to negatively charged surface molecules and polysaccharides in the extracellular matrix.
Qualitative data also can be obtained using fluorescent dyes such as syto9, which binds to DNA
and carbohydrate-specific dyes (e.g., calcofluor and concanavalin A, Con-A) (Braga et al., 2008,
Chandra et al., 2001). Imaging by scanning electron microscopy and measuring the dry weight of
the biomass have also been used to estimate biofilm growth and the architecture (Silva et al.,
2009).
Quantitation of viable yeast cells in biofilms can be determined by utilization of several dyes
such as fluorescein diacetate and tetrazolium salts. This salt is hydrolyzed by the intra- and
extracellular esterases of living cells to free fluorescein which exhibits a strong absorbance at
- 30 -
Chapter2
490 nm as well as an intense fluorescence. The tetrazolium salt XTT [2,3-bis (2-methoxy-4nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide is reduced inside
cells to a water-soluble formazan (da Silva et al., 2008). Another tetrazolium salt is 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) which is colorimetrically
determined in the cell supernatant (Schillaci et al., 2008). In addition, counting of colonies has
been used to count viable cells in biofilms after detachment using sonication and (or) vortex
(Coenye et al, 2011; Honraet et al., 2005; Hawser, 1994; (Honraet et al., 2005, Hawser et al.,
1994).
The last stage of testing biofilms susceptibility is the presentation of data. The terms used to
present biofilm susceptibility data differ from those used for planktonic cells. If minimum
inhibitory concentration (MIC) is defined as the concentration that is required to prevent
establisment of biofilms, this is equal to that of planktonic cells. Therefore, biofilm inhibitory
concentration was defined as the minimum concentration of antimicrobials that inhibits shedding
of planktonic cells rather than inhibiting microbial growth within biofilms (Ceri et al., 1999, Qu
et al., 2010). The shedding can be measured by using CBD assay via counting the number of the
cells which sediment at the bottom of the microwells (Ceri et al., 1999, Qu et al., 2010). In terms
of clinical response, killing of microbial cells is the most meaningful. When targeting biofilms
the minimal biofilm eradication concentration (MBEC) is defined as a reduction in biofilm
growth by 99.9% which is empirically achievable in vitro but not in vivo as biofilms contains
recalcitrant cells. These cells might resume multiplication when the level of antimicrobials drops
in immunodeficient patients (reviewed by Qu, 2010).
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Chapter2
When yeast cells are exposed to harsh conditions and cellular defence mechanisms fail, cells die
by undergoing apoptosis or necrosis. Apoptosis features including autophagy, phosphatidylserine
externalization, DNA condensation and caspase-like processing were observed in yeast (Madeo
et al., 2002). Thus apoptotic death is accepted to occur in yeast (Büttner et al., 2006, Eisenberg et
al., 2007). Apoptosis has been defined as programmed cell death, which is an organized energy
dependent process, where the intracellular content (such as DNA) undergoes ordered
degradation, exposure or secretion of diverse factors promotes phagocytic cell elimination and
the plasma membrane integrity is maintained. An apoptotic ‘dead cell’ will finally suffer from a
decline in metabolism which necessarily causes the breakdown of plasma membrane integrity
and thus necrotic morphology. This phenomenon is defined as ‘secondary necrosis’.
Alternatively, yeast cells can undergo necrosis, a cell death pathway which has been
conveniently dismissed as uncoordinated and accidental death (Eisenberg et al., 2007). Necrosis
(primary necrosis) is defined as the characterization of cells that die as a result of injury or
typically swell and burst, consequently releasing their contents (Eisenberg et al., 2010, Kroemer
et al., 1998). Activation of caspases (cysteine aspartate-specific proteases) has been connected
with apoptotic death in mammalian cells. In yeast, since the open reading frame YOR197 was
desribed as caspase-like protein, there were attempts to characterise the activity of this ORF. A
detailed analysis of the proteolytic activities of YOR197p was demonstrated by Madeo et al.,
(2002) and proposed its function as a metacaspase like protein in S. cerevisiae when it was
treated with H2O2 (Madeo et al., 2002). YOR197 has been named as a metacaspase 1 (MCA1)
(http://www.yeastgenome.org) or as it is called by Madeo et al. (2002) yeast caspase 1 (YCA1)
(reviewed in (Váchová et al., 2007, Madeo et al., 2002). Like mammalian caspases, caspase–like
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Chapter2
proteins in yeast (YCA1) were shown to undergo cleavage to activate their proteolytic activity
toward several synthetic caspase substrates (Madeo et al., 2002).
The investigations of this chapter cover:
(i)
Evaluation of the in vitro activity of thymoquinone against planktonic cells of different
Candida sp. in comparision to their susceptibility to fluconazole.
(ii)
Determination of thymoquinone’s Minimum Fungicidal Concentration (MFC) by broth
assay and monitoring the killing kinetic in C. glabrata.
(iii)
Establishment of a biofilm model of C. glabrata to test its susceptibility to
thymoquinone.
(iv)
Testing the efficacy of thymoquinone in the form of nanoparticles.
(v)
Obtaining data on the death mode in C. glabrata which might be caused by TQ.
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Chapter2
2.2 Materials and methods
2.2.1 Yeast strains and media
The yeast strains employed in these studies are listed in Table 2.1. Candida krusei 6832 was used
as a reference strain according to CLSI standards (CLSI document M27-A3, 2008). Candida
albicans ATCC14053 and Candida glabrata clinical isolates (A2a2, A5d7, A6c7) were kindly
provided by Dr. Danilla Grando (RMIT University). The C. glabrata strains were obtained from
women infected with vaginal candidiasis (Watson et al., 2013). These strains were characterised
using CHROMagar plates (Thermo Fisher, Adelaide, Australia) and their identity confirmed
using API ID 32 C™ (bioMerieux, Lyon, France).
Strains were maintained and grown on YEPD agar medium. The test medium used for the
minimum inhibitory concentration assay was RPMI1640 containing L-glutamine, but lacking
sodium bicarbonate (Invitrogen), buffered to pH 7.0 with 0.165 M MOPS (Sigma) (for media
preparation see appendix A).
2.2.2 Determination of TQ and fluconazole minimum inhibitory concentration
The in vitro antifungal activities of fluconazole (FL) and thymoquinone (TQ) were performed in
accordance with Clinical and Laboratory Standards Institute guidelines (CLSI M27-A3, 2008),
using a broth microdilution method. Minimum inhibitory concentration (MIC) values were
determined in 96 well flat-bottomed plates by measuring the optical density (OD600) of Candida
cultures in RPMI1640. The inocula were prepared from overnight Candida cultures grown on
YEPD agar plates. Candida cell suspensions were prepared in sterilized MilliQ water at ~ 106
- 34 -
Chapter2
CFU/ml and diluted 1:50 followed by 1:20 dilution in RPMI1640 to give a final inoculum of
5x102 - 2.5x103 CFU/ml. Because of the crusty nature of C. krusei 6832, before adjusting the
turbidity, colonies were further broken up in a drop of sterile water using a sterilized inoculation
loop on the surface of the agar plate and then suspended in sterile water. Thymoquinone and
fluconazole (FL) stocks were prepared in DMSO according to the manufacturer’s
recommendations. The stocks were diluted in RPMI1640 medium to achieve a final
concentration of 1 % DMSO. The solvent control wells contained inoculum suspension in 1, 2
and 3 % DMSO in medium to prove that solvent had no inhibitory effect on the investigated
Candida at the applied concentration. Thymoquinone stocks of 20 mg/ml were diluted 1:10 and
a further 1:2.5 in RPMI1640 before adding in equal volume to RPMI1640 in the wells. MIC tests
were set up in a total volume of 0.2 ml/well with 2-fold dilutions of FL or TQ.
The final concentration of TQ and fluconazole in the wells was 6.5-200 µg/ml, and 0.125-64
µg/ml respectively. The 96 well plates were incubated for 24 to 48 h at 35 °C in the dark, and the
absorbance (OD600) was measured with a POLARstar Omega Microplate Reader (BMG Labtech,
Australia). Uninoculated medium was used as the background for the spectrophotometric
calibration for each drug concentration; the growth control wells contained inoculum suspended
in the drug-free medium were set as 100% growth. The MIC was defined as the lowest
concentration of drugs that totally inhibited growth. All experiments were repeated at least three
times with three replicates each time.
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Chapter2
Table 2.1 Strains used in this study
Candida Strain
Source or reference
C. glabrata ATCC 90030
ATCC90030
C. glabrata CBS138 (or ATCC2001)
ATCC2001
C. glabrata (clinical isolate ) A5d7
(Watson et al., 2013)
C. glabrata (clinical isolate) A6c7
(Watson et al., 2013)
C. glabrata (clinical isolate) A2a2
(Watson et al., 2013)
C. albicans ATCC14053
Danilla Grando (RMIT University)
C. albicans WM1172
Helen Williams (RMIT University)
C. krusei ATCC6832
Helen Williams (RMIT University)
C. dubliniensis
Helen Williams (RMIT University)
2.2.3 Testing of thymoquinone fungicidal activity
After MIC determination, the fungicidal kinetics of TQ were determined for C. glabrata ATCC
90030 planktonic cells from three wells in flat bottom 96 well plates by a conventional culturebased method. The protocol followed Pfaller and colleagues (2004b) review. Plates were
prepared as previously described for MIC determination except the inoculum preparation. To
prepare inocula, cells from overnight C. glabrata cultures grown on YEPD agar plates were
suspended in sterilized MilliQ water to reach an OD600 of 1 (approximately equal to 107
CFU/ml), then diluted 1:50 and 1:2 respectively. Suspensions were then added to an equal
volume of media or media with different concentrations of TQ. At each time point 10 µl were
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Chapter2
collected and diluted 1:10 in RPMI1640. A volume of 100 µl was spread on YEPD agar plates.
Plates were incubated for 48 h at 37°C and fungicidal activity was defined as 3-log10-unit
reduction in CFU/ml from the starting inoculum (Klepser et al., 1998, Cantón et al., 2004).
2.2.4 Biofilm establishment
2.2.4.1 Microwell plates
C. glabrata isolates and C. albicans ATCC14053 were examined visually for their adherence
ability to polystyrene surface using 96 or 6 well plates (Nunc, for cell culture). Cell suspensions
of OD600= 1 were prepared from overnight plates. The wells were filled with these suspensions
until their surface were covered (about 1/ 4 of the well volume) and incubated at 30 - 35 ºC. The
ability of these cells to adhere was checked after 1, 2 and 4 h. Shaking was stopped once a
monolayer of cells was observed, then the plates were incubated overnight at 35 ºC.
Siliconized glass cover slips were also used to visually examine the adhesion ability of these
isolates. Slides were sterilised by submerging them in ethanol and passing them on flame. One
sterilised slide was placed in each well and then were merged with cell suspension (OD 600 = 1).
Testing media were including YEPD, YMM or RPMI1640 (appendix A).
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Chapter2
2.2.4.2 Polypropylene biofilm reactor
2.2.4.2.1 Reactor assembly
The reactor was assembled manually using commercially available laboratory materials. The 96
well plate that is used in the CBD was replaced with a pipette tip box and the pegs of the CBD
were replaced with microcentrifuge tubes. In detail, the reactor consisted of a two-part reaction
vessel (Fig. 2.2A) which was a recycled empty 200 µl pipette tip box (Barrier) which can
accommodate 96 capped microcentrifuge tubes (400 µl, 7 mm x 47 mm). These parts are made
from polypropylene (PP) and can be autoclaved.
The box is divided internally into nine
compartments (6 large compartments (105 cm3) and 3 small ones (23.8 cm3) Fig. 2.2B), allowing
for nine simultaneous separate treatments. The surface pipette tip locator plate was modified by
enlarging the holes by drilling with an 8 mm drill bit. This enabled enough space around the
microcentrifuge tubes so that they could be inserted and removed without contacting the surface
of the tubes. The tubes were stabilised in their positions by the addition of tape on the top 5 mm
of the tube (Fig. 2.10). The box with added tubes was autoclaved before use. In addition, empty
tip boxes were also autoclaved for use in second growth rounds if it was needed (see 2.2.4.2.2).
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Chapter2
(A)
(B)
Figure 2.2 Polypropylene Biofilm reactor mimicking CBD consists of a 200 µl pipette tips box and 0.4
ml PP microcentrifuge tubes. (A) The complete reactor with PP tubes. A, B and numbers indicate the
compartments and rows which were used for Fig. 2.9. (B) The tip rack without tip locator showing the
separate compartments, indicates large compartments (*) and small compartments (**). A & B indicate
the compartments that were used for Fig. 2.9.
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2.2.4.2.2 Biofilm establishment
An inoculum of approximately 107 CFU/ml (OD600 = 1) was prepared by obtaining a cell
suspension in YEPD liquid medium. The cells were picked from colonies grew overnight on
solidified YEPD media. The large compartments of the box were filled with 8 ml of the cell
suspension by aseptically lifting the tip locator plate complete with tubes or by pipetting directly
through an empty tip locator plate opening (all small compartments and the big compartments at
the middle raw were not used in this step). The height of medium in each compartment was
about 1.5 cm which is approximately one third of the compartment depth. The complete biofilm
reactor with culture was then incubated at 35 °C with shaking at 220 rpm on a rotary shaker for 2
h (adhesion phase) and then left for ~30 min to sediment planktonic cells. The shaking during
incubation caused fluid flow around the tubes, generating shear forces across all tubes and
allowing cells to either stay in suspension or attach to tubes. One tube was lifted from the
reactor and the surface of the tube was observed using a Leica DM2500 microscope and
photographed with a Leica DFC310 FX camera to visualise the established monolayer.
A second growth cycle was performed in fresh YEPD to form mature biofilms. Briefly, the
clean sterile compartments of the PP device were filled with YEPD liquid media, 12 to 15 ml for
small compartments, 18 ml in the case of larger compartments. The medium volume depends on
the desired amount of biofilm that is wanted to be produced. The most important issue is the
similarity in the height of the media in the compartments. Thus equal areas of the PP tubes are
covered, leading to equal surface area of biofilm. Tubes with established biofilms (2 h) were
transferred (by holding them by their caps using sterile forceps) to the new reactor with fresh
YEPD. The box was covered by its lid and incubated at 35 °C for 24 h. All these steps were
performed aseptically.
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2.2.4.3 Enumeration of CFU in biofilms and testing PP reactor reproducibility
Cells were removed from the surface of the tubes by a 30 sec vortex in 3 ml sterile MilliQ water
or PBS in a 15 ml centrifuge tube (Buck et al., 1999). Viable cell numbers (CFUs) were
determined by subculture after measuring turbidity of the resulting suspension using a
spectrophotometer (Eppendorf). CFU/ml was calculated for the bulk solution which was 3 ml of
MilliQ water. Efficiency of detachment of cells after vortexing was checked by choosing three
PP tubes and repeating a further wash and vortexing followed by colony counting. To determine
the reproducibility of assays, each test was repeated three times in independent experiments.
2.2.4.4 Viewing attached cells by Environmental Scanning Electron Microscopy (ESEM)
The PP tubes incubated in the tip box (24 h) were washed in MilliQ water to remove media.
Washing was performed by dipping the PP tube in and out of 8 ml of sterile MilliQ water in a 10
ml centrifuge tube. The tubes were then immersed in 4 % formaldehyde and stored at 4 °C. For
ESEM, tubes were dried at room temperature and then visualized using a SEM (RMIT facility,
FEI Quanta 200 ESEM (2002)) in low vacuum mode at 10 kV.
2.2.5 Testing TQ toxicity against C. glabrata biofilm
Preliminary testing showed that the C. glabrata A2a2 strain showed a better ability to form
biofilms than other strains. This strain was used to test the biofilm inhibitory effects of TQ.
Biofilms were grown in the polypropylene reactor which included 0.4 ml polypropylene
microcentrifuge (PP) tubes in pipette tip box (section 2.2.4; Almshawit et al, 2014a). Briefly, the
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big compartments in the reactor were each filled with 8 ml of A2a2 cell suspension in YEPD
(OD600 = 1) incubated at 35°C with shaking (220 rpm) for 2 h (adhesion phase). Boxes were left
to rest for approximately 30 min to sediment planktonic cells, then PP tubes were transferred to
sterile small compartments in the same reactor or a new reactor was used for treatment with TQ.
The small compartments were filled with 15.5 ml RPMI1640 medium (same medium that was
used in 2.2.2) containing different concentrations of TQ. The medium was warmed to 45 °C in a
water bath to dissolve the high concentrations of TQ. The established biofilms, which had
adhered to PP tubes, were transferred to TQ filled compartments and incubated for a further 24 h
at 35 °C without shaking. These biofilms were considered as established immature biofilms.
Mature biofilms were tubes with established biofilms (2 h old biofilm) transferred to new YEPD
media (18 ml) in a new big compartment in the reactor and incubated without shaking at 35 °C
for 24 h. The mature biofilms were then treated with TQ as described for the established
biofilms. These steps are summarised in figure 2.3. The number of viable cells was determined
by transferring each PP tube to a 15 ml centrifuge tube containing 3 ml of phosphate buffered
saline (PBS buffer) followed by detachment of the cells by vortexing for 30 sec. Cell suspensions
were spotted, dried partly and spread on YEPD plates after dilution.
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Figure 2.3 Diagram summarising steps of C. glabrata biofilm establishment in PP reactor and
treating them with TQ. Stars indicate the location of PP tubes.
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2.2.6 Susceptibility of C. glabrata to Protein-polymer TQ nanoparticles
Thymoquinone nanoparticles were kindly provided by Vishal Mistry (RMIT University) and
were stored at 37 ºC in the dark. The stocks provided had a concentration of ~ 333 µM (~ 55
µg/ml) and diluted using 2x RPMI1640 medium. Examination of C. glabrata sensitivity to TQ
was performed according to CLSI standard method (2008) as described in 2.2.2.
2.2.7 In vivo staining of caspase activity and membrane integrity
Staining of caspase activity in vivo was analysed by using FITC-VAD-fmk (fluorescein-5isothiocyanate-VAD-fluoromethylketone; CaspACE, Promega) which has been used before to
label YCA1 in S. cerevisiae (Madeo et al., 2002). Cell membrane integrity was tested by staining
cells with propidium iodide (Sigma Aldrich). Caspase activation and cell intact was labelled
following previous described protocol (Madeo, et al., 2002, Wysocki & Kron, 2004) except a
modification in the concentration of PI staining which is lower in this study. Briefly, about 10 6
cell/ml of C. glabrata ATCC2001 from an exponential phase culture (4 h old) in YEPD were
treated with 25 and 50 µg/ml of TQ for 0.5, 1 and 4 h, harvested, washed once in 1 ml PBS and
resuspended in 200µl staining solution containing 10µM FITC-VAD-fmk (CaspACE, Promega).
After incubation for 20 min at 30°C, cells were washed in 1 ml PBS. Then cells were suspended
in 500 µl PBS contains 2 µg/ml (PI) and incubated 15 min at 30 °C. After that cells were
harvested, washed and resuspended in 500 µl PBS. Fluorophore emission was analysed by a BD
FACs Diva flow cytometer using FITC (494/519-nm excitation /emission) and PE-Cy5-A
(482/695 nm excitation/ emission) filters.
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2.2.8 Cell cycle analysis
The cell cycle of cells that were treated with TQ was analysed by flow cytometry. The main aim
of analysing the cell cycle is observing cells that contain degraded DNA. DAPI (4′,6-diamidino2-phenylindole dihydrochloride; Sigma) was used for DNA staining. Candida glabrata cells
from exponential phase culture (4 h old) were washed and suspended in RPMI1640 to the
density of 106 cell /ml. Suspensions were treated with TQ for 4 or 24 h. Cultures with 0, 25, 50
and 100 µg/ml TQ in RPMI1640 were harvested by centrifugation at 5890 g for 1 min, washed
with PBS then fixed and permeabilized with 70 % cold ethanol on ice for 5 min. Pellets were
resuspended in PBS at room temperature for another 5 min and then cells were harvested and
stained with 3 µM DAPI and incubated for 15 min in room temperature in the dark (Li et al.,
2006; Liang & Zhou, 2007). Cells were analysed by flow cytometry using a pacific blue filter
(351/450-65 nm excitation/emission).
2.2.9 Data analysis
GraphPad Prism 6 was used to analyse the data statistically. A one way analysis of variance
(ANOVA) test combined with Tukey's multiple comparisons test and e-test were used. A p
value of < 0.05 was considered significant. Flow cytometer data were analysed by Flowing
software which was created by Perttu Terho (Cell Imaging core, Turku Centre for
Biotechnology, Finland).
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2.3 Results
2.3.1 Thymoquinone inhibits Candida glabrata growth
Exposure of different Candida sp. to TQ resulted in inhibition of growth in a concentrationdependent manner. The NCCLS subcommittee suggest that determination of MIC at 100% or
complete growth inhibition could more reliably detect azole resistance (Espinel-Ingroff et al.,
2001). Therefore, the values depicted in Table 2.2 were obtained by using the 100% inhibition
criterion of MIC compared to the growth in the control to avoid bias. The clinical isolate C.
glabrata A2a2 showed high resistance to fluconazole and needed a higher TQ concentration to
inhibit its growth. The MICs of fluconazole for Candida sp. in this study range from 0.125 to >
64 µg/ml. The MICs of TQ ranged between 25 to 50 µg/ml for most strains except C. glabrata
A2a2 which was fluconazole resistant (MICTQ=100 µg/ml, MICFL> 64 µg/ml). The effect of
fluconazole on other C. glabrata clinical isolates (A5d7 and A6c7) was intermediate (8-16 and
32 µg/ml) and 50 µg/ml of TQ inhibited their growth. However, C. glabrata ATCC2001 and
ATCC90030 strains had a lower MIC to TQ (25 µg/ml). Candida krusei 6832 and C. glabrata
A2a2 exhibit slow growth rates, therefore they were incubated for longer than 24 h before the
readings were performed. In conclusion, the effect of both drugs on C. glabrata strains was strain
dependent; however TQ inhibited growth in a narrow concentration range of 25 to 100 µg/ml.
2.3.2 Thymoquinone time-kill curve study
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The killing activity of TQ for C. glabrata ATCC 90030 (mean and SEM) is presented in Fig. 2.4.
A reduction of 3 log10 units (99.9%) in CFU/ml from the starting inoculum was used to define
the fungicidal activity of TQ (Qu et al., 2010). The fungicidal activity of 100 µg/ml TQ occurred
within 2 h and caused a greater than 3 log10 units reduction. A reduction of 3 log10 was achieved
by 50 µg/ml of TQ within 6 h. The MIC concentration (25 µg/ml) correlated with the extent of
growth inhibition but did not achieve a significant reduction in the viability. Treatment at the
MIC caused a gradual decrease in the colony number at the beginning of the experiment;
however, after 24 h, there was evidence that survivors in the population grew. Since the cell
number of the inoculum in the MIC assay differed from the fungicidal assay, an experiment was
conducted using C. glabrata ATCC90030 to roughly estimate the relationship between TQ
toxicity and cell number in RPMI1640. Figure 2.5 shows a decrease in TQ inhibition of growth
with increasing cell number. The MIC (25 µg/ml) of TQ inhibited the growth of 103 cells/ml and
was sub-inhibitory for cell concentrations >104 cells/ml.
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Candida Strain
MIC (µg/ml)
Thymoquinone Fluconazole
C. glabrata ATCC 90030
25
16
C. glabrata CBS138 (or ATCC2001)
25
4
C. glabrata (clinical isolate ) A5d7
05
8-16
C. glabrata (clinical isolate) A6c7
05
32
C. glabrata (clinical isolate) A2a2*
100
>64
C. albicans ATCC14053
50
0.25
C. albicans WM1172
50
1
C. krusei ATCC6832**
25
32-64
C. dubliniensis
25
0.125-0.25
Table 2.2 Minimum inhibitory concentration of thymoquinone and fluconazole. The values
represent MIC100 of three replicates after 24 h and each experiment was performed at least three
times independently. * after 2 d, ** after 3 d.
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C F U /m l (lo g
10)
6
25
50
4
100
200
2
 g /m l T Q
4
2
9
8
6
4
2
0
0
T im e (h )
Figure 2.4 In vitro kinetics of fungicidal activity of thymoquinone against C. glabrata.
C. glabrata ATCC 90030 cells were treated with TQ in a 96 well plate at concentrations ranging
from 25-200 µg/ml. Viable cells were determined by plating aliquots at each time point to
measure CFU/ml. All incubations were at 35 ºC. This experiment was performed three times
independently. The mean of three independent replicates was shown with the standard error of
the mean (SEM).
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Figure 2.5 Population densities versus concentrations of TQ. Suspensions of C. glabrata
ATCC90030 in RPMI1640 were prepared from an overnight plate and incubated for 24 h at
35°C with shaking. The initial optical density of 106 cell/ml culture at time zero was equal to 0.1.
Error bars indicate the mean and SEM of three replicates.
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2.3.3 Biofilm formation and reproducibility
Adherence of C. glabrata strains and C. albicans on polystyrene in 96 well plates (Nunc, for cell
culture) and 6 well plates was examined using different media including YEPD, YMM and
RPMI1640 media. The cells were not able to attach except for A2a2 strain cells. This strain
could form a thin visible biofilm layer after shaking for 2 h (Fig. 2.6 A) and formed white thick
layer after static overnight incubation (Fig. 2.6 B). In contrast, C. glabrata ATCC2001,
ATCC90030, A6c7, A5d7 and C. albicans ATCC14053 did not form any observable adherence
after 2 h or overnight incubation (Fig. 2.6 B). However, a gentle washing procedure which
included pipetting off the medium and pipetting in PBS caused a notable destruction in biofilm
architecture. Figure 2.7 reveals the effect of the first steps of washing on biofilm topography
with or without TQ treatment. Candida glabrata ATCC2001 was able to form a fine biofilm
layer while A2a2 formed a thicker white biofilm layer on the surface of siliconized glass cover
slips which were located in a 6 well plate. Again these biofilms slipped and were easily
destroyed once they were washed gently with PBS (data not shown). Candida glabrata A2a2
cells were able to adhere to the internal wall surface of 50 ml polypropylene centrifuge tubes
(Sigma-Aldrich) when they were incubated at 30 ºC or 35 ºC with shaking. Therefore the surface
of polypropylene microcentrifuge tubes (PP tubes (Fig. 2.10)) was the preferred option for
establishing C. glabrata biofilms.
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(A)
A6c7
ATCC90030
ATCC2001
C. albicans
A2a2
A5d7
(B)
Figure 2.6 Adhesion of C. glabrata isolates and C. albicans ATCC14053 to polystyrene. (A)
Two micro-wells show the visual observation of monolayer formation by C. glabrata A2a2 after
shaking for 2 h using YEPD medium, (B) Candida spp. suspensions in YEPD (OD600 = 1) were
incubated at 35°C with shaking (220 rpm) for 2 h followed with overnight static incubation.
Polystyrene 6 well cell culture plates were used. C. glabrata isolates (wells with white labels)
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did not show adhesion to the bottom of the well except for A2a2 strain which formed a white
biofilm layer.
(A)
(B)
(C)
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Figure 2.7 Effect of washing process on C. glabrata A2a2 biofilms architecture. The biofilms were
established in 6 well plate (A) Biofilms before washing (B) Biofilms after pipetting off medium (C)
biofilms after pipetting in PBS solution. The three wells in the upper row were not treated with TQ. Wells
in the lower row from right to left were treated with 50, 100 and 200 µg/ml of TQ.
Adhesion of C. glabrata cells on the surface of PP tubes after 2 h was observed by light
microscopy (Fig. 2.8). The clinical isolate A2a2 had more apparent adhesion than the two
ATCC strains after 2 h incubation. Figure 2.9 shows a comparison between cell numbers in
biofilms that were formed after 24 h on different PP tubes in two different compartments, and
between different rows of one compartment. The results show reproducibility of cell counts in
different compartments with means of 2.5 x 107 and 2.2 x 107 CFU/ml for compartment A and B
respectively and for counts between rows of a single compartment (P > 0.05, Fig. 2.9 A & B).
Counting cells from biofilms by vortex disruption has been used in a previous study (Buck et al.,
1999). The efficiency of removing the attached cells by vortexing was checked by repeating a
further wash and vortexing. The mean cell number after the first wash and vortexing was 2 x 10 7
CFU/ml whereas after further washing and vortexing was 0.9 x 104 CFU/ml. This means less
than 0.1% of the original CFU/ml was obtained through this extra wash.
The ability of A2a2 strain to produce biofilm was compared with the control strains (ATCC2001
(also known as CBS138) and ATCC90030) by counting colonies. Table 2.3 compares the mean,
standard error of the mean, P-value, maximum and minimum counts found between biofilms of
these strains in the same conditions. The cell concentrations after detachment from biofilms
were 2.3 x 107, 5.8 x 105 and 1.1 x 105 CFU/ml for C. glabrata strains A2a2, ATCC2001 and
ATCC90030, respectively. The variation between maximum and minimum counts for each
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strain are comparable to those which were achieved by the CBD device (Ceri et al., 1999). This
indicates that the reproducibility of this reactor is comparable to the CBD device. Figure 2.10
demonstrates a visual difference in biofilm producing ability of the clinical and a control strain.
A2a2 was able to build a thick white biofilm layer whereas the control strain tubes looked clear.
This demonstrates that the PP reactor shows a difference between strong biofilm producing
strains and weak or non-biofilm forming strains. The experiment was repeated with RPMI 1640
medium supplemented with 2% dextrose (Kucharikova et al., 2011), and coating the tubes with
lysine to increase the positive charge on the PP tubes surface (Harrison et al., 2006) or by using
foetal calf serum. However, the attachment of C. glabrata ATCC2001 and 90030 could not be
improved and the attachment of the clinical isolate was optimal when YEPD medium was used.
(b)
(a)
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Figure 2.8 Establishment of biofilm by the isolates on the surface of PP microcentrifuge tubes
after shaking for 2 h in media. (a) C. glabrata ATCC2001 scale bar = 35 µm (b) C. glabrata
A2a2 scale bar = 70 µm.
(A)
(B)
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Figure 2.9 Comparisons between biofilm formation of A2a2 strain at different sites in the PP
reactor (A) difference in cell number between two big compartments (B) comparison between
cell number between different rows in each compartment. ns - Insignificant difference (P >
0.05), error bars represent the standard error of the mean (SEM).
Table 2.3 The variation of colonization capability on four PP tubes surface by different C.
glabrata strains after 24 h. Maximum, minimum counts and the calculation of Means and
Standard deviations (SD) were based on log10 of CFU/ml per tube.
C. glabrata
strain
A2a2
Mean SD
7.34
0.05
Maximum
count
7.42
Minimum count
ATCC2001
5.61
0.50
6.07
5.07
ATCC 90030
4.84
0.45
5.36
4.28
7.28
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P value
A2a2 vs ATCC2001 (P <
0.01)
A2a2 vs ATCC 90030 (P
< 0.0001)
ATCC2001 vs ATCC
90030 (P > 0.05)
Chapter2
Figure 2.10 Biofilms of C. glabrata ATCC2001 (left) and the clinical isolate C. glabrata A2a2
(right). PP microcentrifuge tubes after incubation for 24 h in the biofilm reactor. The arrow
indicates the tape which was used to stabilise the tube in the rack.
2.3.4 Candida glabrata biofilm observed by scanning electron microscopy
Scanning electron microscopy analysis was used to examine biofilm structure and to determine
C. glabrata biofilm morphological characteristics. C. glabrata A2a2 (Fig. 2.11) showed a
discontinuous monolayer of large blastospores in clusters adhering to the PP tube surface
associated with an amount of extracellular polymeric substances matrix (EPS). The collapsed
appearance of the yeast could be due to the formaldehyde fixation.
2.3.5 Thymoquinone fungicidal activity against C. glabrata biofilms
Figure 2.12 shows the fungicidal activities of TQ against C. glabrata A2a2 biofilms. The effect
was examined on established, pre-adhered biofilms (2 h biofilm) and mature biofilms (24 h old).
The minimum TQ concentration which could successfully inhibit the growth of pre-adhered
biofilms by 3 log10 CFU/ml in the biofilm was 1.25 mg/ml. This is fifty times higher than the
MIC for planktonic cells. However, this concentration failed to reduce the viability effectively
with mature biofilms (≤ 2 log10 CFU/ml). Higher concentrations could not be tested due to the
low solubility of TQ in the media.
2.3.6 Susceptibility of C. glabrata to TQ nanoparticles
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Candida glabrata ATCC2001 was neither more sensitive nor more resistant to TQ protein
polymer based nanoparticles compared with TQ. The minimum inhibitory concentration of TQ
protein polymer nanoparticle was approximately 24.6 µg/ml similarly the MIC of TQ was 25
µg/ml.
Figure 2.11 Scanning electron micrograph of C. glabrata clinical isolate (A2a2) biofilm formed on the
biofilm reactor. PP microcentrifuge tubes were removed from the device after 24h washed and fixed with
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formaldehyde. Note the discontinuous layer of large blastospores in clusters on the PP surface. The inset
at right corner on the top indicates the small amounts of extrapolymeric substances (white arrows).
10
8
6
****
4
2
5
.2
1
1
0
.5
0
5
.2
.5
0
0
0
T Q ( m g /m l)
Figure 2.12 Activities of different concentrations of thymoquinone (TQ) against C. glabrata A2a2
biofilms. Lined bars show TQ activity against 2 h established biofilms under treatment with various
concentrations of TQ. White bars show activity of TQ against mature biofilm (24 h old biofilm) under
treatment with different concentrations of TQ for 24 h. Results represent the means of two independent
experiments with four replicates each time. Data analysed by using one way ANOVA combined with
Sidak’s multi comparisons test, error bars indicate SEM values ( *** = p < 0.001, **** = p < 0.0001
comparing with untreated control).
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2.3.7 Thymoquinone induces FITC-VAD-FMK activation in dead cells
Fluorescein-5-isothiocyanate-VAD-fluoromethylketone (FITC-VAD-FMK) has been widely
used to detect caspase activation in aged yeast cells and cells treated with H2O2 (Herker et al.,
2004, Madeo et al., 2002, Qi et al., 2003). Exponentially growing C. glabrata cells were treated
with 25 and 50 µg/ml of TQ for 0.5, 1, 4 and 24 h. Then activation of yeast caspsase-1 (YCA1)
was detected using FITC-VAD-FMK combined with PI for cell membrane integrity analysis.
This assay can help to distinguish the sequence of events leading to cell death. Labelling with
FITC-VAD-FMK and PI can distinguish four distinct cell states: (a) live non-apoptotic cells
(FITC-negative/PI-negative), (b) live cells with activated caspases/early apoptotic cells (FITCpositive/PI-negative), (c) dead post-apoptotic cells (FITC-positive/PI-positive), and (d) dead
necrotic cells (FITC-negative/PI-positive) (Madeo et al., 2002). The results in Fig. 2.13
demonstrate that most of the cells which were FITC positive had lost their membrane integrity at
the same time. Microscopic observations showed few of FITC positive / PI negative cells.
However flow cytometric analysis did not show significant number of these cells. An
interpretation of these data might be treated cells picked up caspase fluorophore as a result of
death and not before death, indicative of necrotic death. These samples were analysed after 24 h
as well and the same pattern was observed.
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(A)
(B)
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Figure 2.13 Monitoring of metacaspase1 activation and cell membrane integrity. (A) exponentially growing
cells were treated with 50 µg/ml TQ for 5 h and observed by fluorescence microscopy after labelling with
FITC-VAD-FMK and PI, white arrows indicate FITC positive and PI negative cells (B) Exponentially
growing cells were treated with 25 or 50 µg/ml of TQ then analysed by flow cytometry after labelling with the
same fluorophores; 10,000 cells were analysed for each sample. (N) cells did not emit any fluorescence; error
bars represent the standard error of the mean (SEM) of two independent experiments.
2.3.8 Thymoquinone treated cells did not exhibit DNA degradation
It has been well documented that apoptotic cells show reduced DNA staining with a variety of
fluorochromes, including PI and DAPI. A large portion of DNA in apoptotic cells is of low
molecular weight due to activation of endogenous endonucleases which break the DNA linking
the nucleosomes. These small fragments of DNA can leach out from ethanol-fixed
(permeabilized) cells during rehydration and staining process. Consequently, flow cytometric
analyses detect cells with lower staining intensity than those at G0/1 phase, a phase when live
cells have their minimum content of non-fragmented DNA. These cells can be observed as a
‘sub-G /1’ peak in a DNA histogram. In contrast, necrotic cells generally do not show an
immediate reduction in DNA staining, and sub-G0/1 cannot be discriminated from a DNA
histogram content analysis of live cells (Darzynkiewicz et al., 1992, 2010; Dive et al., 1992;
Riccardi & Nicoletti, 2006; Calvert et al., 2008). Flow cytometry analysis of C. glabrata
ATCC2001 cells which were treated with sub-inhibitory and inhibitory concentrations of TQ (25
and 50 µg/ml) in figure 2.14 showed no events in the sub-G0/1 region after 4 h or 24 h treatment,
which indicates an absence of apoptotic cells in the periods of analysis. Equal numbers of cells
from growth control and treated cells plus same the concentration of DAPI stain were used. An
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artificial shift of the fluorescence intensity peaks may result from these factors (Haase & Reed,
2002).
Figure 2.14 Monitoring the cell cycle of TQ treated cells by using DAPI. (A) 4 h treatment, (B)
24 h treatment. (1) Untreated cells without staining, (2) Stained untreated cells, (3) Stained cells
treated with 25 µg/ml TQ and (4) Stained cells treated with 50 µg/ml. H-1 no fluorescent
emission zone where sub G0/1 phase can be recorded, H-3 fluorescent emission zone.
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2.4 Discussion
2.4.1 Thymoquinone anti-Candida effect
The results of the present study demonstrated the anti-Candida activity of the Nigella sativa
compound thymoquinone. The results show evidence of TQ antifungal activity against different
Candida sp. including C. glabrata. C. glabrata is an agent of human mycoses which is an
important opportunistic fungal pathogen that causes minor infections in many individuals but
very serious infections in those who are immuno-compromised. Considering that C. glabrata is
able to develop resistance against most commercial antifungal agents and many antifungal drugs
have unwelcome side effects (Laniado-Laborín et al., 2009), TQ might be a new option for
treating Candida infections.
Candida glabrata is known to have varying susceptibility to fluconazole (Pfaller et al., 2003,
Bruder-Nascimento et al., 2010). The C. glabrata strains in table 2.2 showed high variability in
their sensitivity to fluconazole (0.25 to > 64 µg/ml), but their susceptibility to TQ had a narrow
range with MICs between 25 and 50 µg/ml in most cases. This was true for the other Candida
spp. as well, indicating that the MIC for many yeasts is around 25-50 µg/ml.
The fungicidal effect which was determined over 24 h by using C. glabrata ATCC90030 showed
an inhibitory effect of TQ rather than a strong fungicidal effect at the MIC concentration of
25µg/ml. In contrast, 50 µg/ml of TQ could decrease the viable number of cells by ≥ 99.9%
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within six hours. According to CLSI M26-A (1999) the antibacterial agents which cause 99.9%
reduction in viability were considered to be bactericidal. Still there is no standardised guideline
to test the fungicidal activity of antifungal agents, however, many studies attempted to
standardise method to determine the fungicidal activity of antifungal agents have been
summarised and reviewed by Pfaller and collegues (2004). In this review a reduction of survivor
cells by ≥ 99.9% or 3 log10 in population of about 105 cells/ml was considered to be an indicator
of the fungicidal activity of antifungal agent. In this assay there was another challenge for TQ
toxicity which is the cell number (about 105 CFU/ml) which was higher than that in the MIC
assay (5x102 to 103 CFU/ml). This might be the reason of less efficient killing by 25µg/ml TQ
which is the MIC value. To highlight the relationship between MIC of TQ and cell density,
another measurement of growth inhibition in C. glabrata cultures with different cell number was
performed. Figure 2.5 showed that the MIC concentration caused a delay in culture growth but
not total inhibition of growth in cultures that have a high cell number (>10 4 CFU/ml). These
results are in agreement with Perumal’s study (2
7) which demonstrated that a correlation
exists between cell density and phenotypic resistance to fluconazole independent of whether the
source of the cells is in a planktonic or biofilm form.
The concentrations which were needed to kill C. glabrata in this study are higher than those that
were needed to kill some types of cancer cell lines. For example, breast cancer and other cancer
cell lines including human glioblastoma cells need between 4.1-16.4 µg/ml to reduce their
viability (Gurung et al., 2010, Motaghed et al., 2013, Attoub et al., 2013). Explanations for this
difference may relate to the permeability of the yeast cell, efflux pumps in yeast and the method
of cell killing. Data reviewed in section 1.2.3 regarding thymoquinone toxicity indicate a wide
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range of safety for TQ therapeutic doses. Thus its anti-Candida MIC range (25-100 µg/ml) is
still acceptable as it is within the estimated maximum tolerated oral dose of TQ (approximately
.6 mg/kg/day (Shuid et al., 2012)). Using TQ cream (10% TQ) to treat vaginal candidiasis
(Abdel Azeiz et al., 2013) is the main study that supports the idea of using TQ safely to treat
such infections.
The results of this study revealed that complete eradication of C. glabrata biofilm by TQ is still
challenging but preventing of biofilm stabilisation and biofilm growth inhibition might be
achievable. Free TQ might be not successful in treating biofilm related infection or systematic
Candida infections due to its binding to serum proteins which reduce its activity in vivo (ElNajjar et al., 2010). Alternative applications to use thymoquinone such as liposomal TQ which
showed high efficiency in treating systematic infection caused by C. albicans (Khan et al., 2015)
or TQ loaded nanoparticles might improve TQ efficiency. In this study TQ protein polymer
nanoparticles were used to increase the sensitivity of C. glabrata biofilms to TQ by increasing
the absorption of this compound. In fact these nanoparticles did not increase sensitivity of
Candida glabrata ATCC2001 to TQ in vitro. This strain showed lower MIC than A2a2 isolate
which forms thicker biofilm. Therefore the ability of these nanoparticles to increase biofilm
sensitivity was not predictable. Despite that, TQ protein polymer nanoparticles might improve
TQ’s anti-candidiasis effect in vivo as it occurred with liposomal TQ (Khan et al., 2015).
Possibly these nanoparticles may add more stability to TQ action by blocking interaction
between TQ and serum proteins. Therefore further investigation for this possibility is required.
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2.4.2 Polypropylene biofilm reactor affordability and reproducibility
Washing process caused destruction of C. glabrata biofilm formed in microwell plates. In
addition, fixation with chemicals such as formaldehyde is not helpful because of the need to
count viable cells. The Calgary biofilm device (CBD) offers less destruction (Ceri et al., 1999)
and consequently more reliability in terms of defining minimum inhibitory concentration of
biofilms. However, its costly features and the combined sonication were considered as
disadvantages in using this model. Sonication was supposed to be relatively inefficient in
dissociating biofilm cells and can cause contamination by spreading droplets between wells (Qu,
2010).
The major advantages offered by the biofilm reactor developed for this study are its affordability
and being a partly re-usable device, leading to less environmental waste. While the CBD device
costs 8.5-19 USD (Nunc-Immuno TSP; Innovatech), the reactor described here costs about 3
USD.
Polypropylene microcentrifuge tubes (PP tubes) are available for 3 USD per 100
(California Pacific Labs) and PP tips boxes are a negligible cost since these have been recycled.
Media are an additional cost for the reactors. The CBD uses 20 ml of media but the device
described here needs 168 ml to be filled completely at a cost of approximately 1.3 USD for
YEPD medium and 0.5 USD for RPMI1640 (according to Sigma Aldrich website (2015)).
In addition, this reactor allows the handling of each biofilm separately without disruption to
other biofilms and enables dissociation of microbial cells aseptically. More important is the
reproducibility of the quantitative counts of the biofilms produced in this reactor. The method
was found to produce uniform and reproducible results with no significant differences between
biofilms formed on PP tubes incubated in various compartments of the device. In addition, the
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difference between maximum and minimum counts for each strain were comparable to those
which have been reported for the CBD device (Ceri et al., 1999) which makes this reactor a
promising method for measuring the susceptibility of biofilms to antifungal or toxic materials.
It is well recognized that the chemical nature of the implanted material plays a critical role in
initial Candida colonization and thus helps define the overall characteristics of the resultant
Candida biofilms (Tournu et al., 2012). The adherence of different C. glabrata strains to
polyvinyl chloride (PVC), Teflon, polyurethane, polystyrene, polymethylmethacrylate,
hydroxyapatite, silicone elastomer and soft denture liner surfaces has been studied (Estivill et al.,
2011, Hawser et al., 1994, Kuhn et al., 2002). Since polypropylene is used widely in medical
devices (e.g. catheters, hernia meshes and sutures) (Nava-Ortiz et al., 2010), the development of
reproducible and cost effective in vitro PP models to study Candida biofilms is important.
Microbial cells which are surrounded by EPS, adhere to each other, to a surface and form
aggregates that can be defined as biofilm. In my study C. glabrata cells adhered to each other
and to the surface. The EPS was not absent, but it was present in small amounts. The thickness of
EPS usually depends on strain type and the environmental conditions. The amount of EPS which
appear in figure 2.11 are similar to those which were obtained in other studies on C. glabrata.
Such studies are (Mota et al., 2015, Silva et al., 2011).
Examination of the adhesion of C. glabrata cells to PP in this study demonstrates that C.
glabrata strains vary in biofilm production on polypropylene under the same conditions of
nutrient, temperature and flow. Biofilms that can be formed on discs that are made from
catheters or other clinical devices (Hawser et al., 1994) can be easily disrupted because of the
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transfer process using forceps. The tubes used in this study have a top section separate from the
biofilm forming site that can be used to handle the tubes without disturbing the biofilm. The
washing after biofilm formation may not be necessary since unattached cells will be lost under
the effect of gravity. However, the washing procedure in this PP reactor is very gentle compared
with the washing procedure used in published methods using plates (Tournu et al., 2012).
Furthermore, the separate sections (compartments) in the box allow eight different treatments
simultaneously for at least eight replicates of one treatment.
Candida biofilms have dramatically reduced susceptibility to antifungal drugs resulting in
increased mortality rates for candidiasis patients (Tumbarello et al., 2007). Because of this the
efficacy of TQ on C. glabrata biofilms was tested. C. glabrata A2a2 immature biofilms needed
50 times the planktonic MIC to inhibit their growth by 3 log10, which is a typical clinical
property of biofilms. Biofilms are famous with their development of antimicrobial resistance that
can be up to 1,000-fold greater than planktonic cells (Mah et al., 2003). Therefore, removal of
devices from patients with candidemia is recommended, as patients with biofilm-infected devices
are rarely cured with only antifungal therapy (Nett et al., 2007b). Perumal and his group (2007)
attribute candida biofilm resistance to antimicrobial agents to the high density of cells in biofilms
rather than protection by the extracellular polymeric substances (EPS). In contrast, Nett et al.
(2007) explained this resistance as a result of the increased β-1,3 glucan content in biofilms
compared with planktonic cells. By referring to figure 2.5 this study supports the idea of high cell
number influence on biofilm susceptibility to TQ; and further investigations on the effect of the
observed EPS matrix on TQ susceptibility are needed (Fig. 2.11). Overall these observations
suggest that concentrations higher than MICs are necessary to disinfect biofilm contaminated
medical devices and equipment, and confirm the importance of early recognition and treatment
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of Candida infection and contamination. Success is possible by using the concentrations guided
by the conventional susceptibility tests against non-attached cells, however, Abdel Azeiz’s study
(2013) in which vaginal Candida albicans infection was treated with 10% TQ indicates the
possible safe elimination of biofilms by this compound.
Despite of TQ protein polymer based nanoparticles did not inhibit C. glabrata growth at
concentrations lower than TQ, these nanoparticles will be good approach if they prevent fast
degradation of TQ in vivo or minimise its binding with serum proteins which makes the free
available amount of TQ higher.
2.4.3 Thymoquinone causes necrotic death in C. glabrata
Activity of caspase-like protein in yeast could be reported by binding of specific fluorochromelabelled caspases FITC-VAD-FMK. This binding was prevented by pretreatment with
pancaspase inhibitor Z-VAD-FMK (Madeo et al., 2002, Silva et al., 2005) or generating of
mca1(yca1) mutant (Madeo et al., 2002, Silva et al., 2005, Wysocki et al., 2004). According to
the Candida Genome Database www.candidagenome.org C. glabrata has YCA1 homolog
(CAGL0I10945g) but this ORF has been not characterised experimentally yet. Because of the
fact of C. glabrata is genetically closely related to S. cerevisiae and previous studies
observations on S. cerevisiae (Madeo et al., 2002; Herker et al., 2004) regarding utilization of
FITC-VAD-FMK were encouraging, this assay was used to detect caspase-like activity in this
study. Emission of green fluorescence was recorded which might be an indicator of caspase-like
activity (Fig. 2.12). However analysing FITC-VAD-FMK emission over different time intervals
did not indicate any significant activation of caspase-like proteins before cells had lost their
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viability. Thymoquinone treated cells were either PI and FITC positive or just PI positive
indicative of post apoptotic or necrotic death. Because live cells were not FITC positive, nonspecific binding of FITC dye to live cells was not estimated. In contrast, it has been shown that
dead S. cerevisiae cells non-specifically bind fluorescein and FITC or FITC-conjugated caspase
inhibitor FITC-VAD-FMK and live cells do not (Wysocki et al., 2004). The counter-argument to
these claims was the cells were exposed to extreme death conditions in that study, which either
induced non-apoptotic cell death or caspase-independent apoptosis (Farrugia et al., 2012). In this
study, because no FITC-VAD-FMK positive intact cells were observed, the post apoptotic phase
might be an artificial labelling. In contrast with Wysocki et al. (2004) C. glabrata cells were
treated with both sub inhibitory and toxic concentrations of TQ but apoptotic cells (FITC
positive/ PI negative) were not observed. Therefore, the initial conclusion of this result is TQ
might cause necrotic death. However further detailed proteolytic analysis of CAGL0I10945g C.
glabrata ORF activity should be conducted to confirm the presence of caspase-like proteins in C.
glabrata. Since the results obtained indicate that FITC-VAD-FMK usage can be rather
controversial, further analysis of the cell cycle was performed to decide whether TQ induces
necrotic death or not. DNA condensation and degradation is a known marker of apoptotic death
in yeast and it has been reported by us in C. glabrata (Almshawit et al., 2014). Analysis of
permeablised DAPI labelled cells showed no events at sub G0/1 zone which indicates DNA
degradation did not occur when treating C. glabrata with sub-toxic or toxic concentrations of
TQ. In conclusion, these results indicate that TQ exerts activity against C. glabrata by direct
killing of cells results probably in necrotic death.
Generally, antimicrobial compounds that cause apoptotic-like death of microbes are preferred
since they do not induce inflammation. Antifungal agents such as caspofungin causes necrotic
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death of C. albicans treated with inhibitory concentrations and apoptosis when sub-inhibitory
concentrations are used, however this agent is considered to be highly safe. Others such as
amphotericin induce apoptotic death in C. albicans in vitro but show side effects in vivo (Phillips
et al., 2003). Although Abdel Aziz’s study demonstrated no side effects such as inflammation
were caused by treating infected C. albicans mice with TQ (Abdel Azeiz et al., 2013), further
studies on treating C. glabrata infected animal models are required to demonstrate such possible
side effects.
2.5 Conclusion
This chapter concludes that thymoquinone is able to inhibit the growth of different Candida
species and has a fungicidal effect. A high density of cells (> 104 cell/ml) reduces the fungicidal
effect of TQ. In addition, thymoquinone can inhibit growth of 2 h old C. glabrata biofilms by 3
log10 but not mature biofilms. The polypropylene biofilm reactor which was developed in this
study is a useful, simple, low cost device for parallel study of C. glabrata biofilms and might be
useful for other microbial species which produce biofilms. Due to the reproducibility of biofilms
formed on each tube in the reactor, this assay can enable rapid and reproductive screening of
Candida biofilms for antifungal resistance testing. It can also help to establish biofilm models for
molecular microbiology investigations. The interest in performing biofilm experiments is
widespread. The Almshawit et al. (2014a) article was amongst the ten most downloaded articles
as it was classified by ScienceDirect in the first six months of 2014 (publisher’s report). Death
mode caused by TQ might be necrotic and further investigations confirming this result are
required.
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CHAPTER 3
Thymoquinone’s effect on the oxidative status of C. glabrata
3.1 Introduction
Several mechanisms have been suggested for the chemopreventative and anticancer activity of
TQ; however a clear mechanism of the action of TQ as an antifungal has not been elucidated. TQ
is a known ROS scavenger at low concentrations (Kruk et al., 2000) and most of the studies in
mammalian cells illustrate the mechanism centers on this antioxidant property (Ragheb et al.,
2009, Abdel-Wahab, 2013). In contrast TQ may act as a pro-oxidant at higher concentrations in
mammalian cells (Zubair et al., 2013). However, other groups show that the anti-oxidant/prooxidant ability of TQ depends on the milieu where it is present (Mansour et al., 2002, Staniek et
al., 2010, El-Najjar et al., 2010). As a quinone, TQ can be reduced by a variety of reductases to
yield semiquinone (one reduction) or thymohydroquinone (two reductions). Thymohydroquinone
is reported to have antioxidant effects, while semiquinone acts as a pro-oxidant and generates
reactive oxygen species. For more details see figure 1.4 (Mansour et al., 2002, Staniek et al.,
2010, El-Najjar et al., 2010).
Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide anion (˙O2-), and
hydroxyl radical (˙OH) arise continually during aerobic metabolism (with an estimated 2% of
oxygen utilized by yeast cell being converted into the superoxide anion) (Shanmuganathan et al.,
2004). These ROS typically arise because of electron leakage from the electron transport chain
onto dioxygen (O2) during aerobic respiration. Therefore, living organisms including yeast have
developed defence mechanisms to protect against ROS. C. glabrata has a well-defined oxidative
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stress response, which include both enzymatic and non-enzymatic mechanisms. Enzymatic ROSscavenging molecules (e.g. superoxide dismutases, catalases and thioredoxin reductase), and nonenzymatic mechanisms such as the S-thiolation of oxidation-susceptible proteins, which prevents
oxidation
by
forming
reversible
mixed
disulfide
bonds
with
glutathione
(L-
glutamylcysteinylglycine; reduced form GSH; oxidized form GSSG) (Briones-Martin-DelCampo et al., 2014, Gutierrez-Escobedo et al., 2013). In addition production of an ascorbic acid
homolog (D-erythroascorbic acid), a five-carbon analog of ascorbic acid, has been described in
Saccharomyces cerevisiae and C. albicans (Huh et al., 2001, Huh et al., 1998). The ALO1 gene
is a gene that encodes D-Arabino-1,4-lactone oxidase catalysing the final step of Derythroascorbic acid (EASC) biosynthesis. The ALO1 gene and the biosynthetic pathway of
EASC from D-arabinose by d-arabinono-1,4-lactone oxidase (ALO) D-arabinose dehydrogenase
have
been
well
described.
According
to
the
Candida
Genome
Data
Base
(http://www.candidagenome.org/) CAGL0H04125g open frame in C. glabrata genome is an
ortholog of ALO1 gene, nevertheless, this gene has not been experimentally characterized to
date.
Accumulation of ROS under external factors such as exposure to drugs, metals or phagocytic
cells, which are the first line of defence against fungal infections, may lead to potential oxidative
damage to proteins, nucleic acids and other macromolecules, which can severely compromise
cell health and viability (Ferreira et al., 2013, Cuéllar-Cruz et al., 2008, Fujs et al., 2005).
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The main aims of this chapter were to:
(i)
Investigate whether induction of oxidative stress by TQ is a cause of fungal cell death.
(ii)
Clone CAGL0H04125g open reading frame, which was predicted to be an ortholog to
ALO1 gene.
(iii)
Examine whether cloning of CAGL0H04125g may contribute to over production of
EASC and whether this will provide protection to C. glabrata cells against TQ toxicity.
3.2 Materials and methods
3.2.1 Chemicals
Thymoquinone, fluconazole, hydrogen peroxide, dimethyl sulfoxide (DMSO), L-reduced
glutathione,
2′,7′
dichlorodihydrofluorescein
diacetate
(H2DCF-DA),
rhodamine
123,
monochlorobimane ascorbic acid and D-isoascorbic acid were purchased from Sigma Aldrich
(Australia).
3.2.2 Yeast strains and media
The yeast strains employed in this study are listed in Table 3.1. Yeast strains were maintained
and grown on YEPD agar medium. Yeast minimal medium without amino acids (YMM w/o AA)
was used for screening of transformants. RPMI1640 containing L-glutamine, but lacking sodium
bicarbonate was used for sensitivity assay and measuring oxidation stress.
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Escherichia coli strains were maintained and grown at 37 ºC on Luria-Bertani medium (LB
medium) containing 1
μg/ml ampicillin for selection of recombinant plasmids.
Table 3.1 Strains used in this study.
Strain
Description
Source or reference
Escherichia coli DHαTM
Wild type
Helen Williams (RMIT university)
Escherichia coli DH10B
E. coli DHα carrying pCU-
(Zordan et al., 2013)
HHT2 empty plasmid.
(Addgene plasmid # 45319)
DHα transformed with HHT2-
This study
Escherichia coli 16a
ALO1 plasmid.
Candida glabrata ATCC2001
Wild type
ATCC
Candida glabrata ATCC90030
Wild type
ATCC
Candida glabrata MH100
ATCC2001 ura3∆
(Muller et al., 2008)
Candida glabrata HHT2
MH100 transformed with pCU-
This study
HHT2 plasmid (empty vector)
Candida glabrata 16a
MH100 transformed with
HHT2-ALO1 plasmid
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3.2.3 Effect of antioxidants
In experiments involving antioxidants, cells of C. glabrata ATCC90030 were used. Reduced
glutathione and ascorbic acid stocks were freshly prepared as 40 mM stocks in RPMI 1640. The
same procedure which was used in preparing 96 wells microplates for determining MICs in
section 2.2.2 was performed except adding of ascorbic acid or L-reduced glutathione to the
medium to achieve final concentration of 10 mM. Addition of antioxidants was performed
before treatment with TQ.
3.2.4 Detecting reactive oxygen species (ROS)
The level of ROS was examined using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA).
This molecule can diffuse freely into cells. The acetate moiety is cleaved by cellular esterases,
leaving impermeable, non-fluorescent, 2′,7′-dichlorodihydrofluorescein (H2DCF). The H2DCF
is oxidized by ROS to dichlorofluorescein (DCF), which emits fluorescence at 530 nm in
response to 488 nm excitation (Martin et al., 2004, Eruslanov et al., 2010).
C. glabrata ATCC2001 cells from a freshly grown YEPD plate, were resuspended in the same
medium that was used in determining MICs. Thymoquinone was added to the cell suspensions at
density of approximately 106 cell/ml and incubated for 2 h at 35 °C with agitation (200 rpm).
2′,7′-dichlorodihydrofluorescein diacetate stocks were prepared freshly in DMSO. H2DCF-DA
(Sigma-Aldrich) was added directly to have a final concentration of 10 µg/ml of cell culture
(Madeo et al., 1999) and incubated further 2 h in the dark. One ml from each sample was
centrifuged at 9000 rpm for 1 min. The pellets were resuspended in RPMI 1640 and left to re-
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grow at 35°C for 1 h. Cells were washed and resuspended in filter sterilized PBS before flow
cytometry analysis using a FITC filter in a FACS Canto II (BD Biosciences). Controls including
untreated cells with and without H2DCF-DA, cells treated with DMSO or TQ with and without
H2DCF-DA.
3.2.5 ROS and cell membrane integrity
Cells in exponential phase (2 h in liquid YEPD medium) were used to examine whether ROS
generation follows cells death or TQ causes ROS generation which then leads to cell death.
About 106 cell/ml were treated with 50 µg/ml TQ for 4 h in total. Untreated cells which were
used as negative control, were stained with H2DCF-DA as described above (section 3.2.4). After
washing with filter sterilized PBS buffer, cell pellets were suspended in 500 µl of 2 µg/ml
propidium iodide solution and incubated 30 min at 30 °C in the dark. Cell suspensions were
washed with PBS and observed with an Epifluorescent microscope (Leica DM 2500) fitted with
a Leica DFC310 FX camera. A GFP filter was used to observe DFC positive cells and Texas red
filter was used to observe PI positive cells and Blue filter was used for imaging both stains.
To obtain data from a higher number of cells, cell suspensions were further analysed by flow
cytometry using a FITC filter in a FACS CantoTM II (BD Biosciences) and perCP-Cy5-5A filter.
Controls including untreated cells with and without H2DCF-DA, cells treated with DMSO or TQ
with and without H2DCF-DA were included.
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3.2.6 Estimation of intracellular reduced glutathione content
The cellular level of reduced glutathione (GSH) was estimated with monochlorobimane (Staleva
et al., 2002, Kwolek-Mirek et al., 2009, Nair et al., 1991). C. glabrata ATCC 90030 cells (OD600
= 0.96) from an overnight plate were suspended in RPMI 1640 (medium as in MIC assay), then
were treated with 0, 25 and 50 µg/ml of TQ for 2 h at 35°C with agitation at 170 rpm. One ml
was washed with PBS, and incubated for 30 min with 4 μM monochlorobimane (SigmaAldrich) at room temperature. Cells were washed again and resuspended in PBS. The
fluorescence of the bimane–glutathione conjugates was observed using Nikon® A1 confocal
laser-scanning microscope equipped with a Plan Apo VC X60 water-immersion objective, an
argon laser (488 nm) and a solid state laser (561 nm) using 488 nm excitation laser (excitation:
36 /4 nm). Flow cytometry data were recorded using a BD FACS Canto™ II equipped with a
pacific blue fluorescent filter (351- 450/65 nm excitation and emission).
3.2.7 Measuring of mitochondrial membrane potential
Staining of mitochondria was performed as described by (Vandeputte et al., 2009). Briefly, cells
of C. glabrata ATCC90030 from overnight YEPD plates were suspended in RPMI 1640 (0x106
cell/ml) and incubated 4 h with TQ at 35 °C with shaking (200 rpm). Cells were harvested then
washed with PBS and incubated for 30 min at 35 °C with 250 µl of 10 µg/ml rhodamine 123
solution in the dark. Cell suspensions were then again washed with PBS and resuspended in 250
µl PBS. Resulting aliquots were analysed by flow cytometry using an FITC filter (494-519 nm
excitation /emission). The cells were observed microscopically using an FITC filter to examine
staining efficiency. An epifluorescence microscope (Leica DM 2500) combined with a Leica
DFC310 FX camera for image visualization was used. Controls included untreated cells with and
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without rhodamine 123, cells treated with DMSO and TQ with and without rhodamine 123.
For all fluorochrome experiments, 50 ml centrifuge tubes were used with working volumes not
more than 4 ml. The tubes were not protected from light after adding of TQ but they were
protected from light after the fluorophore was added.
3.2.8 Amplification of ALO1 gene by polymerase chain reaction
The template genomic DNA for polymerase chain reaction (PCR) was isolated from C. glabrata
CBS138 according to the manufacturing guideline of Isolate II Genomic DNA kit (Bioline). The
concentration of isolated DNA was quantified by measuring absorbance (A260/ 280) using a
NanoDrop spectrometer (NanoDrop Lite™). Prior to running of conventional PCR procedure, a
gradual PCR was carried out with gradual annealing temperature between 45 °C to 70 °C to find
out the suitable annealing temperature. The 25 µl PCR reaction mixture contained 0.5 µl of 10
mM of each primer, 5 µl of 10X Taq polymerase buffer (supplied by Bioline), 0.145 µl My Taq
polymerase (Bioline) and about 75 ng template genomic DNA. The mixture was subjected to 30
cycles of denaturation at 95 °C for 0.5 min, 1 min annealing at 50 °C, 2 min extension at 72 °C
and incubated at 4ºC until further investigation. The sequence of interest was amplified using a
bench top thermocycler (G-STORM, Geneworks, Australia). The oligonucleotide primers for
amplifying the ALO1 gene were designed based on the nucleotide sequence of the open reading
frame (ORF) CAGL0H04125g which was obtained from the CGD. Clone Manager 9 software
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(www.scied.com) was used to check the primers design. The ORF of C. glabrata CBS138 ALO1
was amplified using the primer pair ALO1F and ALO1R (table 3.2).
Table 3.2 Primers used in this study: the restriction sites are underlined and arrows indicate the
restriction sites.
Primer name
Primer Sequence
Source of design
ALO1F
5’-GCCCGGGATGGATTTGAAAACATTTGGTG-3’
This study
ALO1R
5’-GCCGCTCGAGTTATTCGTTTTCATCAATGATACC-3’
This study
ALOiF
5’- ACCACAGACTAAGTCAAG -3’
This study
HHT2F
5’-TGTTATTGATTATTTATTTATTTG-3’
(Zordan et al.,
2013)
3.2.9 Agarose gel electrophoresis
Products of PCR were analysed by agarose gel electrophoresis. One percent of agarose (w/v) was
prepared in 1 x TAE buffer (appendix A) and heated in a microwave with stirring intervals until
it was completely dissolved. Dissolved agarose was left to cool then poured into a gel tray with
a comb. Cool gels were located in a gel tank (Bio Rad) filled with TAE buffer. One microliter of
loading dye (Cat 31022, Biotium) was added to each 10 µl sample before loading to gel wells. A
DNA marker (New England Biolabs® Inc.) was run in parallel with the samples for MW
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comparison. Gel electrophoresis was run at 100 Volts for 65 min, then gels were placed in a 1
mg/ml ethidium bromide bath for approximately 10 min. Agarose gels were then destained by
washing under running tap water for 20 min before viewing. Gels were visualised under UV light
in a Bio-Rad Gel Doc™ UV transilluminator system. Images of gel electrophoresis were
processed using Quantity One 1-D analysis software (Bio-Rad, Australia; version 4.6.8).
3.2.10 Vector digestion and ALO1 ligation
The amplified DNA fragment of 1,595 bp was cloned into pCU-HHT2 vector (Plasmid 45319;
Addgene) at the multiple cloning site between HHT2 promoter, which is a constitutive promoter,
and HIS3 terminator (Fig. 3.1). Plasmids were isolated form E. coli DH10B using Quick plasmid
miniprep kit (Invitrogen). Then 1 µl of XmaI and XhoI restriction enzymes and 5 µl of 10x
NEBuffer 3.1 (NEW ENGLAND BioLabs® Inc.) were added to approximately 1.5 µg of
plasmid DNA. The reaction volume was completed to 50 µl using nuclease-free water and the
digestion reaction mixture was incubated at 37ºC for 3 h followed by heat inactivation of the
enzymes at 65ºC for 20 min. After that digested DNAs were cleaned prior to ligation by utilizing
of ISOLATE II PCR and Gel Kit (Bioline) and kept at -20ºC until use. For ligation T4 DNA
ligase (M0202; New England Biolabs® Inc.) was used to generate HHT2-ALO1. The desired
amount of insert DNA was calculated using Ligation calculator which was developed by
Düsseldorf University website (http://www.insilico.uni-duesseldorf.de/Lig_Input.html). Twenty
microliters of ligation mixture contained vector DNA, insert DNA, T4 DNA ligase and T4 DNA
ligase buffer (New England Biolabs® Inc.) was incubated overnight at 16 ºC. Ligation products
were transformed chemically into E. coli DH5α competent cells which were prepared according
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to a protocol adapted from Mrs Kim Stevenson (RMIT University). Briefly, 5 µl of the ligated
DNA were added to 100 µl of E. coli DH5α competent cells and the mix kept on ice for 1 to 15
min and then heat shocked at 42 ºC for 50 sec. Samples were located back on ice for 2 min, then
1 ml of LB broth medium containing 0.4 % glucose was added. Cells were left to recover for 30
min at 37 ºC then plated on LB agar plates supplemented with ampicillin (appendix A).
Transformants were inoculated into LB broth supplemented with ampicillin and incubated
overnight at 37 ºC. The recombinants were purified and sequenced using primers ALOiF and
HHT2F (table 2 & Fig. 3.1). Sequencing of the cloned DNA was performed at Micromon DNA
sequencing facility (Monash University).
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HIS3 terminator
HHT2 promoter
Figure 3.1 Schematic diagram of ALO1 plasmid developed in this study. The pCU-HHT2 plasmid
backbone caters for expression of ALO1 gene (CAGL0H04125g) in C. glabrata. The graphic map was
generated using Addgene software from the full sequence supplied by Zordan et al., (2013). The
restriction sites of XhoI and XmaI in the multiple cloning site (MCS) between the promoter and terminator
are underlined.
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3.2.11 Overexpression of ALO1 in C. glabrata
A 6.1 kb pCU-HHT2 vector containing ALO1 (HHT2-ALO1) was isolated from E. coli DH5α
using Quick plasmid miniprep kit (Invitrogen). The DNA sequence of interest in the purified
plasmids was sequenced by the Micromon sequencing centre (Monash University, Clayton,
Australia) using the primers that were used for amplification of the PCR product and multiple
samples of plasmids with the gene of interest were also sequenced using ALOiF and HHT2F
primers (Table 3.2). The HHT2-ALO1 plasmid was used to transform C. glabrata HM100 cells
using EZ yeast transformation kit (MP Biomedicals). URA3+ cells were selected using complete
yeast minimal medium lacking uracil (YMM).
3.2.12 Measurement of intracellular EASC
The extraction of EASC from C. glabrata was carried out according to the method proposed by
Huh et al. (1998) with some modifications. C. glabrata cells from overnight culture (about 1 g in
wet mass) grown in a YNB liquid medium were recovered by centrifugation at 5000×g for 10
min at 4 ºC, washed twice with cold MilliQ water and resuspended in 10 % trichloroacetic acid
(1 ml per 1 g of wet weight cells). This suspension was stored for 30 min at 4 ºC with vortexing
every 5 min. The insoluble residue was removed by centrifugation at 10,000 g for 2 min
followed by filtration of the supernatant through a 0.22 µm filter.
20 µl from the soluble extract of each strain were subjected to analytical liquid
chromatography/tandem mass spectrometry (LC-ESI-MS/MS). The LC-MS/MS system was an
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Agilent 1200 liquid chromatograph and a 6410 triple quadrupole mass spectrometer with an
electrospray ionisation source. Separation was performed on a Zorbax SB-C18 column (4.6 mm
x 150 mm, 5 µm particle size) by gradient elution using the following gradient: 0-5 min 2 % B
isocratic; 5-35 min linear gradient to 95% B; 35-36 min isocratic 96% B, 36-40 min to 2 % B
and the column was equilibrated with 2% B for 10 minutes. Eluent A was 0.1 % formic acid and
B was methanol. Mass spectrometry was performed on a triple quad spectrometer in ESI positive
ion mode. High purity nitrogen served as both the nebulising and drying gas, with a drying gas
flow 8 L/min and a nebuliser pressure 15 psi. The capillary voltage was set at 4000 V and the
source at 3000 C. The system was calibrated using D-isoascorbic acid (the supplier synonym of
D-erythroascorbic-acid).
3.2.13 Assay for resistance to oxidative stress
The effect of ALO1 overexpression on the sensitivity of C. glabrata to TQ and H2O2 was
examined. Candida glabrata transformed with ALO1 gene (C. glabrata 16a) and its control
strain C. glabrata HHT2 were used in this assay. Experiments in liquid medium were carried out
according to the method proposed by Izawa et al., (1995) with some modifications. Cells were
grown in minimal defined medium (YMM) to mid-logarithmic phase to obtain the initial OD600=
0.5. Then 2 ml of cell suspensions were treated with 38.8 or 77.7 mM of H2O2 or 50 µg/ml TQ.
The treatment process was performed in YMM media or in PBS after washing the cells with the
same buffer. After incubation for 1 h at 30 ºC with agitation (200 rpm), 100 µl aliquots were
taken from the cell suspensions, diluted appropriately in PBS and plated onto solid minimal
LC-MS/MS data collection and analysis was done kindly by Mr. Sushil Anand (RMIT University)
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defined medium. Colonies were counted after incubation for 72 h at 30 ºC.
In addition, sensitivity of C. glabrata 16a to TQ and H2O2 was compared to the control strain C.
glabrata HHT2 and its parental strain on YMM agar plates. Ten-fold serial dilutions were
processed using colonies grown on YMM plates. Five microliters of cell suspension were spotted
on YMM plates containing 38.8 or 77.7 mM of hydrogen peroxide H2O2 or 50 µg/ml of
thymoquinone. Plates were incubated overnight at 30 ºC.
3.2.14 Data analyses
The corresponding geometric mean (G mean) values of the replicates was calculated using
GraphPad Prism Software. Error bars represent Standard Error of the Mean (SEM) and one way
ANOVA test combined with Tukey's multiple comparisons test and e-test were used to analyse
the data and find p values.
The BLAST analysis tool was used to match the sequence of CAGL0H04125g ORF with cloned
sequence and alignment of ALO1 protein sequence.
Fluorescence distributions were obtained from 10,000 events per sample. The fluorescence of
cell populations was analysed using Flowing software (Cell Imaging Core of the Turku Centre
for Biotechnology, Finland, version 2.5.1).
LC-MS/MS data were analysed using the MassHunter software package (Agilent Corporation,
MA, USA).
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3.3 Results
3.3.1 Antioxidants protect against the toxicity of TQ
To demonstrate the role of oxidative stress in the toxicity of TQ, antioxidant protection of C.
glabrata against this quinone was examined. Ten milimolar of ascorbic acid and L-reduced
glutathione protected C. glabrata ATCC 90030 from TQ toxicity (Fig. 3.2). This indicated the
pro-oxidant effect of TQ.
3.3.2 Thymoquinone stimulates the generation of reactive oxygen species (ROS)
To measure the increase of ROS in cells exposed to TQ, ROS level in the cells treated with TQ
was estimated using H2DCFDA which indicates oxidation by hydrogen peroxide, peroxynitrite,
hydroxyl radical and at lesser extent superoxide anions (Eruslanov et al., 2010). After treating C.
glabrata ATCC2001 for four hours with 25 or 50 µg/ml TQ, the green fluorescence intensity
which corresponds to levels of ROS was found to depend on TQ concentration (Fig. 3.3). About
50% of the cells were stained with H2DFC-DA after 4 h once they were treated with 50 µg/ml
TQ. Sub-inhibitory concentration of TQ (25 µg/ml) did not significantly affect cells. Additional
controls indicated that neither autofluorescence nor nonspecific binding of fluorochromes
contributed significantly to the staining patterns observed.
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Figure 3.2 Elimination of TQ toxicity using 10 mM of reduced glutathione (GSH) and ascorbic
acid (AA).
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0.1% ± 0.1%
3%
± 2%
50% ± 10%
Events
(10,000)
0
3
4
10
10
5
10
DFC fluorescence intensity
Figure 3.3 Thymoquinone induces ROS production in C. glabrata ATCC2001. Cells were
treated for 4 h with TQ and stained with H2DCF-DA. Cells were treated with 0 (black), 25 (blue)
or 50 µg/ml TQ (green). The percentages of fluorescence intensity are averages of three
independent replicates ± SD. Percentages colour correspond to each lines colour.
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3.3.3 Induction of ROS generation causes cell death
To investigate whether ROS generation was the cause of cell death or it was a result of cell
death, C. glabrata ATCC2001 cells were stained by H2DFC-DA to observe ROS generation as
described in section 3.3.2 followed by propidium iodide (PI) staining to detect loss in membrane
integrity. Cells were treated with 25 or 50 µg/ml TQ for 5 h and observed by epifluorescence
microscopy or by flow cytometry. Labeling with H2DFC-DA and/or PI can distinguish four
distinct cell states: (a) live unstressed cells (DFC-negative/PI-negative), (b) live cells that suffer
from oxidative stress (DFC-positive/PI-negative), (c) dead cells (DFC-positive/PI-positive or
DFC-negative/PI-positive). This assay can help to establish the sequence of events leading to cell
death. If all cells are both DFC and PI positive it means ROS was a result of cell death whereas
if only part of the population is DFC positive that means ROS generation leads to cell death.
Figure 3.4 shows cells which have been treated with 50 µg/ml TQ for 5 h. A portion of these
cells was only DFC positive which indicated induction of oxidation in these live cells, others
were both DFC/PI positive or PI positive which indicated the death of cells after suffering from
ROS damage. In addition, there were some live cells which did not stain with either dye. Cells
which were treated with 25 µg/ml TQ did not show any significant number of positive events for
either stain.
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(A)
(B)
(C)
12%
18%
PI
PI
48%
3
10
4
10
5
3
10
10
4
10
5
10
DFC
(D)
(E)
Figure 3.4 Pro-oxidant effect of thymoquinone leads to cell death. Microscopy images of C. glabrata
cells treated with 50 µg/ml TQ for 5 h then stained with H2DFC-DA and PI (A) bright field (B) cells
suffer from oxidative stress (DFC fluorescent positive), (C) cells that lost their cell membrane integrity
(PI positive) merged with DFC positive cells. Scale bars represent 10 µm, (D) Flow cytometry analysis
for DFC and PI stained untreated cells. (E) Flow cytometry analysis for DFC and PI stained cells which
were treated with 50 µg/ml TQ for 4 h. Data are representative of three independent experiments.
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3.3.4 Thymoquinone depletes cellular glutathione
Depletion of the reduced form of glutathione was shown to be another possible marker of
oxidation in yeast (Castro et al., 2010). After treatment of cells with TQ, the level of intracellular
reduced glutathione was determined using monochlorobimane (mBCl), a dye that shows a high
degree of specificity for reduced glutathione (Nair et al., 1991). In this experiment cells from an
exponential phase culture had an insignificant number of bimane–glutathione conjugate positive
cells, while cells that were taken from overnight plates showed higher rate of positive events.
Therefore the latter cells were used. Cells treated with 50 µg/ml TQ showed a reduction in
fluorescence intensity within 2 h compared with untreated cells. Figure 3.5 shows an example of
GSH depletion which was caused by TQ. About 50% of the cells had reduced ability to emit the
fluorescence of bimane–glutathione conjugate after treating them with 50 µg/ml TQ for 2 h
compared with untreated cells. This demonstrates a depletion in the reduced glutathione (GSH)
intracellular content.
Controls of TQ, DMSO and untreated cells indicated that neither
autofluorescence nor nonspecific binding of fluorochromes contributed significantly to the
staining patterns observed.
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85%
40%
0
10
3
5
10
mBCl fluorescence intensity
Figure 3.5 Thymoquinone caused a depletion in reduced glutathione in C. glabrata ATCC90030. Flow
cytometry analysis of monochlorobimane (mBCl) staining. Cells inoculated in RPMI 1640 and stained
with mBCl (black line). Cells were treated for 2 h with 50 µg/ml TQ and stained with mBCl (red line).
Percentages represent the mean of fluorescence intensity of three independent replicates. Percentages
colour correspond to each lines colour. The histogram adapted from one experiment. The inset shows C.
glabrata cells stained with mBCl.
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3.3.5 Thymoquinone causes depolarization of mitochondria
The lipophilic cationic fluorochrome rhodamine123 stains mitochondria selectively in a
membrane potential and mass dependent manner. This fluorochrome incorporation depends on
the electrochemical potential difference between the intermembrane space and the mitochondrial
lumen (Riesbeck et al., 1990). A defect in the electron flow through the mitochondrial
respiratory chain leads to lower incorporation of this dye (Riesbeck et al., 1990, Skowronek et
al., 1990, Vandeputte et al., 2009, Ludovico et al., 2001). Figure 3.6 shows great incorporation
of rhodamine123 by untreated cells whereas TQ treated cells showed a dose dependent decreased
rate of rhodamine123 staining. Compared to the untreated control, treatments with 50 and 100
µg/ml TQ led to approximately 30 % and 49 % decreases in rhodamine123 fluorescent intensity.
These results indicate that in these conditions the membrane potential and the mass of
mitochondria in C. glabrata cells that were treated with TQ was much lower than that of the
control. Similar results were obtained in two independent experiments, and incubation of the
cells without rhodamine123 confirmed the absence of autofluorescence.
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Figure 3.6 Thymoquinone causes a decrease in mitochondrial membrane potential. Flow
cytometry analysis of rhodamine123 staining of C. glabrata ATCC90030. Black line indicates
untreated cells with (right) and without (left) rhodamine123 staining, green line is cells treated
with 0.5% DMSO (the highest concentration used), blue line cells treated with 50 µg/ml TQ and
red line cells treated with 100 µg/ml TQ. Data are representative of two independent biological
replicates. The inset shows fluorescent microscopy image of mitochondria in C. glabrata cells
without treatment after staining with 10 µg/ml Rhodamine 123.
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1
2
3
Figure 3.7 Digestion of HHT2-ALO1 vector with XhoI and XmaI enzymes resulted in a band of
approximately 1.6 kb (lane 2), lane 1 is digested empty HHT2 vector, lane 3 DNA markers.
3.3.6 Characterization of the ALO1 gene in C. glabrata
The ORF CAGL0H04125g from the chromosomal DNA of C. glabrata ATCC2001 was
amplified by PCR using the oligonucleotide primer pair ALO1F and ALO1R. When cloned and
digested the 1,595 bp fragment was observed in plasmid HHT2-ALO1 but absent in the starting
vector pCU-HHT2 (Fig. 3.7). The nucleotide sequence of the cloned ALO1 open reading frame
was compared with that of the same gene sequence obtained from C. glabrata ATCC 2001 in the
Candida Genome Database (CGD, http://www.candidagenome.org). The identity was 99.6%
because six mismatches were found within the coding region of the gene (Fig. 3.8).
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Cg16a: 1
CgCBS: 1
Cg16a: 61
CgCBS: 61
Cg16a: 121
CgCBS: 121
Cg16a: 181
CgCBS: 181
Cg16a: 241
CgCBS: 241
Cg16a: 301
CgCBS: 301
Cg16a: 361
CgCBS: 361
Query: 421
Sbjct: 421
Query: 481
Sbjct: 481
Query: 541
Sbjct: 541
Query: 601
Sbjct: 601
Query: 661
Sbjct: 661
Cg16a: 721
CgCBS: 721
Cg16a: 781
ATGGATTTGAAAACATTTGGTGGTCGGCGGAACTTTGTGTTTCGCAATTGGGCTGGTATC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ATGGATTTGAAAACATTTGGTGGTCGGCGGAACTTTGTGTTTCGCAATTGGGCTGGTATC 60
TATTCCTCAAGACCAGAATGGTACTTTCAGCCTTCCTCAGTCGACGAAGTTGTCGAAATT 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TATTCCTCAAGACCAGAATGGTACTTTCAGCCTTCCTCAGTCGACGAAGTTGTCGAAATT 120
GTCAAAGCTGCTAAACTAAAGAACAAGACTATTGTTACAGTTGGTTCAGGCCACTCCCCT 180
|||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||
GTCAAAGCTGCTAAACTAAAGAACAAGACTATTGTTACAGTTGGCTCAGGCCACTCCCCT 180
AGTAACATGTGTGTTACCGACGAATGGATGATGAACTTGGACAAGATGAACAAGTTACTA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AGTAACATGTGTGTTACCGACGAATGGATGATGAACTTGGACAAGATGAACAAGTTACTA 240
GATTTTGTGGAAAACGAGGATAAGACATACGCAGACGTTACTATTCAAGGTGGTACTAGG 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GATTTTGTGGAAAACGAGGATAAGACATACGCAGACGTTACTATTCAAGGTGGTACTAGG 300
TTGTATGAGATCCACAGAATATTAAGGGAAAAGGGATACGCCATGCAAAGTTTGGGGTCC 360
|||||| ||||||||| |||||||||||||||||||||||||||||||||||||||||||
TTGTATAAGATCCACAAAATATTAAGGGAAAAGGGATACGCCATGCAAAGTTTGGGGTCC 360
ATTTCTGAACAAAGTATTGGTGGTATTATCTCTACTGGTACTCATGGTTCGTCTCCATTC 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ATTTCTGAACAAAGTATTGGTGGTATTATCTCTACTGGTACTCATGGTTCGTCTCCATTC 420
CATGGTCTGGTATCTTCTACTATTGTCAACTTGACTGTTGTCAATGGTAAAGGTGAAGTA 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CATGGTCTGGTATCTTCTACTATTGTCAACTTGACTGTTGTCAATGGTAAAGGTGAAGTA 480
TTATTTTTAGACGAAAAGTCTGACCCTGAAGTTTTCAGGGCTGCTACTTTGTCGCTTGGT 540
||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||
TTATTTTTAGACGAAAAGTCTAACCCTGAAGTTTTCAGGGCTGCTACTTTGTCGCTTGGT 540
AAGATTGGTATTATTGTGGGTGCAACTGTTCGTGTTGTTCCAGCTTTCAACATTAAATCA 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AAGATTGGTATTATTGTGGGTGCAACTGTTCGTGTTGTTCCAGCTTTCAACATTAAATCA 600
ACCCAAGAAGTTATCAAATTTGAAACTCTTTTGGAAAAATGGGACTCTCTCTGGACTTCT 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ACCCAAGAAGTTATCAAATTTGAAACTCTTTTGGAAAAATGGGACTCTCTCTGGACTTCT 660
TCAGAATTTATCAGAATTTGGTGGTACCCATATACACGTAAGTGTATCCTTTGGAGAGGT 720
||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||
TCAGAGTTTATCAGAATTTGGTGGTACCCATATACACGTAAGTGTATCCTTTGGAGAGGT 720
GTAAAGACTAATGAACCACAGACTAAGTCAAGATATTCGTGGTGGGGTTCTACTCTAGGT 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GTAAAGACTAATGAACCACAGACTAAGTCAAGATATTCGTGGTGGGGTTCTACTCTAGGT 780
CgCBS: 781
AGATTTTTCTACCAGACTTTGTTGTTCATATCTACCAAGATTTACCCACCTTTGACTCCA 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AGATTTTTCTACCAGACTTTGTTGTTCATATCTACCAAGATTTACCCACCTTTGACTCCA 840
Cg16a: 841
TATGTTGAAAGATTTGTCTTCAGAAGACAATATGGTGAAGTTGAAACCCTAGGTAAAGGT 900
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CgCBS: 841
Cg16a: 901
CgCBS: 901
Cg16a: 961
CgCBS: 961
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TATGTTGAAAGATTTGTCTTCAGAAGACAATATGGTGAAGTTGAAACCCTAGGTAAAGGT 900
GATGTGGCAATTGAGGATTCTGTTACTGGGTTCAACATGGACTGTTTGTTTTCTCAGTCC 960
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |
GATGTGGCAATTGAGGATTCTGTTACTGGGTTCAACATGGACTGTTTGTTTTCTCAGTTC 960
GTTGATGAATGGGGTTGTCCAATGGACAATGGTTTAGAAGTCTTGCGTTCTCTTGACCAC 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GTTGATGAATGGGGTTGTCCAATGGACAATGGTTTAGAAGTCTTGCGTTCTCTTGACCAC 1020
Cg16a: 1021 TCAATTGCTCAAGCCGCTGCTAACAAGGATTTCTATGTACATGTCCCAGTTGAGGTTCGT 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CgCBS: 1021 TCAATTGCTCAAGCCGCTGCTAACAAGGATTTCTATGTACATGTCCCAGTTGAGGTTCGT 1080
Cg16a: 1081 TGTGCAAACACAACTTTGCCAAAGGAACAACCTGAAACTTCTTTCCGTTCTAACACTAGT 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CgCBS: 1081 TGTGCAAACACAACTTTGCCAAAGGAACAACCTGAAACTTCTTTCCGTTCTAACACTAGT 1140
Cg16a: 1141 AGAGGTCCAGTTTACGGTAACCTATTACGTCCTTACTTGGATAACACCCCATCTCAATGC 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CgCBS: 1141 AGAGGTCCAGTTTACGGTAACCTATTACGTCCTTACTTGGATAACACCCCATCTCAATGC 1200
Cg16a: 1201 TCTTATGCTCCTATCCACAGTGTTACTAATAGTCAGTTGACGCTATACATTAACGCGACA 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CgCBS: 1201 TCTTATGCTCCTATCCACAGTGTTACTAATAGTCAGTTGACGCTATACATTAACGCGACA 1260
Cg16a: 1261 ATTTACAGACCATTCCACACCAATGCCCCAATTCACAAGTGGTTCACCTTGTTTGAAGAT 1320
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CgCBS: 1261 ATTTACAGACCATTCCACACCAATGCCCCAATTCACAAGTGGTTCACCTTGTTTGAAGAT 1320
Cg16a: 1321 ACAATGTCTGCTGCTGGCGGTAAACCACATTGGGCTAAGAACTTCTTGGGCTCTACCTCT 1380
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CgCBS: 1321 ACAATGTCTGCTGCTGGCGGTAAACCACATTGGGCTAAGAACTTCTTGGGCTCTACCTCT 1380
Cg16a: 1381 TTTGCTCAAGGTCAAGTTAAGGCTGAAGGTCAATACCAAGACTATGAGATGAGAGGTATG 1440
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CgCBS: 1381 TTTGCTCAAGGTCAAGTTAAGGCTGAAGGTCAATACCAAGACTATGAGATGAGAGGTATG 1440
Cg16a: 1441 GCTACAAGAGTCAAGGAATGGTATGGTTCAGACTTGGAAACCTTTAAGAAGGTTAGAAGA 1500
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CgCBS: 1441 GCTACAAGAGTCAAGGAATGGTATGGTTCAGACTTGGAAACCTTTAAGAAGGTTAGAAGA 1500
Cg16a: 1501 GAACAAGACCCAGACAACATTTTCTTGGCAAACAAACAGTGGGCCTTGATCAACGGTATC 1560
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CgCBS: 1501 GAACAAGACCCAGACAACATTTTCTTGGCAAACAAACAGTGGGCCTTGATCAACGGTATC 1560
Cg16a: 1561 ATTGATGAAAACGAATAA 1578
||||||||||||||||||
CgCBS: 1561 ATTGATGAAAACGAATAA 1578
Fig 3.8 Alignment of CAGL0H04125g ORF published in the CGD with the sequenced insert (16a) in this study.
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Using the ExPASy gene sequence translate tool http://web.expasy.org/translate/ , cloned ALO1 gene
sequence was translated to its protein sequence (Fig. 3.9). Based on BLAST search prediction the
submitted ALO1 protein sequence had a putative binding site for covalently bound flavin adenine
dinucleotide (FAD) of oxygen-dependent oxidoreductases corresponding to amino acid residues 25-161
and ALO1 domain between intervals 188-510 (Fig. 3.10). Comparing the ALO1 protein in this study
(Cg ALO1) with the sequence available on CGD, the six nucleotide changes result in four amino acid
changes. The first three changes appear conservative, while the fourth is a less conservative change,
resulting into substitution of a serine with a phenylalanine at residue 320 (Fig. 3.10). Comparing the
ALO1 protein sequence in C. glabrata with characterised ALO1p in S. cerevisiae and C. albicans (Fig.
3.11) the first yellow highlighted lysine in CAGL0H04125g was replaced with glutamic acid in the
sequenced Cg ALO1 (C. glabrata 16a in this study) which is similar to C. albicans ALO1p and
asparagine (blue highlight in Fig 3.11) was replaced with aspartic acid which is similar to S. cerevisiae
ALO1sequence.
In order to overexpress ALO1 in C. glabrata, the plasmid HHT2-ALO1 was constructed by inserting the
entire ALO1 gene and its flanking sequences into the plasmid pCU-HHT2, as described in Materials and
Methods. C. glabrata (ΔURA3) cells were transformed with the parental plasmid pCU-HHT2 or HHT2ALO1, and transformants containing either plasmid C. glabrata HHT2 or C. glabrata 16a subsequently,
were selected by plating on minimal media lacking uracil.
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MCVTDEWMMNLDKMNKLTDFVENEDKTYADVTIQGGTRLYEIHRMLREKGYAMQSL
GSISEQSIGGIISTGTHGSSPFHGTVSSTIVNLTVVNGKGEVLFLDEKSDPEVFRAATLS
TGKIGIIVGATVRVVPAFNIKSTQEVIKFETTLEKWDSTWTSSEFIRIWWYPYTRKCITW
RGVKTNEPQTKSRYSWWGSTTGRFFYQTLLFMSTKIYPPLTPYVERFVFRRQYGEVET
TGKGDVAIEDSVTGFNMDCLFSQSVDEWGCPMDNGLEVLRSTDHSIAQAAANKDFYV
HVPVEVRCANTTLPKEQPETSFRSNTSRGPVYGNTLRPYLDNTPSQCSYAPIHSVTNSQ
LTTYINATIYRPFHTNAPIHKWFTLFE
Figure 3.9 ALO1 protein sequence which resulted from translation of the sequenced Cg ALO1 gene
in this study. The highlighted amino acids are different from those in the predicted sequence of C.
glabrata ALO1 protein as it was published by the CGD.
Figure 3.10 Diagram represents FAD binding domain (interval 25-161) and ALO1 (interval 188-510)
domain, which is specific to D-arabinono-1,4-lactone oxidase, in the submitted C. glabrata ALO1
sequence. Conserved domains have been detected in the submitted protein sequence using NCBI Blast
tool http://blast.ncbi.nlm.nih.gov/Blast.cgi.
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Scer|YML086C
Cgla|CAGL0H04125g
Calb|orf19.7551
----------------------------MSTIPFRKNYVFKNWAGIYSAKPERYFQPSSI
--------------------------MDLKTFGGRRNFVFRNWAGIYSSRPEWYFQPSSV
----------------------MTDIPESLKPFVTKKVIHSTWAGTFLCKPQAIFQPRNV
Scer|YML086C
Cgla|CAGL0H04125g
Calb|orf19.7551
*
DEVVELVKSARLAEKSLVTVGSGHSPSNMCVTDEWLVNLDRLDKVQKFVEY--------DEVVEIVKAAKLKNKTIVTVGSGHSPSNMCVTDEWMMNLDKMNKLLDFVEN--------EEIQELIKQARLHGKTIMTVGSGHSPSDLTMTTEWLCNLDKFNHVLLEEPYYAPKSPTDD
Scer|YML086C
Cgla|CAGL0H04125g
Calb|orf19.7551
-PELHYADVTVDAGMRLYQLNEFLGAK-GYSIQNLGSISEQSVAGIISTGSHGSSPYHGL
-EDKTYADVTIQGGTRLYKIHKILREK-GYAMQSLGSISEQSIGGIISTGTHGSSPFHGL
TPEIKFVDLTVEAGTRIFELNEYLKRN-NLAIQNLGSISDQSIAGLISTGTHGSTQYHGL
Scer|YML086C
Cgla|CAGL0H04125g
Calb|orf19.7551
ISSQYVNLTIVNGKGELKFLDAENDPEVFKAALLSVGKIGIIVSATIRVVPGFNIKSTQE
VSSTIVNLTVVNGKGEVLFLDEKSNPEVFRAATLSLGKIGIIVGATVRVVPAFNIKSTQE
VSQQVVSVKFLNSAGELITCSSVDKPEYFRAILLSLGKIGIITHVTLRTCPKYTIKSKQE
Scer|YML086C
Cgla|CAGL0H04125g
Calb|orf19.7551
VITFENLLKQWDTL--WTSSEFIRVWWYPYTRKCVLWRGNKTT--------DA-QNGPAK
VIKFETLLEKWDSL--WTSSEFIRIWWYPYTRKCILWRGVKTN--------EP-QTKSRY
IINFETLLNNWDNL--WLESEFIRIWWFPYTNKCVLWRANKST--------DP-LSDPRP
Scer|YML086C
Cgla|CAGL0H04125g
Calb|orf19.7551
SWWGTKLGRFFYETLLWISTKIYAPLTPFVEKFVFNRQYGKLEKS--STGDVNVTDSISG
SWWGSTLGRFFYQTLLFISTKIYPPLTPYVERFVFRRQYGEVETL--GKGDVAIEDSVTG
SWYGTKLGRFFYESLLWVSVHLFPRLTPFVEKFVFGQQYGEVETL--GKGDIAVQNSVEG
Scer|YML086C
Cgla|CAGL0H04125g
Calb|orf19.7551
FNMDCLFSQFVDEWGCPMDNGLEVLRSLDHSIA-------------QAAINKEFYVHVPM
FNMDCLFSQFVDEWGCPMDNGLEVLRSLDHSIA-------------QAAANKDFYVHVPV
LNMDCLFSQFVNEWSSPLNSGPEILTELKKIIT-------------DASQTGDFFVHAPI
Scer|YML086C
Cgla|CAGL0H04125g
Calb|orf19.7551
EVRCSNTTLPSEPLD---------------TSKRTNTSPGPVYGNVCRPFLDNTPSHCRF
EVRCANTTLPKEQPE---------------TSFRSNTSRGPVYGNLLRPYLDNTPSQCSY
EVRCSNVTYSDEPFT-DDKNQKSLYPSQEWLSNRSKTSAGPIPGNNLRPYLDNSPK-LPY
Scer|YML086C
Cgla|CAGL0H04125g
Calb|orf19.7551
APLEN---VTNSQLTLYINATIYRPFGCNTP---IHKWFTLFENTMMVAGGKPHWAKNFL
APIHS---VTNSQLTLYINATIYRPFHTNAP---IHKWFTLFEDTMSAAGGKPHWAKNFL
SKDGK---ITNDQLTLFINATMYRPFGTNVE---THKWFQLFEDVMSKAGGKPHWAKNFI
Scer|YML086C
Cgla|CAGL0H04125g
Calb|orf19.7551
GSTTLAA--------GPVKKDTDYDD---FEMRGMALKVEEWYGEDLKKFRKIRKEQDPD
GSTSFAQ--------GQVKAEGQYQD---YEMRGMATRVKEWYGSDLETFKKVRREQDPD
GLTQDEKYDKQ----QDLKTQLEFGGKPFYTMLGFKPVMQDWFGKDLVAFNKVRKETDPD
Scer|YML086C
Cgla|CAGL0H04125g
Calb|orf19.7551
NVFLANKQWAIIN----------------------------------------------NIFLANKQWALIN----------------------------------------------GVFLSGKVWAERN-----------------------------------------------
Scer|YML086C
Cgla|CAGL0H04125g
Calb|orf19.7551
---------------------------------GIIDPSELSD-------------------------------------GIIDENE----------------------------------------GILLD----------
*
K = E; K = R; N = D; F = S in C. glabrata 16a
Figure 3.11 Multiple ALO1 protein sequence alignment in C. glabrata ATCC2001 (CAGL0H04125g) with S.
cerevisiae (YML086C) and C. albicans (orf19.7551). Colored amino acids indicate a difference between published
sequence on CDG data base and ALO1 sequence analysed in this study. The regions where the sequences have been
extended to allow optimal sequence alignment are indicated with dashes. Identical residues are shaded. The asterisk
indicates the histidine residue believed to be responsible for covalent attachment of FAD.
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3.3.7 Candida glabrata transformants produce higher amounts of EASC
The presence of intracellular EASC in the resulted transformants C. glabrata 16a (carrying
HHT2-ALO1) and C. glabrata HHT2 (carrying pCU-HHT2) was confirmed and the level
quantified by comparison to a known standard using LC-ESI-MS/MS run in Multiple Reaction
Monitoring (MRM) mode. 20 µl from the soluble extract of each strain were subjected to
analytical liquid chromatography/tandem mass spectrometry to compare the concentration of
EASC at each extract. The MS chromatogram of the strain 16a and HHT2 by LC/MS/MS is
presented in figure 3.12A. The peak identity was determined by comparing the retention time of
the analyte to the standard (EASC) as well as by MS at m/z 177.1 (Fig. 3.12B). Multiple
Reaction Monitoring (MRM) recorded MS/MS ions at m/z 85.1 and 95.1 resulting from
fragmentation of m/z 177.1 (data not shown). Strain 16a showed a notable increase of EASC
production compared to the EASC produced by the control strain HHT2. The content of EASC
in 16a strain was estimated to be about 2.5 fold higher than that of HHT2 strain. Moreover,
MRM analysis of the EASC produced by both strains confirmed the characteristic fragment ion
at m/z 95.1 which would have originated from a direct elimination of heterocyclic ring from the
parent ion.
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(A)
(B)
Figure 3.12 The Extracted Ion Chromatogram (EIC) for protonated EASC and ESI-MS Spectra (a) EASC
in C. glabrata 16a strain (pink) and C. glabrata HHT2 strain (red). (b) ESI-MS Spectra.
3.3.8 Effect of EASC over production on resistance to oxidative stress
The difference in sensitivity of C. glabrata HHT2 and C. glabrata 16a to hydrogen peroxide and
thymoquinone as pro-oxidants was tested in liquid and solid YMM media. Over three biological
replicates in YMM liquid media no significant difference was observed between sensitivity of
both strains to the oxidants. Likewise on YMM agar plates transformants did not show
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differences in their sensitivity to either H2O2 or TQ compared with the strain that carries empty
vector (Fig. 3.13). The wild type strain (C. glabrata ATCC2001) showed less sensitivity to H2O2
than transformants when they were spotted on agar plates carrying the pro oxidants. The results
of C. glabrata sensitivity to H2O2 are in agreement with other results were obtained in previous
studies (Cuellar-Cruz et al., 2008, Roetzer et al., 2011b).
No treatment
38.8 mM H2O2
WT
CgHHT2
Cg16a
5
77.7 mM H2O2
(A)
50 µg/ml TQ
WT
CgHHT2
Cg16a
H H T2
16a
40
5
C F U /m l (x 1 0 )
50
compared with the wild type C. glabrata ATCC2001(WT)
30
and the control strain (C. glabrata HHT2). (A) Ten fold
20
serial dilutions of cell suspensions were spotted on YMM
10
plates supplemented with H2O2 or TQ. (B) Cells were treated
0
5
7
7
8
3
H 2O 2 (m M )
.6
.8
0
0
(B)
Figure 3.13 Sensitivity of C. glabrata 16a to H2O2 and TQ
with H2O2 or TQ for 1 h in YMM liquid medium then viable
T Q (  g /m l)
cells were counted, error bars indicate means of three
replicates and SEM.
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3.4 Discussion
3.4.1 Thymoquinone induces death via oxidation in C. glabrata
An oxidant/antioxidant ability of TQ depends on the milieu where it is present (Mansour et al.,
2002). Based on the possible involvement of pro-oxidant activity in the fungicidal effect of TQ
two well-known antioxidants, GSH and ascorbic acid, were investigated. Both antioxidants
could abrogate drastically the toxicity of TQ (Fig.3.2). Combination of TQ and these
antioxidants was examined to identify the initial products that are responsible for TQ toxicity
which seemed to be reactive oxygen species (ROS). The increased production of ROS by cells
treated with TQ confirmed the oxidative effect of TQ (Fig. 3.3). However, a further experiment
was needed to find out whether TQ induces ROS production which leads to cell death or ROS is
a bystander that accumulates as a result of cellular demise. The results of cell staining with DFC
and PI showed that part of treated population of cells generate ROS but still keep their cell
membrane integrity, confirming the generation of oxidative stress leading to cell death. This
accumulation of cellular ROS results in oxidative damage to important cell biomolecules such as
proteins, DNA and lipids. Failure to avoid such damage is associated with cell death (Farrugia et
al., 2012). Because accumulation of ROS involves a change in the redox state of cells, functions
that are involved in setting redox and maintaining redox homeostasis are relevant to an
understanding of the possible intracellular actions of TQ. For instance, interference with the
electron flow through the mitochondrial respiratory chain is one of the major sources of ROS in
yeast (Eisenberg et al., 2007, Vandeputte et al., 2009). Drugs that cause a damage in the electron
flow of the respiratory chain lead to reduction of mitochondrial dye uptake. Thus the effect of
TQ on depolarization of mitochondrial membrane potential in C. glabrata was monitored by
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rhodamine123 uptake. Figure 3.6 demonstrates that the membrane potential of TQ-treated C.
glabrata mitochondria was much lower than that of the control mitochondria. Significant, yet
incomplete, depolarization could be observed in the presence of 50 µg/ml TQ within 4 h. This
indicates loss in the membrane potential.
3.4.2 Reduced glutathione protects from TQ oxidation
Glutathione has a low redox potential (E0 = 0.24 V) and high concentration (2–3 mM) in yeast
cells. This intracellular reducing power provides an ability to eliminate potentially harmful free
radicals and reactive oxygen species (Fortuniak et al., 1996, Jamieson, 1998, Yadav et al., 2011).
Glutathione plays a major role in determining the cellular redox environment in most yeast
cellular compartments including mitochondria, nucleus, cytosol and the endoplasmic reticulum
which is the most oxidative compartment in the cell. Therefore the redox state of the GSH/GSSG
redox couple provides a good indication of the redox environment of cells and most cell
compartments (Ayer et al., 2013). Thymoquinone (50 µg/ml) at 2 h caused depletion in the GSH
reduced form which indicates the consumption of GSH in an attempt to stabilize the redox
homeostasis in the cell. The decrease in GSH concentration (relating to fluorescence intensity)
was not significant once cells were exposed to 25 µg/ml: this is due to the high density of cells
that was used in this assay (OD600= 1). It has been documented that exponential phase cells have
a lower storage of glutathione than yeast cells in stationary phase (Izawa et al., 1995). In
agreement with this less fluorescent intensity of mBCl was observed once untreated exponential
phase cultures were used (data not shown). Therefore, in this experiment it was decided to use
cells from an overnight plate rather than cells in exponential phase. Cells from a plate have a
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mixture of cells at different growth phases similar in that respect to populations that are found in
the environment. Yadav et al. (2011) showed that C. glabrata is not able to use exogenous
glutathione due to the lack of a glutathione transporter. However, Gutierrez-Escobedo et al.
(2013) showed that C. glabrata gsh1∆ pro2-4 and gsh2∆ mutant strains are able to incorporate
GSH from the medium. This study also shows the protective effect of exogenous reduced
glutathione to rescue C. glabrata from TQ toxicity which indicates the ability of these cells to
incorporate GSH from the medium.
3.4.3 C. glabrata can produce intracellular D-erythroascorbic acid
Supplementation of C. glabrata cells with extracellular ascorbic acid was protective against TQ
toxicity and identifying whether endogenous ascorbic acid homolog would be protective was
asked in this study. In C. glabrata, the ALO1 gene is 1,595 bp in size and encodes 557 amino
acids with a calculated molecular mass of 63,428 Da. It was predicted to encode D-arabinono1,4-lactone oxidase (ALO1p) that catalyzes the final reaction of EASC biosynthesis. This study
confirmed further its sequence and function.
LC-MS/MS analysis indicates intracellular
biosynthesis of EASC in C. glabrata. In addition the increase in EASC production via cloning
of CAGL0H04125g ORF confirms the involvement of this gene in EASC biosynthesis. The
transformants, carrying ALO1 on a multicopy plasmid, showed a 2.5 fold increase in EASC
production compared with the control strain. In S. cerevisiae EASC has been reported to function
as an important antioxidant like ascorbic acid in higher animals and plants (Huh et al., 1998).
The present study did not show a major role of EASC in defence against oxidation. This might
be due to the limited increase (2.5-fold) in EASC production compared with that in S. cerevisiae
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transformants which led to approximately 7-fold increase in EASC (Huh et al., 1998) and 4-fold
increase in C. albicans transformants (Huh et al., 2001). However, further investigation of the
importance of ALO1 in C. glabrata as antioxidant needs to be confirmed by generation of C.
glabrata ALO1 mutant strain.
3.5 Conclusion
In conclusion, these data support the hypothesis that generation of intracellular oxidative stress is
definitely a part of thymoquinone’s mechanism of action as a fungicidal agent. Oxidative stress
caused by TQ leads to loss of cell membrane integrity, reduction in reduced glutathione amount
and reduced mitochondrial membrane potential. D-erythroascorbic acid was identified as an
important antioxidant in other yeast, the over produced amount in this study was not enough to
create a significant difference in overcoming the oxidative stress caused by H2O2 or TQ.
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CHAPTER 4:
A model of hypo-osmotic stress to screen cell wall MAPK inhibitors in C.
glabrata
4.1 Introduction
In yeast, oxidative stress induces several mitogen activated protein kinases (MAPK) and
signalling routes. These include PKC1 MAPK, TOR and RAS-PKA-cAMP. Cross-talk between
these signalling pathways exists to activate transcription factors required for the expression of
response genes (reviewed by (de la Torre-Ruiz et al., 2010). The PKC1 (Protein kinase C)MAPK which is known also as the cell wall integrity (CWI) MAPK is activated usually in
response to stress of the cell surface. Cell wall integrity is essential for viability of fungi and is
an effective drug target in pathogens such as Candida glabrata (Hohmann, 2002). Cell wall
stress sensors pass the signal through CWI MAPK cascade (Fig. 1.2) to activate transcription
factors, mainly Rlm1 and SBF complex (consisting of Swi4 and Swi6) which are required for the
expression of cell wall biogenesis genes (Jendretzki et al., 2011). In Saccharomyces cerevisiae
phosphorylation of the last mitogen-activated protein kinase SLT2 in the CWI MAPK cascade
has been reported under stress conditions such as growth at high temperatures, hypo-osmotic
shock, oxidative stress, polarized growth, actin perturbation or the presence of compounds or
mutations that interfere with cell wall biosynthesis (Davenport et al., 1995, Soriano-Carot et al.,
2012, Krasley et al., 2006b). Therefore, the activation of SLT2 in C. glabrata under hypoosmotic and oxidative stress is proposed in this study. Phosphorylation of SLT2 has been
reported to mediate cyclin C destruction in response to H2O2 treatment which enables the genetic
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response in S. cerevisiae (Krasley et al., 2006a). Likewise, thymoquinone as a pro-oxidant might
induce SLT2 phosphorylation in C. glabrata.
Candida glabrata ∆slt2 strain has been shown to be more sensitive to antifungal agents such as
caspofungin due to its decreased chitin content (Cota et al., 2008). In terms of thymoquinone’s
action, inhibition of CWI MAP kinase might interfere with the normal response to oxidation and
as a result the sensitivity of C. glabrata to TQ will be increased.
As hypotonic shock is an inducer of CWI MAPK, suspending cells in water might be a useful
model to screen inhibitors of this cascade. A considerable amount of work has been carried out
on hyper osmotic stress signalling in S. cerevisiae (Dickinson and Schweizer, 2004), but less
work has been reported on hypo-osmotic stress signalling response. Cells suspended in water
experience hypo-osmolarity that may result in a rapid inflow of water, cell swelling and
increased turgor pressure. Weaknesses in lipid–lipid bonding may also lead to cell lysis due to
chaotropic solutes (Cray et al., 2013). However, activation of protective mechanisms, such as
release of osmolytes and remodelling of the cell wall, plays an important role in preventing cell
rupture (Hohmann, 2002). Efflux of intracellular electrolytes and small organic solutes
‘osmolytes’ is a universal response to cell swelling (Strange et al., 1995). Protozoa and lung
cancer cells release amino acids in response to acute hypo-osmotic stress (Kirk, 1997). In other
organisms such as the algae Dunaliella tertiolecta, intracellular osmolytes are depleted by
metabolism and not released in response to hypo-osmotic stress (Goyal, 1989). In yeast, glycerol,
arabitol and erythritol are released in response to hypo-osmotic stress (Kayingo et al., 2001,
Fuchs et al., 2009). Until recently, there was no clear evidence on whether yeast cells undergoing
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apoptotic or necrotic death in case cellular defence mechanisms failed under hypo-osmotic
stress. This study attempted to define whether death under hypo-osmotic stress would be an
apoptotic or necrotic death (Almshawit et al., 2014).
Cells in water might be a useful model to study CWI MAPK pathway and the physiology of the
cells. Moreover, preparing yeast suspensions in water is a critical step in many experiments,
including the preparation of inocula by standard methods to determine antifungal susceptibility.
In addition, laboratory studies frequently involve yeast, including C. glabrata suspended in water
to monitor the effects of carbon sources, drugs and peptides such as glucose, dopamine and
amyloid beta (Macreadie et al., 2010, Granot et al., 1993, Bharadwaj et al., 2008).
The hypothesis of this chapter is “thymoquinone induces SLT2 phosphorylation, and inhibition
of SLT2 phosphorylation or CWI MAPK cascade activation will result in increasing the
sensitivity of C. glabrata to TQ”. Briefly the present study aimed to:
i.
Determine whether hypo-osmolarity and thymoquinone induce SLT2 phosphorylation in
C. glabrata.
ii.
Monitor survival of C. glabrata in water as a hypo-osmotic environment.
iii.
Use cells in water to screen cell wall MAPK inhibitor(s) which might interact
synergistically with thymoquinone.
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4.2 Materials and methods
4.2.1 Strains and media
The strains used in this study were C. glabrata ATCC2001 and ATCC90030. Yeast minimal
medium (YMM), RPMI1640 and YEPD media (appendix A) were used where they were need.
4.2.2 Detection of Slt2 phosphorylation
Detection of Slt2 phosphorylation by flow cytometry was carried out using Phospho-p44/42
MAPK (Erk1/2) (Thr202/Tyr204) Antibody (Cell Signaling Technology). Cells of C. glabrata
ATCC2001 from an overnight YEPD plate were used to inoculate YEPD liquid medium and
incubated 4-5 h at 30 ºC with agitation (200 rpm). Cells were washed with RPMI1640 and
resuspended in the same medium unless they were washed and suspended in water.
For TQ, H2O2 and water treatment 106 cell/ml were used. Cells were treated with 25, 50 or 100
µg/ml of TQ or 30 mM hydrogen peroxide. Samples were collected after 1 and 2 h including
untreated cells. For testing of Slt2 induction by doxycycline, four millilitres of OD600 = 1 of C.
glabrata ATCC2001 exponential phase culture were collected at 0, 30 and 60 min after adding
doxycycline at a final concentration of 25, 50 or 100 µg/ml. Cells were fixed, permeabilised and
antigen detected according to the manufacturer’s instruction of the primary antibody. Briefly, C.
glabrata ATCC2001 from an overnight plate was inoculated into YEPD liquid medium and
incubated 4 – 5 h with shaking at 30 ºC. The cells were harvested and washed with RPMI1640
medium then the cells were resuspended in the same medium to reach OD6
of ≥ 1. The
suspension was diluted 1:10 and 1 ml was collected at the start (t=0). Part of the cell suspension
was left without treatment and the rest was treated with TQ, H2O2 or washed and resuspended in
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water. At different time intervals 1 ml sample was collected from each treatment and 100 µl of
37% formaldehyde was added. The samples were incubated at 37 ºC for 10 min, chilled on ice
for 1 min and centrifuged (9000 rpm) for 1 min. The pellets were resuspended in 100 µl PBS.
Permeabilisation was carried out by adding 900 µl of 100% cold methanol (-20 ºC) and
incubated on ice for 30 min. For immunostaining, Cells were harvested and washed with PBS.
Pellets were resuspended in blocking buffer by vortexing and incubated for 10 min at room
temperature. Then the cells were harvested and resuspended in blocking buffer contained 1:200
diluted Phospho-p44/42 MAPK primary antibody. Cells were incubated 1 h at room temperature.
After washing with PBS the cells were suspended in 1:1000 diluted an anti-rabbit HRP
secondary antibody (Sapphire, Australia) in blocking buffer and incubated for 30 min in dark at
room temperature. After three times washing with PBS, fluorescence intensity was detected by
AmCyan filter in FACS Canto II (BD Biosciences) flow cytometer.
Detection of SLT2 phosphorylation by TQ was also performed by total protein extraction and
analyzed by western blot as previously described in (Casagrande et al., 2009). Briefly, C.
glabrata ATCC2001 cells were growing exponentially in a flask containing YEPD medium (30
°C, 175 rpm) to OD600nm = 0.2. Four millilitres of OD600= 1 were withdrawn for t = 0.
Thymoquinone was then added to a final concentration of 50 µg/ml and the incubation was
continued. Four OD units of cells were withdrawn after 30 and 60 min from TQ addition. Culture
samples were centrifuged at 4 °C and washed with water. Cells were resuspended in 0.2 ml of 2
M NaOH, 5% β-mercaptoethanol and incubated 10 min on ice. After the addition of 40 µl of
50% trichloroacetic acid, samples were incubated on ice for further 10 min. After centrifugation
(15 min at 13600g), the pellet was dried at room temperature (5 min) and resuspended in loading
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buffer (50 mM Tris-HCl, pH 6.8, 1 % β-mercaptoethanol, 2% SDS, 0.1% bromophenol blue,
10% glycerol). After SDS-polyacrylamide gel electrophoresis fractionation and transfer onto
nitrocellulose membrane by using iBlotTM Dry Blotting System (Invitrogen). Phosphorylated
SLT2 was detected by hybridization with antibody p44/42 (thr202/tyr204; Cell Signaling
Technology #9101) and total SLT2 was detected with antibody Slt2 (Santa Cruz Biotechnology
#sc-6802). Alkaline phosphatase (AP) secondary antibody (anti- rabbit-IgG-AP conjugated
antibody) was used to detect both proteins.
4.2.3 Viability assays
Candida glabrata cryogenic stocks were subcultured at least five times on YEPD plates,
inoculated into 2 ml YEPD liquid medium and incubated for 24 h with agitation (170 r.p.m.).
Cells were collected by centrifugation in a high-speed microcentrifuge (LaboGene ApS,
Denmark) at 8500 rpm for 1 min, washed six times with sterile MilliQ water and suspended in
MilliQ water to a specific cell density. Cultures were then incubated at 30 °C with agitation (170
rpm) in 50 ml centrifuge tubes with a working volume of 2 ml. One hundred microlitre aliquots
of cells were taken at indicated time points to measure cell viability, as colony-forming units on
YEPD agar plates.
4.2.4 Propidium iodide staining
Propidium iodide (PI) was purchased from Sigma-Aldrich (Australia). A modified PI staining
protocol from Invitrogen was used to measure cell viability. Briefly, 3 µM solution of PI was
made by diluting 1 mg/ml (1.5 mM) stock solution 1:500 in phosphate-buffered saline (PBS),
which was then filtered through 0.22 µm syringe filters (Millex). One ml samples of C. glabrata
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cells in water were centrifuged at 8500 rpm. for 1 min, and the pellets were suspended in PBS
containing 3 µM PI. The samples were incubated at 30 °C for 30 min before flow cytometry
analysis using a FACS Canto II (BD Biosciences) flow cytometer.
4.2.5 Bio-protection assays
Materials released by cells were obtained by incubating cells at 106 CFU/ ml for 1–2 days in
water. Cells were removed by filtration through 0.22 µm syringe filters. Aliquots of filtrates were
plated on YEPD to check sterility before further investigations. The filtrates were used either
directly, or they were lyophilized and reconstituted as concentrated samples. Samples were
analysed using spectrophotometer (Shimadzu UV-1800) before using the filtrate for bioprotection assay.
For protection studies using dead cells, 104 CFU/ml were left in water for 4 days to die naturally
(99.7% death as quantified by counting colonies). After that, C. glabrata cells from a YEPD
culture grown for 24 h were washed and mixed with the dead cells at a concentration of 5.7x103
CFU/ml. The growth was monitored by counting the colonies. Two independent experiments
were performed with three replicates each time.
To test whether the effect of hypo-osmolarity on low density cells can be eliminated by sorbitol,
about 2x103 CFU/ml was suspended in 5 µM, 50 µM or 1 M of sorbitol (Sigma) and incubated
for 24 h at 30 °C with shaking (170 rpm). The impact of low concentration of nutrients on the
survival of low-density cells was tested by suspending the cells in diluted yeast minimal media
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(YMM) which was sterilized by filtration through 0.22 µm syringe filters (Millex). Then cells
were suspended in 1:100 or 1: 1000 diluted YMM.
4.2.6 Testing for apoptosis
Due to the difficulty in collection and observation of dead cells in low-density cell suspensions
through the microscope, apoptosis was observed by dialysing high density cell cultures. About
106 CFU/ml was washed as described above, and 4 ml of the cell suspension was dialysed in
dialysis membranes (12 kDa MWCO), surrounded by 3 L of MilliQ water. The number of
colonies per millilitre was determined after 2 or 3 days at 30 °C. The annexin V-FITC Apoptosis
Detection kit (APOAF-20TST; Sigma-Aldrich) was used for detecting apoptotic cells. Two
samples of 1 ml from 3 days dialysed high-density cells (106 CFU/ml) were centrifuged (8500
rpm, 1 min) and resuspended in 1 ml binding buffer. Ten microlitre PI and 5 µl FITC-annexin V
solutions were added to each 500 µl dialysed cell suspension and incubated 10 or 30 min at room
temperature. After that, cells suspensions were centrifuged at 8500r.p.m. for 1 min, and the
content of the tubes (pellet from 500 µl sample each) were combined in one tube by washing
with 500 or 20 µl sterile MilliQ water for flow cytometry or fluorescence microscopy analysis,
respectively. Cells grown in YEPD media to exponential phase were used as a negative control.
Cells were analysed by a FACS Canto II (BD Biosciences) flow cytometer or observed using an
epifluorescence microscope (Leica DM2500) provided with camera (Leica DFC310 FX ).
For DNA microscopic observation, cells were harvested by centrifugation at 8000 rpm. for 1
min, washed, resuspended in 3 µM DAPI (4′,6-diamidino-2-phenylindole dihydrochloride;
Sigma) and incubated for 15 min at room temperature in the dark. Then, cells were washed and
resuspended in PBS. For cell cycle analysis by flow cytometry, cell suspensions, of about 10 6
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cell/ml of overnight culture in YEPD, and dialysed cells were fixed and permeabilized with 70%
cold ethanol on ice for 5 min, rehydrated in PBS at room temperature for another 5 min and then
cells were harvested and stained with DAPI as described above (Liang et al., 2007, Li et al.,
2006). Cells were analysed by flow cytometry using a pacific blueTM filter (Ex-Max 401 nm).
4.2.7 Toxicity of compounds on cell’s survival in water
One mg/ml stausporine stocks were prepared in ethanol and stored at -20°C. Sorafenib and
XMD8-92 (Santa cruz, USA) stocks were prepared freshly in DMSO and doxycycline was
dissolved directly in water. Cells in water were prepared as in 4.2.2. Thymoquinone stocks were
prepared freshly in DMSO or stocks were kept at -20 °C for no longer than 2 weeks.
Susceptibility testing to TQ, stausporine and doxycycline was performed using the standardized
broth microdilution assays as defined by CLSI (see 2.2.1).
4.2.8 Statistical analysis
For statistical analysis, One-way ANOVA with Tukey’s multi comparisons test was performed
using GraphPad Prism 6 software. The control groups were the cultures at time zero. Error bars
represent the standard error of the mean (SEM). Differences were considered significant for P <
0.05. WEASEL software (WEHI, Parkville, VIC, Australia) or Flowing software (Cell Imaging
Core of the Turku Centre for Biotechnology, Finland, version 2.5.1) were used for flow
cytometry data analysis.
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4.3 Results
4.3.1 Thymoquinone, hydrogen peroxide and hypo-osmolarity induces SLT2
phosphorylation in C. glabrata
Many pro-oxidants including H2O2 and diamide were found to be inducers of SLT2
phosphorylation in S. cerevisiae (Vilella et al, 2005). In this study H2O2 and TQ induced SLT2
phosphorylation in C. glabrata. Flow cytometry data analysis showed no difference between the
fluorescence intensity after 1 h and 2 h treatment of TQ or H2O2. The shift in the fluorescence
intensity was not too high in cells which were treated with 50 µg/ml of TQ (~ 9%) but enough to
be statistically significant. Higher concentration of TQ (100 µg/ml) result in higher fluorescent
intensity (~34%) (Fig. 4.1A). Treatment with water caused insignificant increase in intensity
after 1 h (~4%) but the intensity was increased to be about 17 % after 2 h. Additional
examination was carried out to observe SLT2 phosphorylation under TQ stress. Western blotting
analysis of the total protein showed bands indicating the phosphorylation of SLT2 after treatment
with 50 µg/ml of TQ for 30 min (Fig. 4.1B).
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(A)
1h
2h
No treatment (0%)
TQ=50 µg/ml
(9%)
TQ=100 µg/ml
Events (10,000)
No treatment (0%)
TQ=50 µg/ml (7%)
TQ=100 µg/ml
(34%)
No treatment
(0%)
30 mM H2O2
(9%)
No treatment
30 mM H2O2
Cells in water
Fluorescence intensity
t=
(0%)
(10%)
(17%)
Fluorescence intensity
0’
30’
60’
0’
30’ 60’
75 KDa
(B)
50 KDa
Total SLT2
Phosphorylated SLT2
Figure 4.1 Induction of SLT2 phosphorylation by TQ, H2O2 and water treatment. (A) Representative
FACS histograms of C. glabrata without treatment or with TQ, H2O2 or water. Cells were analysed after
1 h and 2 h. Data were analysed using Flowing software: the percentage represent the fluorescence
emission mean of two independent experiments. (B) Western blot analysis for C. glabrata cells treated
with 50 µg/ml TQ; samples were taken at 0, 30 and 60 mins. This figure represents one of two
independent experiments.
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4.3.2 Cell viability and cell density
The effect of cell density on the viability of C. glabrata in water was examined by plating cells
to measure the number of colony-forming units (CFUs). As shown in Fig. 4.2, cultures with an
initial high cell density (more than 8x104 CFU/ml) maintained their viability over 3 days. In
contrast, cells at low density (≤ 8x104 CFU ml) progressively lost their viability. In summary,
cells at low density lost viability, while a quorum of more than 105 CFU/ml allowed viability to
be maintained. This experiment has been performed three times independently with four
replicates each time.
Cell death was also measured using flow cytometry to examine the proportion of cells that could
be stained with PI, an indicator of cell death. After 24 h, cells in water at high density resulted in
an average of 1.5 % of cells exhibited red fluorescence indicative of cell death (Fig. 4.3 A and
B). Thus, the population had 98.5% viability. At low cell density (103 CFU/ml), 44.4% of the
population were PI-stained after 24 h (Fig. 4.3 C and D). Of the cells that became stained with
PI, populations exhibited either moderate or higher levels of PI staining. Analysis of PI staining
vs. forward light scatter which measure cell size (Fig. 4.3 D) indicates that the more highly
stained cells were larger and therefore able to incorporate more PI.
The question was then asked, could cells at high density be protected by materials from dead
cells? It has previously been shown that yeast cells can proliferate by utilizing nutrients released
from cells where 90–99% of the population has died (Fabrizio et al., 2004). In this study, the
percentage of dead cells was very low. To determine whether such a population of dead cells
could protect a low-density population, dead cells from an initial population of 104 CFU/ml
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(99.7% dead after 4 days in water) were added to low-density cell suspension (5.5x103 CFU/ml).
The suspension of dead cells was incapable of providing protection (data not shown).
Figure 4.2 The relationship between cell density and cell survival in water. Bars represent SEM.
*P< 0.05; **P<0.003. This experiment was repeated at least three times independently with four
replicates each time, the results of one experiment are shown.
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A
Events
10,000
Events
10,000
B
D
Events
10,000
Events
10,000
C
103
104
105
103
104
105
PI Fluorescence intensity
Figure 4.3 Analysis of cell viability by flow cytometry. (A) Cells at high density (106 CFU/ml)
in water for 24 h without staining with PI. (B) Same cells as A were stained with PI, and then
analysed for PI staining versus forward light scatter. (C) Analysis of cells at low density (10 3
cells/ml) after 24 h for PI staining, (D) PI staining versus forward light scatter. This experiment
was performed three times independently.
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4.3.3 Bio-protection
Filtrates of about 106 cell/ml were taken after 1-2 days and were analysed by the
spectrophotometer. The spectrum showed a hump of peaks at wave length between 200 and 300
nm (Fig. 4.4). To determine whether a soluble agent(s) could be involved in protection of cells
from hypo-osmotic stress, a cell filtrate from high-density cells (3x106 CFU/ml) which had been
analysed by spectrophotometer was added to cells at low cell density (7x103 CFU/ml). Cells at
low density, suspended in the filtrate from high density cells (3x106 CFU/ml) maintained
viability (Fig. 4.5A). In the control (without the filtrate from the high density suspension),
viability decreased to 50% after 24 h. This indicates that the high-density cells release material(s)
that protect the population against hypo-osmotic sensitivity and starvation. When this protective
filtrate was concentrated 10-fold by lyophilization and added to a suspension of cells at 7x103
CFU/ml, the filtrate enabled cells to multiply 40-fold in 2 days (Fig. 4.5B). This experiment was
performed three times with independent filtrates. The dry weight of exuded materials is < 5 mg/L
of filtrate. Our efforts to identify the protective agent(s) by lyophilization followed by mass
spectroscopy and NMR have been unsuccessful due to these low levels and the diversity of
materials in the filtrate. Many studies use 1 M sorbitol as an osmotic stabilizer (Soriano-Carot et
al., 2012, de Nobel et al., 2000). Here, figure 4.6 shows that a low concentration of sorbitol (50
µM) can provide protection to cells at low density, but not 5 µM sorbitol. In contrast, the
addition of 1:100 diluted YMM media induced growth of the cells, whereas a dilution of 1:1000
did not contain enough nutrients for growth and did not provide protection.
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Water
5
Filtrate of 10 cell/ml after 1d
6
Filtrate of 10 cell/ml after 1d
6
10X concentrated filtrate of 10 cell/ml after 1d
Figure 4.4 UV-CD spectra of high cell density filtrates. The CD spectra were measured at room
temperature in a cuvette with a path length of 1 mm.
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Viability (%)
100
50
1
0
0
Time (days)
7x103 CFU/ml in water
7x103 CFU/ml in 1X filtrate of 3x10 6 CFU/ml
(A)
Viability (%)
4000
3500
3000
800
600
400
200
2
1
0
0
Time (days)
7x103 CFU/ml in 10X filtrate of 3X10 6 CFU/ml
7x103 CFU/ml in water
(B)
Figure 4.5 Bio-protection of low cells at low density by filtrates of high cell density (A) Cells were
suspended at 7x103 CFU/ml in the filtrate from 3x106 CFU/ml, obtained after one day. (B) The same
filtrate was concentrated 10-fold and cells added to cells at a density of 7x103 CFU/ml. This experiment
was performed three times with independent filtrates. Bars represent SEM of four replicates of one batch
of cells.
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CFU/ml (x10 3 )
2.5
2.0
1.5
1.0
0.5
1
0
0.0
Time (day)
No additives
Sorbitol (1M)
Sorbitol (50M)
Sorbitol (5M)
CFU/ml (x10 3 )
(A)
24
22
20
18
16
3
2
1
1
0
0
Time (day)
YMM (diluted 1:100)
YMM (diluted 1:1000)
(B)
Figure 4.6 Protection of cells against hypo-osmolarity. (A) by sorbitol, (B) by Yeast Minimal
Media (YMM) diluted 1:100 and 1:1000. Data represent the mean of two independent
experiments with three replicates each time. The error bars indicate SEM values.
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The compounds conferring survival were found to pass through 12-kDa MWCO dialysis
membranes. After 2 days, about 80% loss of viability was observed in 7x105 CFU/ml cultures
after dialysis in 12-kDa MWCO dialysis sacs (Fig. 4.7). This indicates that the bioactive
compounds have molecular weights ≤ 12 kDa, and these compounds have been diluted around
the cells. As a result, they have lost their resistance to hypo-osmolarity. The loss of viability was
similar to death rates seen when the population is at 7x103 CFU/ml (Fig. 4.7). Because of the
difficulty in examining cells when they are at low density, especially for microscopic
observation, dialysed cells at high density were used to study the mechanism of cell death.
Viability (%)
150
100
50
2
0
0
Time (days)
7x105 CFU/ml
(without dialysis)
7x105 CFU/ml
dialysed cells
7x103 CFU/ml
(without dialysis)
Figure 4.7 Effect of dialysis on survival of high cell density in hypo-osmolarity. Cells at 7x105 CFU/ml
were dialysed in 14000 MWCO dialysis membranes. Cells from 24 h YEPD liquid media were washed
as described and resuspended in water to have 7x105 CFU/ml and incubated at 30°C as indicated.
Dialysis sacs were dialysed against 100 times the sample volume. This experiment has been performed
more than three times with two replicates each time.
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4.3.4 Apoptotic cell death
The analysis of flow cytometric data demonstrated that about one quarter of high density
dialysed cells show FITC-annexin V staining after 3 days dialysis in water (Fig. 4.8). The cell
staining by FITC-annexin V was further observed by epifluorescence microscopy which showed
green fluorescence in the periphery of the cytoplasm, indicating phosphatidylserine
externalization (Fig. 4.8c upper right). PI staining was used in combination with annexin V
staining to distinguish between apoptotic and necrotic cells. Around 8% of the collected cells
exhibited annexin V and PI staining, indicative of late apoptosis, while 21% of cells exhibited
only PI staining. DNA staining shows that in growing cells, DAPI stains the single, large,
homogeneous, round-shaped nucleus as well as the peripheral mitochondrial DNA (Fig. 4.9A
and B). In contrast, dialysed cells in water show small condensed nuclei or distributed nuclear
fragments (Fig. 4.9 C–H), which indicate chromatin condensation, DNA damage and nuclear
fragmentation: these are features of apoptosis (Madeo et al., 1997, Andres et al., 2008). These
cells were not fixed to avoid the loss of small DNA fragments, which can occur due to increase
in the permeability of fixed cells. Figure 4.9 A–H also shows a difference in cell size. Cells in
water are smaller compared to those from growth media. Flow cytometric analyses of the cell
cycle using DAPI detect fixed cells, which are more permeable, with lower staining intensity
than those at G0/1 phase, a phase when live cells have their minimum content of non-fragmented
DNA. These cells can be observed as a ‘sub-G0/1’ peak in a DNA histogram. In contrast, necrotic
cells generally do not show an immediate reduction in DNA staining, and sub-G0/1 cannot be
discriminated from a histogram DNA content analysis of live cells (Riccardi et al., 2006, Dive et
al., 1992, Darzynkiewicz et al., 1992, Calvert et al., 2008). In Fig. 4.9 I-J, about 20% of the
population are in sub-G0/1 region and are comprised of apoptotic cells, apoptotic bodies and
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debris. The accuracy of DNA content measurement was reflected by variation in fluorescence
intensity between individual cells with identical DNA content in G0/1 cell population. The
coefficient of variation (CV) of the mean value was about 3%, which is within the acceptable
range (Darzynkiewicz et al., 2010). We have also used equal numbers of control and treated cells
plus the same concentration of DAPI stain to overcome the drawbacks that may be a
consequence of these factors (Haase et al., 2002).
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(A)
(B)
Stained cells
Counts
10,000
Unstained cells
0
2
10
3
4
10
10
5
10
(C)
Annexin V fluorescence
intensity
Figure 4.8 FITC-annexin V staining on high density cells dialysed for 3 days to examine externalization
of phosphatidylserine. (a) Flow cytometry analysis of cells without staining. (b) Flow cytometry analysis
of cells in A stained with PI and FITC-annexin V. (c) Flow cytometric analysis of unstained and FITC
annexinV-stained cells. The inset shows a fluorescence microscopy image of FITC-annexin V-stained
cells. This analysis was performed more than three times: the result of one experiment has been presented.
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Figure 4.9 DAPI staining of growing and
dialysed
cells.
Fluorescence
and
light
microscopy analysis of cells growing in
(A)
(B)
YEPD (A) and cells dialysed in water (C, E
and G), after DAPI staining, b, d, f and h are
bright field for DAPI staining, respectively.
Flow cytometric analysis of DNA content for
cells growing in YEPD (I) and cells dialysed
(C)
(D)
for 3 days (J). This experiment has been
performed three times independently.
(E)
(F)
(G)
(H)
(I)
(J)
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4.3.5 Effect of PKC1 inhibitor on C. glabrata viability in water
Staurosporine is an antifungal compound that inhibits PKC1 phosphorylation, a control kinase
affecting the CWI cascade in yeast (LaFayette et al., 2010). Therefore this compound was chosen
to examine the effect of CWI inhibition on survival of high density cells of C. glabrata in water.
Figure 4.10A demonstrates 50 % or more loss of viability within 24 h in cultures treated by 1 to
4 µg/ml stausporine. However in RPMI1640 medium the minimum inhibitory concentration of
staurosporine was 4 µg/ml and the fungicidal concentration was 8 µg/ml (Fig. 4.10 B-C).
Toxicity of PKC1 protein inhibitor is four times higher than that in medium, indicative of the
vital role of this protein in preserving cells from hypo-osmotic conditions. Toxicity of
staurosporine in water could be eliminated by sorbitol which is an osmotic stabilizer (Fig. 4.10
D). This confirms that the death caused by staurosporine is due to the hypo-osmotic stress.
Testing combinations between staurosporin and TQ in RPMI1640 medium indicated neither
synergy nor antagonism between these two compounds (FIC = 0.5).
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25
1
20
6
C F U /m l (x 1 0 )
0
(A)
2
15
4
S ta u r o s p o r in e
10
(  g /m l)
5
1
0
0
T im e (d a y s )
Sts (µg/ml)
Sts (µg/ml)
8
4
2
1
8
4
2
1
0.5
0.5
(B)
(C)
2 .0
6
C F U /m l (x 1 0 )
n o tr e a tm e n t
(D)
s ta u r o s p o r in e
1 .5
s ta u r o s p o r in e + s o r b ito l
1 .0
**
0 .5
1
0
0 .0
T im e (d a y s )
Figure 4.10 Low concentrations of staurosporine (Sts) kills C. glabrata ATCC2001 in water but not in
RPMI1640 media. (A) Cells in water were treated with Sts, (B) Broth microdilution assay of Sts in
RPMI1640, the arrow indicates the MIC value after 24 h of incubation at 35 ºC (C) Five microliter from
wells in B were spotted on YEPD plates (D) Abrogation of 1 µg/ml stausporine toxicity in water by 1M
sorbitol.
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4.3.6 Toxicity of XMD8-92, sorafenib and doxycycline in water
XMD8-92, sorafenib (Nexevar) and doxycycline were chosen to examine their properties to kill
C. glabrata cells in water. The selection of XMD8-92, sorafenib (nexevar) and doxycycline was
based on the bioinformatic database analysis (Candida genome database) which indicates 48%
identity and 65% similarity between SLT2 protein in yeast and ERK5 (also known as BMK1)
and ERK2 in Homo sapiens. XMD8-92 was shown to be an ERK5 inhibitor (Yang et al., 2010).
Sorafenib is an oral multikinase inhibitor that was originally developed because of its inhibitory
effects on the serine/threonine kinase Raf and several RTKs that induce cell proliferation and/or
angiogenesis. The ability of sorafenib to inhibit ERK1/2 in cancer cell lines was demonstrated by
(Manov et al., 2011). Doxycycline also was shown to be an ERK1/2 inhibitor (Kim et al., 2005).
Based on that, the effect of these compounds on C. glabrata cell viability in water was tested.
Beside these compounds staurosporine was included as a positive control.
XMD8-92 with concentrations of up to 50 µM did not reduce viability in water nor inhibited
growth in RPMI medium (data not shown). Sorafenib (up to 10 µM) also was not effective
against C. glabrata in water (data not shown) nor in medium. In contrast, doxycycline caused
loss of viability in cells in water. Loss in viability of high density cells in water was caused by 40
µg/ml of doxycycline whereas the minimum inhibitory concentration of doxycycline in
RPMI1640 medium was 200 µg/ml (Fig. 4.11 A,B). The toxicity of doxycycline in water could
be abrogated by adding 10 mM of the antioxidant ascorbic acid but not reduced glutathione.
Sorbitol also could not save cells from doxycycline toxicity in water (Fig. 4.11C).
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4.3.7 Doxycycline induces Slt2 phosphorylation
Experiments were designed to find whether toxicity of doxycycline in water was due to
inhibition of CWI cascade. Concentrations of 25, 50 and 100 µg/ml of doxycycline were tested
for their ability to inhibit Slt2 phosphorylation. The phosphorylation of Slt2 in C. glabrata
ATCC2001 was measured using flow cytometry. Within 30 min incubation time with the lowest
concentration of doxycycline (25 µg/ml), phosphorylation of Slt2 protein was detected, whereas
untreated cells showed no detectable positive events indicating no Slt2 phosphorylation in these
cells (Fig .4.12).
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C.glabrata 90030 & Dox in water
4
200 100 50 25 12.5 6.2
CFU/ml (x 10 6)
DOX (µg/ml)
0
40 g/ml
400 g/ml
3
2
1
1
0
0
Time (day)
(A)
(B)
D o x in w a t e r 2
400
C F U /m l (x 1 0
3
)
ns
300
T im e = 0 d a y
200
T im e = 1 d a y
100
o
l
o
it
c
rb
s
o
s
x
+
o
D
o
x
D
D
o
x
+
+
A
G
D
S
o
H
x
0
0
(C)
Figure 4.11 Susceptibility of C. glabrataATCC90030 to doxycycline. (A) Checking viability of
C. glabrata ATCC2001 cells after MIC assay by spotting 5 µl from each micro well, (B) in
water, (C) protection of cells against doxycycline toxicity in water using 1 M sorbitol, 10 mM of
ascorbic acid (AA) or reduced glutathione (GSH).
- 138 -
10,000
Events no.
Chapter4
0%
3%
22%
0
102
10
3
4
10
5
10
SLT2 phosphorylation
Figure 4.12
Doxycycline induces SLT2 phosphorylation. C. glabrata cells growing
exponentially were treated with 25 µg/ml doxycycline (red) and 50 µg/ml DOX (blue) after 30
min in media. Black is untreated cells. The percentages indicate the fluorescence intensity.
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4.3.8 Toxicity of thymoquinone on cells in water
Since both thymoquinone and doxycycline induced SLT2 phosphorylation and doxycycline
caused death of cells in water, this led to examination of TQ toxicity on C. glabrata ATCC90030
cells in water to see if it acts like doxycycline. The results showed that thymoquinone was not
toxic in sub-inhibitory concentration to cells in water whereas doxycycline was. Thymoquinone
caused killing of high density cells (2x107 CFU/ml) in water at the same concentration (25
µg/ml) which inhibited growth in RPMI1640 medium (Fig. 4.13 A, C). In addition, toxicity of
sub-inhibitory concentrations did not increase the death rate in low density cultures in water
compared with untreated cells in water (Fig. 4.13 B). Sorbitol did not protect high density cell
cultures from TQ toxic concentration (25 µg/ml) in water, on the other hand ascorbic acid was
protective. In contrast to treatment of cells with TQ alone, reduced glutathione provided some
protection against TQ toxicity in water. But the difference in the number of viable cells was not
statistically significant to consider reduced glutathione as a protector in these conditions (Fig.
4.13 C).
Notably, addition of reduced glutathione did not protect C. glabrata in water from TQ or
doxycycline effect whereas ascorbic acid did.
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(A)
25
1 2 .5
6
C F U /m l ( x 1 0 )
6 .2
20
25
T Q (  g /m l)
15
10
5
1
0
0
T im e (d a y s )
6
0 .1
3
(B)
C F U /m l (x 1 0 )
0
4
0 .4
1 .6
2
10
50
 g /m l T Q
4
2
4
0
0
T im e (h o u r s )
1 .5
**
6
C F U /m l (x 1 0 )
ns
****
****
(C)
T im e = 0 d a y
T im e = 1 d a y
1 .0
0 .5
e
n
io
th
ta
lu
g
+
d
Q
T
Q
+
re
d
u
c
e
T
T
Q
a
+
s
S
c
o
o
rb
rb
it
a
o
te
l
Q
T
0
0 .0
Figure 4.13 Effect of thymoquinone on cells survival in water. (A) Cells at high density. (B) Cells at low
density. (C) High density cells were treated with 25 µg/ml of TQ, TQ and antioxidants or sorbitol. (ns) not
4.4 Discussion
significant,
(**) p < 0.01, (****) p < 0.0001 according to Tukey’s multi comparisons test.
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Chapter4
4.4 Discussion
4.4.1 The hypothesis of inhibiting CWI MAPK to improve TQ efficacy.
The pro-oxidants TQ and H2O2 activated SLT2 phosphorylation in C. glabrata. Hydrogen
peroxide is reported to induce activation of the whole cascade of CWI MAPK in S. cerevisiae
(Krasley et al., 2006a, Vilella et al., 2005). More investigations are needed to know whether TQ
induces the upstream cascade of CWI MAPK signalling pathway as well. Inhibition of SLT2
phosphorylation or this signal pathway might delay the response of the transcription factors and
cells would not be able to resist TQ toxicity. In this stage of the study inhibition of SLT2 or CWI
MAPK by chemical compounds was assumed to act synergistically with TQ. The first challenge
to examine this hypothesis was choosing the environment to screen inhibitors. It is known that
hypo-osmotic stress and heat shock induce activation of SLT2 in baker’s yeast (Fuchs et al.,
2009, Truman et al., 2007). Would hypo-osmolarity affect the survival of C. glabrata?
4.4.2 Candida glabrata survival in hypo-osmolarity
In previous studies of Saccharomyces cerevisiae, cells suspended at high density (2x107 cells/ml)
in water maintained high viability for 20 days (Granot et al., 1993). My study demonstrates that
survival of C. glabrata cells in hypo-osmotic conditions depends on the number of cells. At low
cell density C. glabrata is sensitive to the hypo-osmolarity and starts to lose viability within one
day. At high density major loss in viability occur after 3 or 4 days (Fig. 4.2).
As the concentrated filtrate of high density cells was enough to stimulate divisions in the low
density cell population, it can be concluded that it contains all necessary nutrients for C.
glabrata.
Herker et al., (2004) induced growth and survival in aged cells by addition of
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secretions from high-density, aged yeast cells exhibiting massive cell death. In contrast, in this
study, the bioactive materials leading to survival in hypo-osmotic conditions were obtained from
populations with high viability (> 97% viable, Fig. 4.3). Since the inoculum was washed six
times before adjusting the final cell suspension in water, the possibility of remaining nutrients
from YEPD is excluded. The 40 fold increase in cell numbers is remarkable. The experiment has
been reproduced three times with independent filtrates. It therefore appears that the material
released from cells at 3x106 cells/ml can support cell re-growth from 7x103 cells/ml to 3x105
cells/ml after 48 hours. In others words, material released from 10 cells can support the growth
of one new cell.
Sorbitol protected low density cells very well from hypo-osmolarity. Because protection in high
density cells is provided by < 5 mg of solid matter per litre (after filtration and lyophilisation),
the protection is unlikely to be ‘sorbitol-like’ osmotic protection. In addition, 1:100 diluted
YMM media is a hypo-osmotic condition (Kayingo et al., 2001), but it was protective. This
indicates miniscule amounts of nutrients in the milieu surrounding cells that appear sufficient to
protect against death due to hypo-osmolarity.
4.4.3 Apoptotic cells death in water
There is a high probability that hypo-osmotic conditions cause necrotic death due to increasing
fluidity in the cell membrane and rapid inflow of water which causes cell swelling and increased
turgor pressure. The death mode was investigated in this study by observing some of the
apoptosis markers, mainly, phosphatidylserine exposure, chromatin condensation and DNA
degradation. First of all, shrinkage and not swelling of cells in water was noted (Fig. 4.9a-h),
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which is a primary indicator of apoptosis. In general, necrotic cells show increased size and
swelling (Elmore, 2007). Exposure of phosphatidylserine at the outer leaflet of the cytoplasmic
membrane is an early morphological marker of apoptosis which is conserved from yeast to
mammalian cells (Martin et al., 1995, Madeo et al., 1997). In yeast, phosphatidylserine can be
detected by FITC-annexin V staining after digestion of the cell wall. However, in this study,
annexin V was applied directly without any treatment to remove the cell wall. Unlike other
studies of apoptosis in yeast, in this study, the production of protoplasts was not necessary to
observe FITC-annexin V staining. In this study, annexin V staining of the phosphatidylserine
was probably enabled by the hypo-osmotic conditions leading to increased permeability of the
cell wall. Externalization of phosphatidylserine occurred in cells that were propidium iodide
negative, indicative of early apoptosis.
DNA fragmentation is also another marker of apoptosis. It has been well documented that
apoptotic cells show reduced DNA staining with a variety of fluorochromes, including PI and
DAPI. A large portion of DNA in apoptotic cells is of low molecular weight due to activation of
endogenous endonucleases which break the linkers between the nucleosomes. These small
fragments of DNA can leach out from ethanol-fixed (permeabilized) cells during rehydration and
the staining process. Consequently, flow cytometric analyses detect cells with lower staining
intensity than those at G0/1 phase, a phase when live cells have their minimum content of nonfragmented DNA. These apoptotic cells can be observed as a ‘sub-G0/1’ peak in a DNA
histogram. In contrast, necrotic cells generally do not show a ‘sub-G0/1’ peak because there will
be no immediate reduction in the fluorescence (Darzynkiewicz et al., 1992, Darzynkiewicz et al.,
2010, Dive et al., 1992). Figure 4.9 shows a significant proportion of the population (20%) with
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Chapter4
reduced fluorescence intensity indicating loss of DNA integrity. DNA degradation and chromatin
condensation in the cells exposed to hypo-osmolarity was observed also using an epifluorescence
microscope with unfixed cells. This indicates apoptotic death.
There is evidence of apoptotic death in yeast treated with death inducers such as H2O2, acetic
acid or amphotericin B in Saccharomyces cerevisiae when applied at low concentration, but
necrotic death was observed when used at higher doses (Ribeiro et al., 2006, Phillips et al., 2003,
Eisenberg et al., 2010). DNA degradation, an indicator of apoptosis in S. cerevisiae was
observed under hyper-osmotic stress (60% glucose) by Ribeiro et al. (2006). In this study, we
demonstrate preliminary evidence of hypo-osmotic stress-induced apoptosis in C. glabrata, and
further investigations to confirm this phenomenon may be required.
4.4.4 Cells in water as a model for screen CWI MAPK inhibitors
4.4.4.1 Cell wall integrity MAPK is necessary for maintaining viability in water
It is well known that cell wall integrity MAPK activation is important for yeast cell survival
under hypo-osmotic stress. Previous studies used diluted rich media as hypo-osmotic conditions
to examine this theory (Kayingo et al., 2001, Davenport et al., 1995). In this study MilliQ water
was chosen as an extremely hypo-osmotic medium. The loss in viability of the wild type strain
treated with staurosporine, a known inhibitor of PKC1 phosphorylation in yeast indicates the
importance of this upstream MAPKKKK protein in CWI to protect against hypo-osmolarity (Fig.
4.10). Staurosporine at low concentration (1 µg/ml) was not toxic in RPMI1640 but reduced
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viability in water. This indicates that inhibiting of PKC1 activation in CWI MAPK pathway is
crucial in maintaining viability of C. glabrata cells under hypo-osmotic stress.
4.4.4.2 Screening of CWI MAPK inhibitor
The selection of XMD8-92, sorafenib (nexevar) and doxycycline was based on the bioinformatic
database analysis (Candida genome database) which was discussed before in 4.3.6. Based on
that, the effect of these compounds on C. glabrata cell viability in water was tested. Beside these
compounds staurosporine was included as a positive control. Exposing C. glabrata to 50 µM of
XMD8-92 did not cause loss of viability. This concentration was chosen as the maximum
concentration tested because the most tolerated dose of XMD8-92 in mice was 10 µM (Yang et
al., 2011). Survival of C. glabrata cells in water was also not affected by 10 µM of sorafenib
which is the achievable concentration of sorafenib in the plasma (Manov et al., 2011). The lack
of effect might be due to low concentrations which were used in this study, or different factors
such as permeability of cells. It might also be due to differences between the structures of target
proteins in yeast. In contrast, doxycycline affected survival rate of C. glabrata cells as much as
staurosporine. The same concentrations (≤ 2
µg/ml) were not fungicidal against C. glabrata in
the MIC assay. A concentration of one fifth of the MIC of DOX was enough to kill this fungus in
water. Since doxycycline has been demonstrated as an ERK1/2 inhibitor (Kim et al., 2005), the
question was asked, could this antibiotic act as a CWI MAP kinase inhibitor or might it alter the
composition of cell wall or membrane? The toxicity of doxycycline in water was not abrogated
by adding sorbitol. This indicates DOX has a target other than CWI-MAPK. In addition flow
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cytometry analysis of SLT2 phosphorylation indicated activation of this protein once cells were
treated with sub inhibitory concentrations of DOX in RPMI1640 medium. This means DOX is a
Pkc1 MAPK activator and not an inhibitor. All together, the fungicidal effect of DOX in water is
not due to inhibition of the CWI cascade. Doxycycline is an iron chelator leading to depletion of
intracellular iron (Fiori and Van Dijck, 2012). Iron depletion has been proposed to decrease
ergosterol content in C. albicans and S. cerevisiae, leading to higher fluidity in cell membranes,
with consequent increased passive diffusion (Prasad et al., 2006, Shakoury-Elizeh et al., 2010).
Water and DOX might diffuse passively and caused cell death. The elimination of DOX toxicity
with ascorbic acid might indicate an interference with the oxidative homeostasis due to iron
depletion. In addition, doxycycline was shown to be able to inhibit mitochondrial function in C.
albicans, which eliminates the diauxic shift (Oliver et al., 2008). Lack of diauxic shift, or the
lack of functional mitochondria alters sterol metabolism, resulting in lower ergosterol levels,
consistent with the need for 12 molecules of oxygen to synthesize one molecule of ergosterol
(Oliver et al., 2008). In addition, Saccharomyces cerevisiae was shown to require antioxidant
function to resist the toxicity of doxycycline (Angrave et al., 2001) which has been demonstrated
in this study as well via the addition of ascorbic acid. In conclusion this study supports the
proposed mechanism of killing of doxycycline may be attributed to an alteration of oxidative
homeostasis in C. glabrata which alters the cells permeability. However, the effect of efflux
pumps which might be poorly activated in water because of nutrient limitation should not be
ignored and should be further studied. Not every pro-oxidant increases permeability of cells.
Thymoquinone which has been demonstrated to be a pro-oxidant and alter the membrane
potential of mitochondria (see chapter 3), did not show the same phenomenon in water.
Thymoquinone was not toxic to cells in water at sub-inhibitory concentrations and its toxicity
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could not be eliminated by sorbitol which indicates it does not interfere with CWI-MAPK
cascade activation or permeability of cells. In contrast, staurosporine caused loss in viability of
cells in water at sub-inhibitory concentrations and its toxicity could be abrogated by sorbitol
(Fig. 4.10), indicating its inhibitory effect on the CWI- MAPK cascade.
4.5 Conclusion
In conclusion, these preliminary data demonstrate that high cell density of C. glabrata provides
protection towards hypo-osmolarity but not low cell density (< 105 cell/ml). Death of C. glabrata
caused by hypo-osmolarity is an apoptotic death. In addition, this study proposes a bio-protection
through releasing of nutrients. Cells in water might be a useful model to screen and test CWI
inhibitors. Compounds which inhibit the CWI MAPK cascade are toxic in water but they need
higher concentrations to become toxic in media. The toxicity of the inhibitor can be abrogated by
an osmotic stabilizer such as sorbitol. However, other factors such as efflux pumps, which might
be inactivated in water, should be considered. There was no observed synergy between TQ and
the Pkc1 inhibitor. This indicates that inhibition of the upstream proteins of PKC1-MAPK might
not assist in improving TQ efficacy.
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CHAPTER 5
Effect of thymoquinone on the cell wall
5.1 Introduction
The fungal cell wall is the essential cellular boundary and the first communication point with the
environment. It provides protection from cell lysis and keeps cell integrity. In addition, several
cellular transports, metabolism and sensing processes are controlled via the cell wall (ReinosoMartín et al., 2003). Proper cell wall architecture consists mainly of an external layer of
mannoproteins and an internal layer of β-1,6-glucan and β-1,3-glucan which are linked via β-1,4glucan to chitin chains (reviewed by Cabib et al., 2001). Chitin is a linear polymer of β-1,4-Nacetylglucosamine (GlcNAc) units and spreads all over the cell wall at low levels (1.4–7.1% of
the entire cell wall mass (Aguilar-Uscanga et al., 2003)), with more intensity at the septation
region (Santos et al., 2000). The septum is a cross-wall between the mother and daughter cell
formed during the budding process (Cabib et al., 1996).
Despite its low amounts, chitin is essential for yeast cell survival and its synthesis is highly
regulated. Three chitin synthases have been identified in S. cerevisiae: CHS1, CSH2 and CSH3
The genes which encode the catalytic subunits of these enzymes have been cloned and are
known as CHS1 to CHS7 (reviewed by (Lesage et al., 2006). CHS3 mediates chitin deposition at
the cell membrane after its synthesis in the endoplasmic reticulum (ER). To export CHS3 from
the ER, CHS7 is required. Chitin synthase 3 can then reach the next station which is the Golgi
apparatus where CHS3 seems to be associated with CHS5. CHS5 and CHS6 are necessary for
transferring CHS3 to vesicles (chitosomes) which finally carry CHS3 to the plasma membrane.
CHS3 does not associate with CHS5 until it reaches the ring-like area at the emerging bud,
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where the chitin ring is localized. As a substitute, CHS4 the putative activator of CHS3,
maintains CHS3 in this location. CHS4 can also bind to BNI4, which in turn attaches to the
septin microfilament ring at the bud neck. There is less information regarding CHS1 activity and
CHS2, however CHS2 has been shown to be associated with building of the primary septum
(reviewed by (Cabib et al., 2001). Under non-stress conditions, CHS3 is maintained mostly as
chitosomes within the Golgi apparatus as early endosome compartments. In case of cell wall
stress, CHS3 quickly redistributes to the plasma membrane (Levin, 2011).
The transport of CHS3 from the intracellular pool to the plasma membrane occurs upon
activation of the CWI-MAPK signalling pathway (Valdivia and Scheckman, 2003). The CWIMAPK pathway might be induced upon exposure to cell wall stretching (e.g. budding, heat
shock) or cell wall damage which might be caused by antifungal action (e.g. caspofungin), hypoosmotic stress, calcium signalling-induced stress and oxidative stress (reviewed by (Howard et
al., 2013) and (Fuchs et al., 2009). As described in section 1.1.4 environmental stress is detected
mainly via the plasma membrane-localized sensors Wsc1 and Mid2. Then Rom1/2, the guanine
nucleotide exchange factors are recruited and consequently trigger GTPase Rho1. Rho1 activates
Pkc1 which triggers the MAPK cascade. The MAPK pathway comprises MAPKKKK Pkc1,
MAPKKK Bck1, MAPKKs Mkk1/2 and finally the mitogen activated kinase, Slt2, which
activates the transcription factors Rlm1 and Swi4/Swi6 (reviewed by(Levin, 2011) (Fig. 1.2).
Several studies have explored the biosynthesis of chitin because of the central role of this
polysaccharide in septum formation (Cabib et al., 2001). Studying this region has provided
information on fundamental cellular processes and defined new targets for antifungal agents. As
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a consequence, inhibitors of chitin (e.g. polyoxins and nikkomycin) and β(1-3) glucan synthesis
(e.g. echinocandins) have become available (Cabib et al., 2001).
The aims of this chapter are:
1. As the cell wall integrity (CWI) pathway plays an important role in the oxidative stress
response (Pujol-Carrion et al., 2013) and TQ causes oxidative stress in C. glabrata, this
study investigates the role of CWI-MAPK components in the sensitivity of C. glabrata to
TQ through mutants in CWI-MAPK.
2. The cell wall in C. glabrata is the first interaction point between TQ and the yeast cell.
This study aims to obtain information about this interaction which might define new
approaches to improve the efficacy of TQ as an antifungal compound.
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5.2 Materials and methods
5.2.1 Yeast strains and media
The yeast strains which were employed in this study are listed in table 5.1. TG11 and TG152
strains were maintained and grown on yeast minimal medium lacking tryptophan (YMM- trp
medium). Other strains were maintained on yeast minimal medium (YMM) or YMM-AA. The
test medium which was used for serial dilutions spotting assay was RPMI1640 containing Lglutamine, but lacking sodium bicarbonate (Invitrogen), buffered to pH 7.0 with 0.165 M MOPS
(Sigma). For media preparation see appendix A.
Table 5.1 Strains used in this study
Candida glabrata strain
Strain description
Strain source or reference
ATCC2001
Wild type
www.atcc.org
HTL reference
Control strain of Δbck1and Δmkk2.
(Schwarzmuller et al., 2014)
his3∆::FRT leu2∆::FRT trp1∆::NAT1
Δbck1
BCK1 was replaced with NAT1 marker
(Schwarzmuller et al., 2014)
Δmkk2
MKK2 was replaced with NAT1 marker
(Schwarzmuller et al., 2014)
TG11
The parent strain of TG152. Δtrp1 (a
(Miyazaki et al., 2010)
derivative of ATCC2001) containing
pCgACT-P
TG152
Δslt2∷HIS3, Δtrp1 containing pCgACT-P
(Miyazaki et al., 2010)
KUE100_chr464
Control strain, CgHIS3 marker was integrated
(Ueno et al., 2011)
into an unrelated locus of chromosome I
KUE10269
Δchs3A strain, CHS3A (CAGL0B04389g) was
(Cg Δchs3A)
replaced with the CgHIS3 marker
KUE10362 (Δchs3B)
Δchs3B strain, CHS3B (CAGL0I04840g) was
replaced with the CgHIS3 marker
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(Ueno et al., 2011)
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5.2.2 Testing sensitivity of CWI-MAPK mutants to thymoquinone and hydrogen peroxide
After steam sterilization of RPMI 1640 agar medium (appendix A), the medium was left to cool
down (~50ºC). Thymoquinone or H2O2 were then added with mixing until the mixture became
homogenous. Solid media supplemented with TQ or H2O2 were left for about 2 h to dry in the
dark in a biohazard cabinet before spotting.
Five microliters of 1:10 serial diluted cell
suspensions were spotted on the agar plates and were left to dry in the dark in a clean biohazard
cabinet. Plates then were incubated at 30ºC for 2 d.
5.2.3 Observation of TQ’s fluorescence and PI staining
Candida glabrata ATCC2001 from an overnight YEPD plate was used to inoculate RPMI1640
liquid medium containing 25, 50 or 100 µg/ml TQ. The working volume was 4 ml in each 50 ml
centrifuge tube. Cultures were protected from light and incubated at 30ºC with agitation (200
rpm). Samples of 1 ml were collected by centrifugation and washed with MilliQ water, then the
cells were examined with an Epi-fluorescent microscope (Leica DM 2500) using an I3 FLUOFilter cube (Leica; 450-490 nm excitation) or analysed by Flow cytometry using a BD FACS
Canto™ II equipped with a pacific blue fluorescent filter (351- 450/65 nm excitation and
emission). For propidium iodide staining, cells were harvested, suspended in 500 µl PBS
contains 2 µg/ml (PI) and incubated for 15 min at 30°C in the dark. After that cells were
harvested, washed and resuspended in 500 µl PBS. Propidium iodide fluorescence was observed
by using Epi-fluorescent microscope fitted with a TexasRed filter (540 - 580 nm excitation).
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5.2.4 Qualitative analysis of chitin levels
Exponential phase cultures of C. glabrata ATCC2001 in YEPD liquid media (5 h incubation
time at 30°C with agitation at 200 rpm) were washed with RPMI1640 medium followed by
suspension of the cells in RPMI1640 medium. The initial optical density of these suspensions
was about 0.2. Five millilitres of the cell suspensions were treated with different concentrations
of thymoquinone (25 or 50 µg/ml) in 50 ml centrifuge tubes. Solvent controls were also
included. The cultures were incubated at 30 °C with agitation (200 rpm) and samples were
collected after 3 or 24 h. Before staining, the optical density (OD600) of different samples was
adjusted to about 0.2. Then 1 ml samples of cell suspensions were exposed to light for at least 5
min. The samples were centrifuged and pellets were resuspended in 1 ml of 2.5 µg/ml calcofluor
white (CFW) (Costa-de-Oliveira et al., 2013). Samples were incubated at room temperature in
the dark for 15 min. Then they were washed with filtered PBS buffer and analysed by a BD
FACS Canto™ II flow cytometer equipped with a pacific blue fluorescent filter (351- 450/65 nm
excitation and emission).
5.2.5 Testing the interaction between thymoquinone and a chitin inhibitor
Sensitivity of C. glabrata ATCC2001 to nikkomycin Z (Sigma-Aldrich) and thymoquinone was
examined via checkerboard assay using a standardised broth microdilution assay as defined by
CLSI (CLSI, 2008) (also see 2.2.1) in a 96 micro-well plate. After incubation for 24 h the wells
contents were mixed using a pipetting action. Then 5 µl aliquots from each well were spotted on
YEPD agar plates. The plates then were incubated for 1-2 d at 30ºC.
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5.3 Results
5.3.1 Activity of SLT2 is required for hydrogen peroxide resistance but not thymoquinone
resistance
The results of the last chapter reported that testing of PKC1 (the upstream kinase of CWIMAPK) inhibitor with TQ showed no interaction. To investigate the importance of other
members of the CWI-MAPK cascade in TQ resistance, mutants of BCK1, MKK2 and SLT2 were
tested for their susceptibility to TQ. Spotting dilutions of these mutants on RPMI1640 plates
containing 50 and 100 µg/ml TQ showed no differences compared to their parental strains (Fig.
5.1). The parental strains were more resistant to TQ than the wild type strain C. glabrata
ATCC2001. Although TQ caused SLT2 phosphorylation (section 4.3.1) C. glabrata Δslt2
(TG152) was not sensitive to TQ. This mutant showed a different pattern in its susceptibility to
hydrogen peroxide. It was more sensitive to hydrogen peroxide than its parental strain (Fig. 5.1).
These results indicate a difference in the importance of SLT2 activation in cell protection against
H2O2 and TQ stress. Cells spotted on plates contained 25 µg/ml TQ showed similar growth to
those which grew on untreated plates.
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No treatment
50 µg/ml TQ
100 µg/ml TQ
ATCC2001
HTL
Δbck1
Δmkk2
No treatment
50 µg/ml TQ
100 µg/ml TQ
ATCC2001
TG11
Cg Δslt2
No treatment
30 mM H2O2
60 mM H2O2
ATCC2001
TG11
Cg Δslt2
Figure 5.1 Susceptibility of C. glabrata Δbck1, Δmkk2, Δslt2 and their parental strains HTL and TG11 to
TQ (The two upper panels) and sensitivity of C. glabrata Δslt2 (Cg Δslt2) to hydrogen peroxide (The
panel at the bottom).
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5.3.2 Thymoquinone generates a blue fluorescence in the C. glabrata cell wall
As thymoquinone caused SLT2 activation, the effect of this compound on the cell wall was
further investigated by examining the interaction between TQ and the cell wall microscopically.
After incubation of C. glabrata with 25 or 50 µg/ml TQ for at least 9 h in the dark, a blue
fluorescence was observed surrounding the cells when they were examined microscopically. The
fluorescence was detectable by flow cytometry (Fig. 5.2 C). Epi-fluorescence microscopy images
showed more details of the binding site of TQ. The images demonstrate that TQ fluorescence
was more intense and specific at the bud scars on the cell wall (Fig. 5.2 A). The emission of blue
fluorescence was not observable in the whole area of the bud scar but it was specific at rims of
the bud scars (Fig. 5.2 B). Staining of treated cells (50 µg/ml TQ) with propidium iodide after 9
h treatment showed loss of cell membrane integrity in some of them. Other cells displayed TQ
fluorescence whilst keeping their membrane integrity (Fig. 5.3). This confirms that binding of
TQ to the cell wall leads to cell death.
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Chapter6
(A)
(B)
(C)
TQ fluorescence intensity
Figure 5.2 Candida glabrata ATCC2001 treated with TQ emits blue fluorescence. A and B epi-fluorescence
microscopy images of C. glabrata cells after 10 h treatment with 50 µg/ml TQ. The cells were visualized using an
I3 filter. White arrows indicate TQ fluorescence at the bud scars and specifically at rims of the bud scar. The inset
in A represents a phase contrast image of the cells, scale bars equal to 5 µm. (C) C. glabrata ATCC2001 cells were
treated with 50 µg/ml TQ overnight then were analysed by flow cytometry.
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Figure 5.3 Epi-fluorescence microscopy images demonstrate the binding of 50 µg/ml thymoquinone to C.
glabrata cell wall which leads to
cell death. (A) Bright field, (B) thymoquinone fluorescence
visualization using an I3 filter, (C) Propidium iodide fluorescence was visualized using a TexasRed filter.
Scale bars represent 10 µm.
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5.3.3 Thymoquinone stimulates chitin production
Chitin distributes over all the cell wall, however Cabib and Arroyo (2013) reviewed several
studies and concluded that there is more accumulation of chitin at the bud scars (Cabib and
Arroyo, 2013). As the fluorescence of TQ was observed at the bud scars, further analysis was
carried out to determine whether TQ affects chitin production in C. glabrata. Chitin levels were
analysed qualitatively by use of CFW staining which targets chitin and flow cytometry analysis.
After 3 h, 6 x 106 cells/ml which were treated with 25 or 50 µg/ml TQ showed no significant
difference in the median of CFW fluorescent intensity (data not shown).
However after
overnight incubation of C. glabrata cells with 50 µg/ml TQ, CFW fluorescent intensity was
increased by 76%, indicating increased chitin (Fig. 5.4). In contrast, cells which were treated
with 25 µg/ml TQ (sub-inhibitory concentration) did not show any difference from the control
cells. To avoid the interference between TQ fluorescence and CFW fluorescence, TQ treated
cells were exposed to light before CFW staining: they showed no TQ fluorescence.
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(A)
(B)
250
200
150
100
50
0
103
104
105
0
103
104
105
Figure 5.4 Thymoquinone induces chitin production in C. glabrata. Untreated cells without calcofluor
staining (black). Cells were treated with TQ and stained with calcofluor white (A) 25 µg/ml TQ (red) or
solvent (green); or (B) 50 µg/ml TQ (blue) and corresponding concentration of solvent (green). Cells
were treated with TQ for 24 h before analysis.
5.3.4 Chitin mutants are more resistant to thymoquinone but not hydrogen peroxide
An advantage was taken of observing thymoquinone’s fluorescence around the bud scars to
further investigate the role of chitin in C. glabrata sensitivity to TQ. Chitin synthase III mutants
have an altered cell wall composition and show a response to TQ stress that is different to their
parental strain. The susceptibility of two C. glabrata chitin synthase III mutants (CHS3A and
CHS3B) to TQ was checked by spotting serial dilutions of the cells on RPMI1640 agar medium
supplemented with TQ. Candida glabrata Δchs3B did not show a significant difference in its
sensitivity to TQ from the control strain but Δchs3A was TQ resistant. Colonies of the Δch3A
strain have a dry and rough surface whereas Δchs3B morphology does not differ from the wild
type, it has shiny and smooth colony surface (Fig. 5.5). The phenotypic characteristics of each
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strain were also confirmed through personal communication with the provider (Ueno et al.,
2011). In terms of their sensitivity to the known pro-oxidant hydrogen peroxide, both strains
were the same as the parental strain (Fig. 5.5B). Microscopic examination of Δchs3A showed
growth defects including formation of aggregates and failure in bud scar septum formation which
resulted in failure of buds to separate from the mother cell and formation of pseudo-hyphae like
structures (Fig. 5.6A). These cells were stained by calcofluor to observe clearly the lack of
separation septum between daughter cells and mothers (Fig. 5.6B). In contrast, C. glabrata
Δchs3B did not form clumps and it showed normal budding pattern (data not shown).
5.3.5 Chitinase inhibitor antagonise with thymoquinone
Previous studies demonstrated that Saccharomyces cerevisiae mutants with lowered chitin levels
show resistance to calcofluor. This compound is supposed to target chitin and compounds that
inhibit chitin synthase III in vivo reverse calcofluor’s inhibitory effect (Gaughran et al., 1994).
Therefore a broth micro-dilutions checkerboard assay followed by spotting 5 µl from each
micro-well was carried out to examine the interaction between TQ and the selective inhibitor of
chitin synthase III nikkomycin Z. Figure 5.7 demonstrates that the micro-wells which contained
25 µg/ml TQ with nikkomycin Z concentrations up to 256 µg/ml exhibited enhanced growth.
This chitin inhibitor rescued C. glabrata from the adverse effect of TQ.
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(A)
C. glabrata
No treatment
50 µg/ml TQ
ATCC2001
KUE100_chr464
Δchs3A
Δchs3B
(B)
No treatment
30 mM H2O2
ATCC2001
KUE100_chr464
Δchs3A
Δchs3B
Figure 5.5 Sensitivity of C. glabrata CHS3 mutants to TQ and H2O2. (A) Deletion of chs3A but
not chs3B in C. glabrata resulted in sensitivity to thymoquinone. (B) Both mutants were not
hydrogen peroxide sensitive. KUE100_chr464 is the control strain.
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(A)
(B)
(C)
Figure 5.6 Microscopic examination of C. glabrata Δchs3A demonstrate failure in bud scar
septum formation. (A) Bright field image of C. glabrata Δchs3A, (B) Calcofluor staining of C.
glabrata Δchs3A observed by utilizing I3filter in epi-fluorescence microscope, (C) Bright field
image of the control strain (C. glabrata KUE100_chr464).
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100
50
25
TQ
12.5 TQ (µg/ml)
Nikkomycin Z (µg/ml)
256
128
64
32
16
0
Figure 5.7 Nikkomycin Z rescues C. glabrata from TQ adverse effect. A combination between TQ and
nikkomycin Z was examined by checkerboard assay in 96 micro-wells plate using C. glabrata
ATCC2001. The plate was incubated 24 h at 35 ºC then 5 µl from each well were spotted on YEPD plate
which was incubated for further 24 h and the image was taken.
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5.4 Discussion
Thymoquinone and hydrogen peroxide induce oxidative stress in C. glabrata, however part of
TQ’s mechanism of action differs from H2O2. Although both of them induced SLT2 activation,
the susceptibility pattern of Δslt2 and Δchs3A to each compound indicated different interaction
with the cell wall.
5.4.1 Cell wall integrity MAPK and TQ antifungal effect
Phosphorylation of SLT2 kinase proposed that SLT2 and maybe the upstream kinases are
required for cell viability upon TQ treatment. Indeed, Candida glabrata Δbck1, Δmkk2 and the
downstream protein Δslt2 were neither sensitive nor resistant to TQ. In addition, inhibition of
PKC1 by staurosporine which is known as a PKC1 inhibitor did not enhance TQ’s fungicidal
effect (section 4.3.5). On the other hand C. glabrata Δslt2 was sensitive to H2O2. Previous
studies on the sensitivity of S. cerevisiae Δslt2 to H2O2 showed variable data. My results are in
agreement with the previous findings of Krasely et al., (2006) which reported sensitivity of S.
cerevisiae Δslt2 to H2O2. In contrast, Vilella et al., (2005) showed no sensitivity of this mutant or
Δbck1 to H2O2 but Δpkc1 and upstream sensor mutants were sensitive to H2O2. This indicates
that TQ and H2O2 are different oxidants, acting on different targets in the yeast cells and induce
cellular responses involving cell wall integrity MAPK activation. According to Vilella et al.,
(2
5) “Hydrogen peroxide is highly diffusible and could enter cells passively and act on
internal targets”. Thymoquinone might act on external targets and diffuse inside the cells to
cause oxidation. Although TQ induces the phosphorylation of SLT2, the CWI-MAPK module
seems to be not vital to the cellular response in defence of TQ stress.
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5.4.2 Thymoquinone targets chitin
The observation of blue fluorescence in TQ treated samples and absence of this fluorescence in
DMSO treated cells was an indicator of TQ fluorescence. A previous study has also reported
fluorescent emission from TQ and quantitated TQ by HPLC combined with fluorescence
detectors (Iqbal et al., 2013). Botnick et al., (2012) observed pink fluorescence in longitudinal
sections of N. sativa seeds under UV or blue light and proposed that the fluorescence is due to
hydrophobic materials including thymoquinone and thymohydroquinone (Botnick et al., 2012).
Observation of TQ’s fluorescence binding to dead cells in figure 5.3 suggests a role of TQ as a
cause of cell death. However, TQ fluorescence was similarly observed on the cell wall of viable
cells which might create a doubt about the influence of dead cells with TQ. In fact, there are
several observations support the idea of cell death was caused because of TQ. First, it is known
that any fungicidal agent will cause the death gradually. The delay in a complete killing in this
population (Fig. 5.3) can be referred to the relatively high inoculum size (106 cell/ml) which was
higher than that was used to monitor the fungicidal kinetics of TQ in chapter 2. Moreover, the
experiment of monitoring the relationship between oxidative stress induction and cell killing by
TQ in chapter 3 (Fig. 3.4) showed that untreated cells did not show a significant increase in
intracellular reactive oxygen species generation and death rate, whereas TQ treated cells suffered
from intracellular oxidative stress and increased death rate. Yet many of TQ treated cells were
suffering from oxidative stress without showing loss of their membrane integrity. The
comparison between untreated cells and TQ treated cells indicates the influence of TQ on C.
glabrata death rate.
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In this stage it is not clear if overloading of cell walls by TQ caused the oxidation which led to
cell death or TQ penetrates into the cells and caused increase in ROS generation regardless of
TQ’s amount which binds to the cell wall.
Thymoquinone’s fluorescence on the cell wall of C. glabrata was more intense at the bud scars.
Indeed, the fluorescence was specifically emitted from the rim of bud scars in a similar way to
the CFW staining pattern (see Fig.2 in (Hausauer et al., 2005). Several studies have concluded
that more accumulation of chitin occurs at the bud scars (reviewed in (Cabib et al., 2013, Molano
et al., 1980) ) and CFW has been known to bind chitin in that area and inhibit chitin polymer
assembly (Roncero et al., 1985). The question is if chitin accumulates at the bud scars, why is
TQ and CFW fluorescence more intense at the rims and does not cover the whole surface of the
bud scar? In S. cerevisiae, chitin is laid down all along the cell wall however it forms mainly
two structures at the neck between mother cell and the bud, which later becomes the bud scar
after separation of daughter cell: (i) the chitin ring that forms at budding and (ii) the primary
septum that forms at cell division. The chitin ring is considerably thick whereas the primary
septum is very thin and therefore the bud scar shows a raised ring (Cabib et al., 2013, Cabib et
al., 1971). Thus, the sensitivity of C. glabrata to thymoquinone could be related to chitin.
Analysing chitin content showed increased levels of chitin in the cells which were treated with
an inhibitory concentration of TQ but not a sub-inhibitory concentration (Fig. 5.4). Those cells
which were treated with an inhibitory concentration of TQ should have less budding and
consequently less chitin accumulated at the bud scars. However those cells seemed to be induced
to produce more chitin as a response to TQ treatment. Sub-inhibitory concentration of TQ did not
elevate chitin which means TQ susceptibility was associated with enhancement of chitin levels.
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Roncero and Duran’s study (1985) demonstrated enhancement in the rate of chitin synthesis in
Geotrichum lactis after CFW or congo red treatment. In addition they have observed abnormally
thick septa and incomplete separation of S. cerevisiae buds which were treated with CFW.
Calcofluor white has been shown to alter the assembly of chitin fibrils in S. cerevisiae and C.
albicans cells (Elorza et al., 1983). However in my study there was no observation of abnormal
aggregates resulting from separation defects of C. glabrata buds after TQ treatment. One
explanation might be that TQ binds to chitin without affecting chitin polymerization or affecting
the process of septum formation.
Production of excess chitin is a common response to cell wall damage to strengthening the wall
by the production of excess chitin, initially by the class IV enzymes such as ScCHS3
and CaCHS3 (Lenardon et al., 2010). Genetic manipulation that leads to depletion of CHS3A
(but not CHS3B) the major homolog of C. glabrata chitin synthase gene resulted in TQ
resistance. This protein is required for synthesis of chitin rings at the bud scar but not formation
of septa (Bulawa et al., 1995), which confirms further the importance of this protein to TQ action
which was mainly observed at the rings. Saccharomyces cerevisiae chs3 mutants have failure in
the transport of CHS3 to the cell surface and produce less chitin at the bud ring (reviewed in
(Lesage et al., 2006). Therefore, mutations in CHS3 or other genes required for CHS3 protein
function result in resistance to calcofluor, the toxic compound that binds to chitin (Reviewed in
(Imai et al., 2005). The same principle might be applicable to the observed resistance of C.
glabrata ∆chs3A to TQ. Candida glabrata Δchs3 strains have been shown to have a thickened
mannoprotein layer in Δchs3A and a thickened chitin-glucan layer in Δchs3B strain (Uneo et al,
2011). Uneo et al., (2011) did not demonstrate a difference in chitin levels between Δchs3A and
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the control strain. Attempts to analyse the amount of chitin levels by flow cytometry in the
present study to compare it with the parental strain were not successful. When these cells were
examined microscopically, they showed formation of aggregates due to the lack of budding septa
(Fig. 5.6) which makes flow cytometry an unsuitable approach to analyse this population. The
lack of budding septa indicates a decrease in chitin synthesis levels, and probably that is why
these cells are more resistant to TQ. In contrast, C. glabrata Δchs3A was neither resistant nor
sensitive to H2O2. This means the decrease in chitin synthesis did not influence sensitivity of this
strain to H2O2 maybe because it diffuses at higher rates than TQ. However, the association
between TQ toxicity, chitin synthesis induction and the antagonism between TQ and the chitin
inhibitor nikkomycin Z put more emphasis on the necessity of chitin to TQ antifungal property.
The action of cell wall polysaccharides including β-1,6-glucan and chitin as receptors for
external materials is widely accepted. For instance, β-1,6-glucan in Candida boidinii works as
receptor of
Pichia membranifaciens killer toxin (Santos et al., 2000) and chitin binds to
calcofluor white stain (Roncero et al., 1985). The present study suggests that chitin might
facilitate the transport of TQ or work as a receptor of TQ because of the following facts:

The toxicity of TQ was associated with an increase in chitin levels, and it is known that
chitin locates at the cell wall which is the first contact barrier of the yeast cell (Fig. 5.4).

The mutant of CHS3A is TQ resistant. This protein is responsible for locating chitin at
the ring in the bud scar where TQ fluorescence was observed (Fig. 5.6).

The mutant of CHS3A was neither sensitive nor resistant to H2O2 which is known as a
quick diffuser, indicating the importance of chitin to TQ action.

Inhibition of chitin production by nikkomycin led to a decrease in the antifungal efficacy
of TQ ( Fig. 5.7).
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5.4.3 Chitin, CWI-MAPK and TQ sensitivity
Activation of the cell wall integrity pathway components is supposed to stimulate cell surface
transport of CHS3 (Munro et al., 2007). As a result, CWI-MAPK mutants would have less chitin
and would be resistant to CFW as well as TQ. Candida glabrata Δbck1, Δmkk2 and Δslt2 have
shown similar growth rates to the wild type when they were treated with CFW (Schwarzmuller et
al., 2014). This is in agreement with the sensitivity of these mutants to TQ. Indeed a further
analysis of chitin content in these mutants after TQ treatment might explain further the relation
between induction of these kinases and cellular response to TQ stress. Cota et al., (2008) showed
that C. glabrata Δslt2 had a similar amount of chitin to the parent strain but C. glabrata Δslt2
was not able to enhance its chitin content after caspofungin exposure compared with the parent
strain; therefore C. glabrata Δslt2 was slightly sensitive to caspofungin (Cota et al., 2008).
Activation of other signalling pathways which might induce chitin production should not be
ignored. Intracellular oxidative stress, which can be caused by TQ treatment, induces several
mitogen activated protein kinase (MAPK) routes in yeast: these include CWI, TOR , HOG and
RAS-PKA-cAMP (reviewed by (da Silva Dantas et al., 2015, de la Torre-Ruiz et al., 2010).
Chitin synthesis can be upregulated co-ordinately via the CWI, HOG and Ca2+ signalling MAPK
pathways in Candida albicans. The Ca2+-induced upregulation of CHS promoters can be
independent of the CWI pathway (Munro et al., 2007). Cross-talk between these signalling
pathways also exists (reviewed by (de la Torre-Ruiz et al., 2010). Therefore, inhibition of CWIMAPK components might be compensated by the activity of other signalling pathways.
However, as mentioned before, further analysis of chitin levels in CWI-MAPK mutants is
required to confirm that their susceptibility pattern is related to the similar level of chitin
compared with the control strain.
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5.5 Conclusion
In conclusion, it seems that treatment of C. glabrata cells with thymoquinone increases the
rigidity of the cell wall by increasing chitin levels in a try to overcome TQ’s stress. The increase
in chitin synthesis might be occurred after stimulation of CWI-MAPK pathway. In addition, the
resistance of C. glabrata Δchs3A to TQ and the interference between TQ and nikkomycin Z
indicate an important role of chitin in TQ mechanism of action.
Chapter 6: Final conclusions
There are many studies investigating the mechanism of thymoquinone as an anticancer
compound but, there are no published data on its mechanism of action as an antifungal or even
an antimicrobial compound. The present study of thymoquinone’s effect on oxidative status and
the cell wall integrity pathway in Candida glabrata will advance the understanding of
thymoquinone tolerance mechanisms and help to develop new approaches to the treatment of
fungal infections with thymoquinone.
The antifungal effect of thymoquinone towards C. glabrata, C. krusei and Candida spp. biofilms
has not been investigated before. Chapter 1 showed that thymoquinone was able to inhibit the
growth of different Candida species including C. glabrata and C. krusei. In addition,
thymoquinone showed a fungicidal effect rather than fungistatic effect in a cell-density
dependent way. In terms of its effect on biofilms, thymoquinone can inhibit the growth of 2 h old
C. glabrata biofilms by 3 log10 but not mature biofilms. The biofilms were established using a
polypropylene biofilm reactor (PP reactor) which was developed in this study to mimic the
Calgary biofilm device. It is a simple, low cost device and provides valid data for studying C.
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Chapter6
glabrata biofilms and might be useful for other microbial species which produce biofilms. The
similarity of biofilms produced at different sites of the PP reactor enables rapid and reproductive
screening of Candida biofilms for antifungal resistance testing. It can also help to establish
biofilm models for molecular microbiology investigations. Finally the death caused by
thymoquinone in C. glabrata seemed to be necrotic death and further investigations to confirm
this result are recommended.
It has been concluded from testing thymoquinone on mammalian cell lines that thymoquinone
can act as antioxidant or prooxidant depending on the milieu. The question of whether
thymoquinone acts as prooxidant or antioxidant in yeast was addressed in chapter 2. The
obtained data demonstrate that the generation of intracellular oxidative stress is definitely part of
thymoquinone’s mechanism of action as a fungicidal agent.
Oxidative stress caused by
thymoquinone led to loss of cell membrane integrity and affected intracellular functions
including reduction in reduced glutathione amount and reduced mitochondrial membrane
potential. D-erythroascorbic acid was identified as an important antioxidant in other yeast
species such as S. cerevisiae and C. albicans. This study identified for the first time the synthesis
of D-erythroascorbic acid by C. glabrata. Two isolates of the ALO1 gene were cloned and
sequenced on both strands of DNA, and the sequence of the clone (Cg16a) with less changes is
shown. The differences are likely being PCR errors. Since there was a 2.5 fold increase in
dehydroascorbate production I assumed the changes did not affect the ALO1 enzyme function.
The further completion of the study for publication will involve the construction of an ALO1
disrupting mutant.
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Chapter6
Overexpressing of D-arabino-1,4-lactone oxidase (ALO1) was demonstrated by measuring a 2.5
fold increase in D-erythroascorbic acid production in C. glabrata clones. However, the produced
amount in this study was not enough to significantly alleviate the oxidative stress caused by
H2O2 or thymoquinone.
As a pro-oxidant, thymoquinone induced the CWI-MAPK cascade. This study hypothesised that
inhibition of this cascade might weaken C. glabrata cell wall integrity as well as its response to
the oxidative stress caused by thymoquinone. In fact, utilisation of a PKC1 inhibitor did not
improve thymoquinone efficacy. Furthermore, the mutants of downstream CWI-MAPK
components did not show any difference in thymoquinone sensitivity compared with their
parental strains. That indicates the minor role of this cascade in protection from the
thymoquinone fungicidal effect. In contrast, the downstream kinase SLT2 was crucial in
protection against H2O2 toxicity which indicated a different interaction between C. glabrata cells
and these two pro-oxidants.
This difference was confirmed by discovering the binding of thymoquinone to chitin on the
surface of C. glabrata cells. The results of chapter 5 describe virtual and genetic evidence to
support the idea that cell wall chitin plays a role in the interaction between thymoquinone and C.
glabrata. Observations of thymoquinone fluorescence at the rims of bud scars where more chitin
is accumulated and induction of chitin synthesis by thymoquinone indicated an interaction
between TQ and chitin. Moreover, the antagonism between thymoquinone and the chitin
inhibitor, nikkomycin Z and the resistance of C. glabrata Δchs3A to thymoquinone emphasises
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Chapter6
the relationship between TQ and chitin. However, it is not clear why it took over 9 h for the TQ
staining of bud scars to become apparent and further work to clarify this observation is important
to elucidate further TQ’s mechanism of action as an antifungal agent.
In conclusion, treatment of C. glabrata with thymoquinone activates the CWI-MAPK cascade
which leads to an increase in chitin synthesis. As a consequence, the rigidity of the C. glabrata
cell wall increases to overcome thymoquinone’s stress. On the other hand, there seems to be a
decrease in the amounts of cellular chitin causing a decrease in TQ’s antifungal effect and
increased chitin amounts, increasing TQ’s efficacy as antifungal agent. Since the fluorescence of
TQ needs a long time to be observed at the bud scars, it is not clear whether TQ’s binding to
chitin at the bud scars contributes to overwhelming of C. glabrata cell wall with TQ, causing an
oxidative stress and then cell death or TQ might diffuse, causes oxidative stress and binds to the
cell wall later. The antagonism between chitin inhibitors and thymoquinone should be considered
in selecting agents for clinically treating Candida infections. In addition, the interaction between
TQ and chitin might help in development of TQ nanoparticles.
Chapter 4 also showed data on the survival of C. glabrata in water and demonstrated for the first
time the relationship between cell number and survival rate of this yeast in a hypo-osmotic
environment which was associated with starvation. A high cell density of C. glabrata cells
provides protection towards hypo-osmolarity but not low cell density (<105 cells/ml). In
addition, dead cells in water do not lyse because of the inflow of water inside the cells but they
shrink and die via apoptosis. This study also proposed a bio-protection through releasing of
nutrients from these cells at high density. Low cell density of C. glabrata can survive at hypo-
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Chapter6
osmotic environment in the presence of osmo-stabiliser or low concentrations of nutrients. Such
factors might be important to consider in environmental studies which focus on eradication of
microbial cells (e.g. in water systems).
Cells in water might be a useful model to screen and test CWI-MAPK inhibitors. Compounds
which inhibit the CWI-MAPK cascade such as staurosporine are toxic in water but higher
concentrations are needed to become toxic in media. The toxicity of the CWI-MAPK inhibitor
can be abrogated by an osmo-stabilizer such as sorbitol. Although this study was not able to
screen novel CWI-MAPK inhibitors due to the limited number of tested compounds, the known
PKC1 inhibitor staurosporine has supported this hypothesis.
This study also showed for the first time phosphorylation of SLT2 in C. glabrata as a result of
doxycycline treatment. Doxycycline is used as an inducing agent for some expression systems,
with the thought that it has no side effect. However, this unreported side effect should be
considered when yeast is used as protein expressing system in studies involving doxycycline.
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Appendix A
A.1 Growth media and general solutions preparation
Growth media preparation and general solutions’ recipes were prepared following published
recipes or manufacturer’s guidelines.
YEPD medium contents (1% yeast extract, 2% bacteriological peptone, 2% dextrose) were
dissolved in distilled water followed by steam sterilisation. To prepare YEPD solid medium 1.5
% of bacteriological agar was added. All the components were purchased from Oxoid Ltd or Cell
Biosciences Pty Ltd.
Yeast minimal medium (YMM) was prepared by adding 0.67% yeast nitrogen base (with amino
acids (Sigma-Aldrich)) and 2% dextrose to distilled water. To prepare YMM solid medium 1.8
% of bacteriological agar was added. This medium lacks uracil.
The yeast minimal medium without amino acids (YMM w/o AA) contains yeast nitrogen base
without amino acids (Difco) and 2% dextrose. Solid YMM w/o AA was prepared by adding 1.8
% bacteriological agar. This medium lacks uracil.
RPMI1640 medium 3.04 g (0.165 M) MOPS (Sigma-Aldrich) was added to 1.04 g of RPMI1640
medium (with glutamine and phenol red, without bicarbonate (Invitrogen)) in 100ml of distilled
water. The acidity of this medium was adjusted while stirring to pH 7 using 1 mol/L sodium
hydroxide (CLSI, 2008). This medium was filter sterilised before it was used as a liquid medium.
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Alternatively RPMI1640 medium supplied with 1.8 % bacteriological agar was steam sterilised
to prepare RPMI1640 agar plates.
Phosphate buffer solution (PBS) was prepared by dissolving one tablet (Sigma-Aldrich) in 200
ml of MilliQ water. The solution was sterilised by autoclave and was filtered through 0.22 µm
filter (Millex) prior flow cytometry experiments.
TAE Buffer 0.04M Tris- acetate, 0.001 EDTA and the acidity was adjusted to 8.3.
A.2 Yeast strains maintaining
Candida cryogenic stocks were stored at -80 ºC in YEPD medium with 25 % sterilised glycerol
and streaked onto YEPD plates as working stocks. The working stocks were stored at 4ºC and
replaced at most after two months. Before each experiment candida from working stocks were
sub-cultured twice onto YEPD plates or selective medium with overnight incubation at 30 ºC.
Candida glabrata ura3 HM100 mutant cryogenic stocks were stored at -80 ºC in yeast minimal
medium lacking uracil (YMM-ura3) without amino acids and plated onto the same medium.
Escherichia coli DH5α and E. coli HHT2 were stored in Luria-Bertani LB medium with 20%
sterilised glycerol at -80 ºC. To prepare working stocks, cells were streaked from -80ºC onto the
same LB agar and incubated overnight at 37 ºC. Cultures for plasmid transformation and
selection were grown in LB contain 100 µg/ml of penicillin.
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A.3 Antimicrobial agents used in this study
The antifungal agents including caspofungin stocks were dissolved in sterilised MilliQ water and
stored at -20 ºC. Nikkomycin and doxycycline were dissolved in sterilised RPMI1640 medium
at the time of use. Fluconazole was dissolved in Dimethyl sulfoxide (DMSO) and used
immediately.
Stausporine stocks of 1 mg/ml were prepared in ethanol and stored at -20 °C. Sorafenib and
XMD8-92 (Santa Cruz, USA) stocks were prepared freshly in DMSO.
Thymoquinone stocks were prepared by dissolving thymoquinone in DMSO at concentration of
20 mg/ml and usually were used immediately or stored into Eppendorf tubes at -20 ºC for not
longer than two weeks. Before use, TQ stocks of 20 mg/ml were diluted 1:10 and further 1:2.5 in
RPMI 1640 before adding equal volume of RPMI 1640 to make serial dilutions.
A.4 General procedures
All the practical operations were carried out aseptically on clean bench wiped with 70% ethanol.
Work was kept near from flaming Bunsen burner or in a biohazard cabinet wiped with ethanol
and exposed to UV-light for at least 20 min. Most of the reagents, media and consumables (e.g.
tips) were steam sterilised at 121ºC for 15 min.
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A.5 Spotting of yeast on agar plates
Serial dilutions of 1:10 were done in 96 micro-plates by picking up some of yeast colony from
overnight plate using tooth picks. The biomass was suspended in the first well followed by serial
dilutions. The medium was used for serial dilutions was same as the agar plates. Immediately
after the serial dilutions or after overnight incubation 5 µl from each well were spotted on the
agar plates and left to dry in a biohazard cabinet.
A.6 Microscopy
Epi-fluorescence microscope (Leica DM2500, Germany) was used to visualise fluorescent stains
and Differential interference contrast (DIC) or the bright field . Electronic Images were captured
using a DFC310 FX Leica camera and processed using the Leica Application Suite (LAS, V4.3,
Switzerland). This microscope was equipped with three filter cubes: Filter cube A (UV
excitation) is 340-380 nm, Filter cube I3 (blue excitation between 450-490 nm), GFP filter
(excitation 469 nm), Texas Red filter (Excitation 559 nm).
A.7 Flow cytometry
FACS Canto II flow cytometer (BD Biosciences, Becton-Dickinson, Franklin Lakes, USA) flow
cytometer equipped Different laser filters was utilised to obtain flow cytometry data. The filters
which were used are:

Pacific Blue™, 45 /5 filter (351-450/65nm Excitation max – Emission max).
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
AmCyan, 525/20 filter (458-489 nm Excitation max –Emission max).

PerCP-Cy™5.5, 695/4 filter (482/695 nm Excitation max –Emission max).

FITC, 530/30 filter (494/519 nm Excitation max –Emission max).
Ten thousand cells were collected at the most of experiments and data files were saved as FCS3,
then were analysed whether by Flowing software which was created by Perttu Terho (Cell
Imaging core, Turku Centre for Biotechnology, Finland) or trial version of WEASELTM software
(WEHI, Australia).
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