22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Atmospheric pressure non-thermal plasma-mediated attenuation of acyl
homoserine lactone-dependent bacterial cell-cell communication (quorum
sensing): a possible anti-virulence approach in chronic infection
P.B. Flynn1, A. Busetti1, N.H. Alshraiedeh1,2, W.G. Graham3, S.P. Gorman1 and B.F. Gilmore1
1
2
School of Pharmacy, Queen’s University Belfast, Northern Ireland
Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan
3
Centre for Plasma Physics, Queen’s University Belfast, Northern Ireland
Abstract: To date, the vast majority of antimicrobial studies in the field of plasma
medicine, have examined the susceptibility to, and cellular interactions of, non-thermal
plasmas with bacteria, for infection and contamination control applications. However, few
studies have examined the potential role of non thermal plasmas in the control of key
microbial virulence factors, including the sophisticated bacterial intracellular system of
communication, known as Quorum Sensing (QS). QS is a population density-dependent
cell-cell communication system which coordinates gene expression and controls key
behaviours in bacteria including virulence, biofilm formation and motility. QS relies on
small diffusible signals, termed autoinducers, the most extensively studied of which are the
Gram negative signalling molecules, the acyl homoserine lactones (AHLs). This study
reports for the first time the ability of non-thermal plasma to degrade these molecules and
in so doing, disrupt this critical bacterial signalling pathway.
Keywords: quorum sensing, acyl-homoserine lactones, atmospheric pressure non-thermal
plasma
1. Introduction
The rapidly growing field of plasma medicine has
experienced an explosion of interest within the past two
decades. In particular, atmospheric non-thermal plasma
(APNTP) has shown tremendous antimicrobial efficacy
against microorganisms, [1] suggesting its potential
applicability in a plethora of clinical applications, settings
and scenarios. Whilst still a relatively nascent and rapidly
expanding field, the antimicrobial potential of APNTP has
attracted greatest interest within the fields of infection and
contamination control, with sterilization and disinfection
amongst the earliest applications described [2].
Within a host, bacterial cells produce a variety of
extracellular molecules responsible for mediating
bacteria-host interactions. The term ‘autoinducers’ is
used to describe a class of extracellular molecules
synthesised by bacteria which are integral to cellular
communication, a process termed quorum sensing (QS).
QS is fundamental for the regulation of pathogenicity and
virulence in numerous human pathogens [3]. This study
represents the first investigation on the effects of APNTP
on the biological activity of a class of autoinducers
employed by several Gram negative human pathogens,
the acyl homoserine lactones (AHLs).
QS in bacteria relies on the production, detection and
response to auto inducers. There are different autoinducer
molecules and pathways for detection between bacteria.
Gram negative bacteria typically use the LuxI/LuxR QS
pathway. The LuxI system is responsible for synthesising
autoinducer molecules called acyl homoserine lactones
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(AHL). These molecules are composed of a lactone ring
ligated to an acyl carbon chain through an amide bond.
Examples of these molecules are shown in Fig. 1. These
AHL molecules bind to the cognate LuxR receptor and
activate transcription of target genes.
As well as
activating the production of genes, the bound AHLs also
activate a feedback loop that increases the production of
more AHLs [3]. The transcription of QS-controlled genes
regulate key behaviours in these bacteria, including
antibiotic resistance, biofilm formation, motility and
virulence factor production. QS systems are critical in
control of virulence in many clinically important Gramnegative human pathogens including Pseudomonas
aeruginosa, Acinetobacter baumannii and Burkholderia
species.
Using four acyl homoserine lactones (Fig. 1) as model
autoinducer signalling molecules, we examined the effect
of plasma on the ability of these molecules to induce
phenotypic responses (pigment/ bioluminescence) using
several bacterial bio-reporters. This study describes, for
the first time, the effect of atmospheric plasma exposure
the biological activity of these molecules and its
consequential effect on the QS pathway. This study
demonstrates that, in addition to direct bactericidal
activity, short non thermal plasma exposures have the
ability to disrupt key virulence pathways in bacteria thus
elucidating further the potential biological effects of
atmospheric plasma in the biological milieu of chronic
infections, and highlights the potential of APNTP as an
‘anti-virulence’ approach.
1
and diluted in phosphate buffer saline (PBS) to achieve a
working concentration of 100 µM.
Fig. 1. Structures of acyl homoserine lactone molecules
used in this study. (A) N-butyrl-homoserine lactone, (B)
N-hexanoyl-homoserine lactone, (C) N-octanoylhomoserine
lactone,
(D)
N-(3-oxododecanoyl)homoserine lactone.
2. Materials and Methods
2.1. Plasma Source and Exposure Protocol
The atmospheric plasma jet used in this study has
previously been described in [4, 5] and a diagram of the
experimental set up is shown in Fig. 2. Briefly 20 µl
samples of each molecule were exposed to a 2 standard
litre per minute (SLM) helium/oxygen 0.5% plasma
plume, 15 mm from the nozzle exit. Correction of the
evaporation was considered through weighing before and
after plasma exposure samples. Five replicates were
exposed for 0 (control), 30, 60, 120, 240 seconds for each
molecule.
Fig. 2. Diagram of plasma jet and exposed samples.
2.2. Acyl Homoserine Lactones
A stock 10 mM concentration of the following
molecules N-butyrl-dl-homoserine lactone (C4 AHL).
N-hexanoyl-dl-homoserine
lactone
(C6
AHL),
N-octanoyl-dl-homoserine lactone (C8 AHL) and
N-(3-oxododecanoyl)-homoserine lactone (C12 AHL)
(Sigma Aldrich, Dorset, UK) was prepared in acetonitrile
2
2.3. Bio-reporter strains and Growth Conditions
Chromobacterium violaceum (CVO26) and Escherichia
coli pSB401 were used to report the QS activity of
C6 AHL and C8 AHL. CV026 produces a strong purple
pigment in the presence of C6 and C8 AHLs. E. coli
pSB401 utilises the luxCDABE system from Vibrio fisheri
and produces bioluminescence when exogenous C6 or C8
AHL are added to the culture. Another bioluminescent
strain, E. coli pSB 1142, was used to report the biological
activity of C12 AHL. Agrobacterium tumefaciens ATCC
BAA-2240 was used to report the biological activity of all
AHLs, this system produces an enzyme, β-galactosidase,
in the presence of threshold concentrations of AHL. The
produced enzyme metabolises the chromogenic substrate,
X-Gal (5-Bromo-4-chloro-3-indolyl β-D-galactoside) to
an insoluble blue pigment in the presence of exogenous
AHLs. These bio-reporter strains were grown in Luria
Bertani (LB) broth with 25 µg/ml kanamycin added to
CV026, 5 µg/ml tetracycline added to both E. coli strains
and 25 µg/ml gentamicin added to A. tumefaciens. CV026
and A. tumefaciens were grown at 28 °C and the E. coli
strains at 37 °C.
2.4. Thin Layer Chromatography (TLC) and Bio- reporter
Overlays.
AHLs were spotted on RP-C18 aluminium backed silica
TLC plates and developed in a 60:40% v/v methanol:
water mobile phase. TLCs were air-dried and placed on
LBA. CV026 overlays were prepared by adding 50 µl of
an overnight culture to 10 ml of LB + 1% agar and poured
over the plates. To prepare the A. tumefaciens overlays,
X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactoside) was
added to 10 ml of LB + 1% agar to make a final
concentration of 60 µg/ml along with 1 ml of an overnight
culture, which was poured over the TLC plates. Plates
were incubated at 28 °C for 48 hours allowing the
pigments to develop. The plates were then photographed
using an Olympus E-600 digital camera.
2.5. Bioluminescence Assay
10µl of AHL samples were added to 90 µl of a 1 in 100
dilution of an overnight culture of E. coli pSB401
(C6 & C8) or E. coli pSB1142 (C12). Luminescence was
measured using a BMG Fluostar Optima Fluorescence
plate reader (BMG,Labtech Ltd, Aylesbury, UK) over
14 hours at 37 °C. Luminescence values were normalised
according to growth (OD 550 ) over the same period of time
for each sample.
3. Results and Discussion
TLC overlays were carried out for all AHL molecules
using CV026 and A. tumefaciens. CV026 is specific to
both C6 AHLs and C8 AHLs with C6 eliciting the
strongest response. A. tumefaciens is less selective
responding to a range of AHLs [6]. Fig. 3 shows CV026
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Bioluminescence (RLU/OD 550nm)
response to plasma exposed n-hexanoyl-homoserine
lactone over various times. It is clear from Fig. 3 that
with increasing plasma exposure of C6 AHL, the
phenotypic response (pigment production) of CV026
decreases indicating attenuation of QS.
(A)
10 6
Control 3-oxododecanoyl HL
30 seconds exposure
60 seconds exposure
120 seconds exposure
240 seconds exposure
Negative Control
10 5
10 4
10 3
10 2
10 1
(B)
(A)
10 0
0
6
4
2
10
8
14
12
Bioluminescence (RLU/OD 550nm )
Growth time (hours)
(B)
10 6
Control N-hexanoyl-HL
30 C6 AHL
60 C6 AHL
120 C6 AHL
240 C6 AHL
Negative Control
10 5
10 4
10 3
10 2
(C)
(D)
10 1
10 0
0
2
4
6
8
10
12
14
Fig. 3. TLC overlays of CV026 and 100µM C6 AHL
treated for various times. A, positive control (0 second’s
exposure). B, 30 seconds exposure. C, 60 seconds
exposure. D, 120 second’s exposure. E, 240 seconds
exposure. The black line represents were 1 µl of
N-hexanoyl homoserine lactone was spotted (1 x 10-9 M).
Fig. 4, presents the bioluminescence assays for plasma
treated C12, C6 and C8 AHLs. It is clearly shown that
with increased plasma treatment the phenotypic response
decreases, confirming that plasma has altered the
biological activity of these QS molecules. The negative
control (no AHL added) is present indicating the “no
response” limit approx. <102 RLU/OD 550 . From this
description it can be put that there is no phenotypic
response at 4 minutes exposure indicating complete QS
inhibition.
These curves were normalised to the
corresponding growth curve. Bacteria entered the log
phase after four hours and accounts for the near plateau
shape of each exposure time.
4. Conclusion
In this study we have, for the first time, demonstrated
that APNTP in addition to eliciting a direct a biocidal
effect on microorganisms, is also capable of interfering
with the key QS pathways of Gram negative bacteria,
potentially leading to attenuation of bacterial virulence, as
well as reducing the bacterial load. This is particularly
relevant to the study of APNTP in chronically infected
wounds were the production of virulence factors delays
wound repair and normal healing. Further work is
however required to elucidate the potential of this
approach in vivo, but this is the first report of the potential
for APNTP to be used as an ‘anti-virulence’ approach.
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Bioluminescence (RLU/OD 550nm)
Growth time (hours)
(C)
10 6
Control N-octanoyl-HL
30 seconds C8 AHL
60 Seconds C8 AHL
120 Seconds C8 AHL
240 Seconds C8 AHL
Negative control
10 5
10 4
10 3
10 2
10 1
10 0
0
2
4
6
8
10
12
14
Growth time (hours)
Fig. 4. Bioluminescence activity of bioreporter strain
after
plasma
treatment
of
AHLs.
(A)
N-(3-oxododecanoyl)-homoserine lactone using E.coli
pSB 1142, (B) N-hexanoyl-homoserine lactone using
E. coli pSB 401, (C) N-octanoyl-homoserine lactone
using E. coli pSB 401. Each point represent five
replicates with error bars representing the standard
deviation.
5. References
[1] M.Y. Alkawareek, Q.T. Algwari, S.P. Gorman,
W.G. Graham, D. O’Connell and B.F. Gilmore.
"Application of atmospheric pressure nonthermal
plasma for the in vitro eradication of bacterial
biofilms". FEMS Immunol. Med. Microbiol., 65,
381-384 (2012)
[2] M. Laroussi. "Sterilization of contaminated matter
with an atmospheric pressure plasma". IEEE Trans.
Plasma Sci., 24, 1188-1191 (1996)
[3] S.T. Rutherford and B.L. Bassler.
"Bacterial
quorum sensing: its role in virulence and
possibilities for its control". Cold Spring Harb.
Perspect. Med., 2, a012427 (2012)
[4] M.Y. Alkawareek, Q.T. Algwari, G. Laverty,
S.P. Gorman, W.G. Graham, D. O’Connell and
B.F. Gilmore.
"Eradication of Pseudomonas
3
[5]
[6]
4
aeruginosa biofilms by atmospheric pressure nonthermal plasma". PLoS One, 7, e44289 (2012)
J.S. Sousa, K. Niemi, L.J. Cox, Q.T. Algwari,
T. Gans and D. O’Connell. "Cold atmospheric
pressure plasma jets as sources of singlet delta
oxygen for biomedical applications". J. Appl. Phys.,
109, (2011)
L. Steindler and V. Venturi. "Detection of quorumsensing N-acyl homoserine lactone signal molecules
by bacterial biosensors". FEMS Microbiol. Lett.,
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