University of Iowa
Iowa Research Online
Theses and Dissertations
2007
Targeted antimicrobial activity of SMAP28
conjugated to IgG antibody
Michael Ryan Franzman
University of Iowa
Copyright 2007 Michael Ryan Franzman
This thesis is available at Iowa Research Online: http://ir.uiowa.edu/etd/140
Recommended Citation
Franzman, Michael Ryan. "Targeted antimicrobial activity of SMAP28 conjugated to IgG antibody." MS (Master of Science) thesis,
University of Iowa, 2007.
http://ir.uiowa.edu/etd/140.
Follow this and additional works at: http://ir.uiowa.edu/etd
Part of the Oral Biology and Oral Pathology Commons
TARGETED ANTIMICROBIAL ACTIVITY OF SMAP28 CONJUGATED TO IgG
ANTIBODY
by
Michael Ryan Franzman
A thesis submitted in partial fulfillment
of the requirements for the
Master of Science degree in Oral Science
in the Graduate College of
The University of Iowa
July 2007
Thesis Supervisor: Professor Kim A. Brogden
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
___________________________
MASTER’S THESIS
_________________
This is to certify that the Master’s thesis of
Michael Ryan Franzman
has been approved by the Examining Committee
for the thesis requirement for the
Master of Science degree in
Oral Science at the July 2007 graduation.
Thesis Committee:
_____________________________
Kim A. Brogden, Thesis Supervisor
_____________________________
Deborah V. Dawson
_____________________________
David R. Drake
_____________________________
Janet M. Guthmiller
TABLE OF CONTENTS
LIST OF TABLES
iv
LIST OF FIGURES
v
CHAPTER
I. INTRODUCTION
1
II. REVIEW OF THE LITERATURE
3
Innate Immune System
Microbial Community Associated with
Periodontal Disease
III. SIGNIFICANCE AND SPECIFIC AIMS
Hypothesis
Aim 1
Aim 2
3
6
10
10
10
10
IV. MATERIALS AND METHODS
Synthesis of SMAP28
Bacterial Species and Growth Conditions
Preparation of Antiserum
Isolation of Microorganism-Specific Antibody
Whole Cell ELISA Analysis
SMAP28 and IgG Conjugation Procedure
Mass Spectrometry
HPLC
Spot Blot
Radial Diffusion Antimicrobial Assay
Conjugate-Specific Killing
in a Mixed Culture Suspension
Statistical Analyses
V. RESULTS
11
11
11
12
13
15
16
17
17
18
18
19
20
22
E. coli and P. gingivalis Antibodies were Isolated
and Purified
The Antimicrobial Activity of SMAP28 Conjugated
to Antibody Reagents was Determined
ii
22
23
The Antimicrobial Specificity of SMAP28 Conjugated
to Organism Specific Antibodies in a Community of
Microorganisms was Determined
The Antimicrobial Activity of the
SMAP28-E. coli IgG Antibody Conjugate was Determined
The Antimicrobial Activity of the
SMAP28-P. gingivalis Antibody Conjugate was Determined
VI. DISCUSSION
25
26
27
33
Antimicrobial Activity of SMAP28 with
Attached Maleimide and IgG Antibody
Alternate Carriers
Applicability
Future Directions
VII. SUMMARY AND CONCLUSIONS
37
39
40
40
42
APPENDIX A. TABLES AND FIGURES
43
REFERENCES
77
iii
LIST OF TABLES
Table A1. Antimicrobial Proteins and Peptides Found in Human
Mucosa and Mucosal Secretions
43
Table A2. Antimicrobial Proteins and Peptides Found in Human
Oral Mucosa and Oral Secretions
45
Table A3. Characteristics of SMAP28
46
Table A4. Intraclass Correlation Coefficients for Rater
Reliability in Counting Colony Forming Units
47
Table A5. Intraclass Correlation Coefficients for Rater
Reliability of Base 10 Log Transformations of Colony
Forming Units
48
Table A6. Estimate of the Average Rate of Change in the Colony
Forming Units Over Time
49
Table A7. Descriptive Statistics of the Mean and Standard Deviation
for the Number of Colony Forming Units for the Three Organisms
by Treatment and Day of Experiment
50
iv
LIST OF FIGURES
Figure A1.
Overview of Methods
52
Figure A2.
SMAP28 Peptide
53
Figure A3.
MALDI-TOF Assessment of the Purity of the SMAP28
Sample
54
Reversed Phase-High Performance Liquid
Chromatography (HPLC) Assessment of the Purity
of the SMAP28 Sample
55
Figure A5.
Isolation of E. coli Specific Antibody
56
Figure A6.
Isolation of P. gingivalis Specific Antibody
57
Figure A7.
The SMAP28-IgG Antibody Conjugate
58
Figure A8.
Dot Blot Analysis
59
Figure A9.
Mixed Microbial Culture Testing the Specificity and
Activity of the E. coli IgG-SMAP28 Conjugate
60
Mixed Microbial Culture Testing the Specificity and
Activity of the P. gingivalis IgG-SMAP28 Conjugate
61
MALDI Assessment of Maleimide Linker(s)
Added to SMAP28
62
Figure A12.
HPLC Separation of SMAP28+1, 2, 3 or 4 Linkers
63
Figure A13.
E. coli IgG Conjugate Too Concentrated Showing
Non-Specific Killing
64
Figure A14.
E. coli IgG Conjugate Specific Killing
65
Figure A15.
Photo of Aerobic Plates
66
Figure A16.
SMAP28-IgG P. gingivalis Antibody Conjugate
Mediated Killing in Mixed Microbial Culture
(100 ug/ml) Method IIA
67
SMAP28-IgG P. gingivalis Antibody Conjugate
Mediated Killing in Mixed Microbial Culture
(100 ug/ml) Method IIB
68
Figure A4.
Figure A10.
Figure A11.
Figure A17.
v
Figure A18.
P. micros Viability in Reduced, Anaerobic Saline
69
Figure A19.
SMAP28-IgG P. gingivalis Antibody Conjugate
Mediated Killing in Mixed Microbial Culture
(40 ug/ml) Method IIA
70
SMAP28-IgG P. gingivalis Antibody Conjugate
Mediated Killing in Mixed Microbial Culture
(40 ug/ml) Method IIB
71
Pre-Immune IgG Antibody Control Mediated Killing
in Mixed Microbial Culture (40 µg/ml).
72
The Increased Antimicrobial Activity of the SMAP28P. gingivalis IgG Antibody Conjugate Option IIB
(CONJ_IIB) for P. gingivalis
73
The Rapid Drop in Viability of P. micros in the Test
Conjugates and the Control Solutions
74
The Decreased Antimicrobial Activity of the SMAP28P. gingivalis IgG Antibody Conjugate Option IIB
(CONJ_IIB) for A. actinomycetemcomitans
75
The Decline of P. gingivalis is Visible After Incubation
in the SMAP28-P. gingivalis IgG Antibody Conjugate
Option IIB for 30 minutes
76
Figure A20.
Figure A21.
Figure A22.
Figure A23.
Figure A24.
Figure A25.
vi
1
CHAPTER I
INTRODUCTION
The innate immune system plays many roles in first line immune defense. In the
human oral cavity, innate immune elements include lysozyme and antimicrobial peptides
such as defensins and cathelicidins. Other cathelicidins in sheep, like SMAP28, have
potent antimicrobial activity. This research assessed the antimicrobial activity of
SMAP28 conjugated to IgG antibodies specific for Escherichia coli and Porphyromonas
gingivalis. Our hypothesis is that this conjugated peptide will selectively kill E. coli or P.
gingivalis in mixed microbial cultures. This technique may be a step towards developing
a selective antimicrobial agent.
Antibiotic therapy, although effective, is often broad spectrum in nature,
eliminating both pathogenic microorganisms as well as the reducing the concentrations of
normal commensals in the oral cavity. As a result, opportunistic microorganisms may
become resistant, or at least thrive due to a change in the ecosystem, leading to oral yeast
infections or thrush, 1 or an inability to eradicate pathogens with standard antibiotics.
There is a clear advantage of narrow-spectrum antimicrobial therapy with regards to
treatment of periodontal diseases where a group of offending organisms has been
identified 2and could be targeted without affecting normal commensals or potential
opportunistic organisms like Candida albicans.
These peptides are fast acting, broad-spectrum, do not induce bacterial resistance,
are easy to synthesize in large quantities, work in synergy with other antimicrobial
agents, and have very few side effects, characteristics that are highly desirable for
pharmacological development 3.
Sheep myeloid antimicrobial peptide SMAP28 was synthesized. Specific E. coli
and P. gingivalis antibodies were isolated from rabbit antisera using an immunoaffinity
column and concentrated using a protein G column. A maleimide linker was attached to
2
one or more of the eleven free amino groups on SMAP28. The minimum inhibitory
concentrations (MIC) of SMAP28 alone and the SMAP28+maleimide linker (preconjugation) complex were determined using broth microdilution assays. The
SMAP28+maleimide linker complex was then conjugated to E. coli and P. gingivalisspecific antibodies. The selective activity of the E. coli-targeted conjugate was assessed
by adding it to an artificially generated microbial community containing equal
concentrations of four common laboratory strains of aerobic bacteria: E. coli,
Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus. These
microorganisms were selected due to differences in size and color of the colonies when
grown on agar plates, thus allowing easy counting. At 0, 30, 60, 120, and 240 minutes,
aliquots were removed and grown on agar. After 24 hours, colony forming units were
counted. Selective activity of the P. gingivalis-targeted conjugate was also assessed in an
artificially generated microbial community containing equal concentrations of three
bacteria associated with periodontal disease: P. gingivalis, Aggregatibacter
actinomycetemcomitans, and Peptostreptococcus micros. At 0, 10, 20 and 30 minutes,
aliquots were removed and grown anaerobically on blood agar. Colony forming units
were counted at one week. Control assays using SMAP28 alone and IgG alone were also
completed.
If species-specific killing of microorganisms in a community of microorganisms
can be obtained, it would potentially open new therapeutic avenues for treatment and
prevention of infectious diseases. Epidemiological studies indicate that 5-20% of the
general population suffers from severe forms of periodontitis. 4 This approach may be an
initial step for developing a selective antimicrobial agent capable of eliminating specific
periodontal pathogens (e.g., P. gingivalis) from patients with periodontal disease without
harming the normal commensal population.
3
CHAPTER II
REVIEW OF THE LITERATURE
Innate Immune System
The innate immune system in mammals and other vertebrates plays a number of
crucial roles 5, 6. Specifically, it: (a) provides first line recognition of microorganisms 7,
8
, (b) contains the infection prior to the induction of adaptive immune responses, which
can take 4 to 5 days, and (c) controls the activation of adaptive immunity and determines
the type of effector responses that are appropriate for the infecting pathogen 5, 9, 10. These
functions critically depend on the ability of the innate immune system to detect the
presence of infectious microorganisms and to induce a set of endogenous signals such as
secretion of cytokines by macrophages and natural killer cells 11, 12, attachment of a
complement protein to antigen 13-15, preferential uptake of microbial antigen by lectin
receptors on cells that specialize in presenting antigen to lymphocytes 16, and
maintenance of antigen-reactive T lymphocytes 12, 17. Co-stimulatory membrane proteins
B7.1 (CD80) and B7.2 (CD86) on antigen-presenting cells interact with receptors on T
cells (CD28) to deliver a signal separate from that delivered by the T cell receptor with
peptide complexed to major histocompatability complex class II molecules on the surface
of antigen-presenting cells 18-21. The finding that the human homologue of Toll induces
B7.1 on macrophages required for lymphocyte activation also places it in the
communication network between innate and adaptive immunity 5, 12, 22.
Innate host defense elements range from simple inorganic molecules (e.g.,
hydrochloric acid, peroxidases, and nitric oxide) to complement 14, C-reactive protein,
extracellular collectins (lung surfactant proteins A and D, conglutinin, and collectin-43)8,
and germline-encoded receptors recognizing pathogen-associated molecular patterns
(e.g., lipopolysaccharide, lipoteichoic acid, lipoarabinomannan, mannans, etc.)7, 22-24.
Epithelial cells, intraepithelial lymphocytes, phagocytic cells, and NK cells are also
included as innate defenders 10, 25-27.
4
Antimicrobial proteins and peptides are innate immune elements found in human
oral mucosa and oral secretions and include lysozyme 28, 29, lactoferrin 30, 31, acidic
proline-rich proteins 29, 32, plunc 33-35, salivary mucin glycoprotein 30, 36, histatin 29, 32, 37,
HBD1 38, 39, HBD2 38, 40, 41, HBD3 42, 43, HBD4 44, and hCAP18/LL-37 41, 45. Table A1
outlines innate peptides found in mucosa and mucosal secretions and Table A2 lists those
peptides found in oral mucosa and oral secretions. In additional to their direct
antimicrobial activity, many of these proteins and peptides participate in other aspects of
innate immunity 46, 47.
Adaptive host defense elements involve antibodies (humoral immunity) and/or
immuno-reactive lymphocytes (cell mediated immunity) 48. Antibodies agglutinate or
precipitate pathogens or their antigens, enhance phagocytic engulfment of bacteria or
their antigens, block or prevent bacterial attachment to susceptible cells on mucosal
surfaces, neutralize bacterial toxins or block their interaction with specific cell targets,
and activate the complement cascade, which facilitates complement-mediated bacterial
lysis, chemotaxis of phagocytes, and phagocytic engulfment of killed bacteria or their
antigens.
Cell-mediated immune responses utilize various subsets of T lymphocytes that are
activated and develop into cytotoxic T lymphocytes and T helper cells of the TH1 and
TH2 subsets 48-50. Cell-mediated immune responses are important to clear infections
where bacteria grow or multiply intracellularly. This is particularly important in
infections caused by Mycobacterium tuberculosis 51, Brucella spp., 52, and others 53.
Recovery is associated with development of a pronounced cell-mediated immune
response, even though cell-mediated immunity also contributes to the pathology of the
disease. Cytotoxic lymphocytes kill cells that are harboring intracellular bacteria as long
as the infected cell is displaying a microbial antigen on its surface. Lymphocytes
produce lymphokines that are "helper" factors for development of B-cells into antibodysecreting plasma cells and also produce cytokines which stimulate the differentiation of
5
effector T lymphocytes and the activity of macrophages 54. Macrophages are also
involved in expression of cell-mediated immunity since they become activated by gamma
IFN produced in a cell-mediated immune response. Activated macrophages have
increased phagocytic potential and release soluble substances that cause inflammation
and destroy many bacteria and other cells.
Cathelicidins are a group of antimicrobial peptides that differ greatly in their
sequences, structures, and sizes 55, 56. Generally, they all have a high content of the basic
amino acids arginine and lysine 57, 58; a common N-terminal preproregion of about 100
residues that is homologous to the cysteine protease inhibitor cathelin 59, and a highly
variable C-terminus that contains the cationic antimicrobial domain. After synthesis, the
C-terminus is cleaved off, forming the mature antimicrobial peptide. Cathelicidins are
often found in the secondary granules of neutrophils and expressed early in myeloid
differentiation. The human cathelicidin LL37 is present in saliva and gingival tissues
(Table A2) and has antimicrobial activity.
Sheep myeloid antimicrobial peptide SMAP28, SMAP29, and SMAP34 are
sheep-derived cathelicidins 60. They have potent activity against a variety of
microorganisms and are potential candidates for the therapeutic treatment of acute and
chronic respiratory infections including P. aeruginosa associated with chronic respiratory
inflammation in cystic fibrosis 58, 61-65. They are also active against many oral bacteria
and clinically important oral yeasts including A. actinomycetemcomitans, Fusobacterium
nucleatum, P. gingivalis, Streptococcus sanguis, Candida krusei, Candida tropicalis and
C. albicans 66, 67 SMAP28 is a 28 amino acid residue cationic peptide with an average
mass of 3,198.95 Da, net charge of +11, and theoretical pI of 12.31 (Table A3).
The structure and composition of SMAP29 (also known as SC5) was first
deduced from sheep myeloid DNA 62, 63 and later synthesized to assess its antimicrobial
activity 58, 61, 64. SMAP29 has broad-spectrum antimicrobial activity (Table A3), is active
in both low and high ionic strength conditions 58, and induces significant morphologic
6
alterations in bacterial surfaces 64, 68. SMAP28-AMID is thought to be the native form of
the peptide 63, 64 and synthesized SMAP28-AMID and many synthetic congeners are
antimicrobial 68. The antimicrobial activity of this group is strongly dependent upon their
size, amino acid composition, and charge 68.
SMAP29 induces extensive ultrastructural damage in bacterial cells 65, 68.
SMAP29 induces bacterial membrane blebs within 1 minute, kills P. aeruginosa within 1
hour, and causes a dose-dependent, reversible decrease in transepithelial resistance within
5 hours 65. In transmission electron microscopy, SMAP29 induces damage characterized
by rough surfaces containing extracellular debris and outer membranous blebs, thickened
cell walls, and electron dense cytoplasmic material. Interestingly, the ultrastructural
changes induced by SMAP29 were different from that induced by other peptides like
CAP18 68. In addition to severe membrane changes, SMAP29 rapidly penetrated the
outer and inner membranes and entered into the bacterial cytoplasm as early as baseline
time 0 68.
Microbial Community Associated with
Periodontal Disease
Microorganisms rarely grow as a single species in nature. Rather they live as
members of extensive microbial communities in unique niches 69, often as biofilms 70.
These niches include body surfaces and cavities open to the environment: the nasal
cavity, the oral cavity, the respiratory tract, the gastrointestinal tract, and the urogenital
tract 71. These mucosal surfaces contain extensive communities with a large diversity of
phylotypes. In the oral cavity, for instance, the subgingival microbial community is
estimated to contain more than 700 phylotypes 72; saliva can contain ~108 total
microorganisms/ml 73, plaque can contain ~1011 total microorganisms/gm 73, and the
microbiota of the subgingival environment differs from the microbiota of the tongue 74.
Periodontal disease is a polymicrobial infection 75. Polymicrobial infections are
characterized by the presence of several microbial species. In the oral cavity, the
7
subgingival microbial community contains commensals and opportunistic pathogens with
the potential to cause periodontal or systemic diseases. They include Veillonella atypica,
Actinomyces naeslundii, F. nucleatum, A. actinomycetemcomitans, P. gingivalis,
Treponema denticola, numerous Streptococcus spp., Haemophilus parainfluenzae,
Propionibacterium acnes, P. micros, and P. intermedia 76, 77. These bacteria occur in
distinct subgingival microbial profiles in a) individuals with refractory periodontitis, b)
periodontally healthy individuals, c) periodontally well-maintained elder individuals, and
d) untreated periodontitis subjects 78. Profile I is characterized by high proportions of
‘yellow’ and ‘green’ complex species. These include Streptococcus sanguis,
Streptococcus oralis, Streptococcus intermedius, Streptococcus gordonii, Streptococcus
mitis, and Streptococcus constellatus. Profile II is characterized by high proportions of
‘orange’ and ‘purple’ complex species. These include Fusobacterium nucleatum ss
vincentii, P. micros, Prevotella nigrescens, F. periodonticum, and Prevotella intermedia.
Profile III is characterized by high total counts and counts of Actinomyces and ‘purple’
complex species. Profile IV is characterized by high proportions of ‘red’ and ‘orange’
complex species. These include F. nucleatum ss vincentii, P. micros, P. nigrescens, F.
nucleatum ss polymorphum, Campylobacter showae, Fusobacterium periodonticum, F.
nucleatum ss nucleatum, Prevotella intermedia, P. gingivalis, Treponema denticola, and
B. forsythus.
Antibiotic therapy has been a hallmark of periodontal disease treatment for many
years. 79 Systematic reviews of this literature have shown that systemically administered
antibiotics provided a clear clinical benefit in terms of mean periodontal attachment level
gain post-therapy when compared with groups not receiving these agents. 80, 81
Combined with non-surgical and surgical treatment, chemotherapeutics provide another
mode for eliminating pathogenic microorganisms from patients suffering from
periodontal disease. However, they are not without problems.
8
Antibiotic therapy, although effective, is often broad spectrum in nature,
eliminating both pathogenic microorganisms as well as the reducing the concentrations of
normal commensals in the oral cavity, gastrointestinal tract, and urogenital tract. As a
result, opportunistic microorganisms may become resistant, leading to oral yeast
infections or thrush; vaginal yeast infections, and gastrointestinal infections including
diarrhea 1. The advantage of narrow-spectrum antimicrobial therapy is clear with regards
to periodontal disease where a group of offending organisms has been identified and
could be targeted without affecting normal commensals or potential opportunistic
organisms like C. albicans.
Resistance to antibiotics has become a significant problem when treating bacterial
infections. Qiu, et al. reported on the ability of a staphylococcal pheromone fused to a
channel-forming peptide to kill vancomycin-resistant E. faecalis 82. Vancomycin has
been increasingly used to treat enterococcal infections, which, through conjugal transfer,
has led to multidrug resistance among those pathogens. Periodontal disease causing
microorganisms have also recently shown increasing resistance to antibiotic therapy. 83
Treatment of these antibiotic-resistant bacteria will require antimicrobial agents with a
different mechanism of action than standard antibiotics.
It is thought that natural antimicrobial peptides or synthetic congeners with potent
in vitro and in vivo antimicrobial activity are the next potential generation of
pharmaceuticals for the treatment of antibiotic-resistant bacterial infections or septic
shock 3, 84, 85. They could possibly suppress microbial infections in vivo if given
topically, orally, or systemically, similar to conventional antibiotics. These peptides are
fast acting, broad-spectrum, do not induce bacterial resistance, are easy to synthesize in
large quantities, work in synergy with other antimicrobial agents, and have very few side
effects, characteristics that are highly desirable for pharmacological development 3.
Although active against a variety of microorganisms in vitro, many peptides cannot be
used to prevent or treat microbial infections and sepsis in vivo. Some have undesirable
9
characteristics that include hemolysis and cytotoxicity for host cells of some species and
decreased activity in host environments containing high ionic strength conditions or
serum.
Novel uses of antimicrobial peptides have been considered to prevent or treat
microbial infections and sepsis 3. For example, antifungal peptides have been linked to
recombinant antibodies and shown to provide resistance to fungal diseases in plants 86.
Fusarium is a notorious plant pathogen that produces human harming mycotoxins, and is
responsible for crop destroying diseases such as cereal scab. Current treatment is limited
to chemical control, similar in theory to broad-spectrum antibiotic therapy in humans.
This chemical control can have harmful environmental effects, thus driving the effort
towards development of a narrow-spectrum agent. Another model has been proposed by
Eckert where the caries causing bacteria Streptococcus mutans was targeted by
combining a pheromone produced by S. mutans to a known antimicrobial peptide 87. Due
to their many benefits and relatively few side effects, narrow-spectrum antimicrobial
agents provide an exciting avenue for possible elimination of pathogenic
microorganisms.
If targeted killing of specific microorganisms in a community of microorganisms
can be obtained, it would potentially open new therapeutic avenues for treatment and
prevention of infectious diseases. Epidemiological studies indicate that 5-20% of the
general population suffers from severe forms of periodontitis 4. This approach may be an
initial step for developing a selective antimicrobial agent capable of eliminating specific
periodontal pathogens (e.g., P. gingivalis) from patients with periodontal disease without
harming the normal commensal population.
10
CHAPTER III
SIGNIFICANCE AND SPECIFIC AIMS
Hypothesis
My central hypothesis was that SMAP28, conjugated to E. coli or P. gingivalisspecific IgG antibodies will selectively kill E. coli or P. gingivalis, in mixed aerobic or
anaerobic microbial cultures, respectively. To test this hypothesis, the following specific
aims were proposed.
Aim 1: Determine the Antimicrobial Activity
of SMAP28 Conjugated to Antibody Reagents
My working hypothesis is that SMAP28 with an attached maleimide linker will
retain its antimicrobial activity comparable to SMAP28 alone. I further hypothesize that
SMAP28 with an attached maleimide linker and an attached antibody will retain its
antimicrobial activity comparable to SMAP28 alone.
Aim 2: Determine the Antimicrobial Specificity
of SMAP28 Conjugated to Organism-Specific
Antibodies in a Community of Microorganisms
My working hypothesis is that antimicrobial activity will be specific, not broadspectrum, and targeted to the specificity of the antibody.
This project is innovative. If species-specific killing of microorganisms in a
community of microorganisms can be accomplished, it would potentially open new
therapeutic avenues for treatment and prevention of infectious disease. This technique
may be an initial step for developing a selective antimicrobial agent capable of
eliminating specific periodontal pathogens (e.g., P. gingivalis) from patients with
periodontal disease without harming the normal commensal population.
11
CHAPTER IV
MATERIALS AND METHODS
Synthesis of SMAP28
An overview of the methodology of this project is presented (Figure A1).
SMAP28 was synthesized by NeoMPS, Inc. (San Diego, CA). Stock solutions (1.0
mg/ml) of SMAP28-AMID were prepared in 0.01 M sodium phosphate buffer, pH 7.2
with 145 mM NaCl (PBS). A schematic diagram of SMAP28 is shown (Figure A2). The
purity of SMAP28 was assessed by MALDI-TOF (Figure A3) (High Resolution Mass
Spectrometry Facility, University of Iowa, Iowa City, IA) and reversed phase-high
performance liquid chromatography (HPLC) (Figure A4) and was found to be within the
specifications of the manufacturer.
Bacterial Species and Growth Conditions
My pilot work to develop a ‘targeted’ antimicrobial peptide utilized aerobic,
laboratory strains of bacteria. E. coli ATCC 12795, P. aeruginosa ATCC 47085, S.
marcescens ATCC 14756, and S. aureus ATCC 29213 were purchased from the
American Type Culture Collection (Manassas, VA) and grown aerobically at 37º C in
Trypticase Soy Broth (TSB). Stock cultures were plated on Trypticase Soy Agar (TSA)
containing 5% defibrinated sheep blood (Remel, Lenexa, KS) and stored at 4º C. Cultures
were plated on blood agar to quantitate microbial numbers in the assays below. P.
gingivalis strain 381 was obtained from Ann Progulske-Fox (Department of Oral
Biology, University of Florida, Gainesville, FL) and A. actinomycetemcomitans FDC-Y4
and P. micros were obtained from Janet M. Guthmiller (Department of Periodontics and
Dows Institute for Dental Research, The University of Iowa, Iowa City, IA). All three
organisms were grown as previously described at 37º C in an atmosphere that contained
85% N2-10% H2-5% CO2 88. P. gingivalis strain 381 was grown in tryptic soy broth
(Difco Laboratories, Detroit, MI) supplemented with 5 µg/ml hemin (Sigma, St. Louis,
12
Mo.) and vitamin K (Sigma, St. Louis, Mo.); and A. actinomycetemcomitans, was grown
in tryptic soy broth supplemented with 0.6% yeast extract (Difco Laboratories, Detroit,
MI); and P. micros was grown in brain heart infusion (Difco Laboratories, Detroit, MI)
supplemented with 0.5% neopeptone and 5 µg/ml of hemin. Cultures were plated on
anaerobic blood agar (CDC formulation, Remel, Lenexa, KS) to assess purity and to
quantitate microbial numbers in the assays below.
Preparation of Antiserum
E. coli was grown aerobically for 24 hours in TSB and P. gingivalis was grown
anaerobically for 48 hours in TSB supplemented with hemin and vitamin K. Bacterial
cells were pelleted from the TSB at 6,000 x g (6,831 RPM) for 10 minutes at 4° C. The
bacterial cell pellets were washed twice in 140 mM NaCl with 0.3% formalin each time
pelleting the cells at 6,000 x g (6,831 RPM) for 10 minutes at 4° C. After the second
wash, the bacterial cells were suspended in 25 ml of 140 mM NaCl with 0.3% formalin
and incubated overnight at room temperature. The bacterial suspension was adjusted to
0.108 OD at 600 nm by adding cells drop wise to a tube of 140 mM NaCl with 0.3%
formalin in the spectrophotometer (Spectronic 20D+, Thermo Fisher Scientific, Inc.,
Waltham, MA). Suspensions of bacteria at 0.108 OD at 600 nm consistently contain 1.0
x 108 CFU/ml. Each suspension was adjusted to contain 4.0 X 108 CFU/ml by putting 20
ml of the 1.0 X 108 CFU/ml into a tube, pelleting the cells and suspending them into 5 ml
of 140 mM NaCl with 0.3% formalin.
A water-in-oil emulsified bacterin was prepared as follows. Solution A
containing 5 ml of bacterial suspension (4 X 108 CFU/ml) in 140 mM NaCl with 0.3%
formalin, solution B containing 0.5 ml of muramyl dipeptide (1 mg/ml in 140 mM NaCl
with 0.3% formalin), solution C containing 4.5 ml of 140 mM NaCl with 0.3% formalin,
and solution D containing 10 ml of Freunds incomplete antigen (FIA) were all chilled on
ice. Solutions A, B, and C were combined and added down the sonicator probe into D
13
while D was being sonicated. Each 1 ml dose contained 1.0 X 108 CFU and 25 ug MDP
in a 50% oil emulsion.
Four rabbits were immunized 7 times over a 13 week period with these whole cell
bacterins (IMGENEX Corp., San Diego CA). Pre-bleed serums were collected before
immunization and antiserum samples were collected at weeks 9, 11, and 13. Antibody
titers were determined by ELISA as described below.
Isolation of Microorganism-Specific Antibody
Rabbit IgG antibody to whole cell surface-specific antibodies of E. coli or P.
gingivalis were isolated from rabbit antiserum by affinity chromatography. The
immunoaffinity column was prepared using the Pierce AminoLink® plus immobilization
kit (Product Nos. 44894 and 20394, Pierce, Rockford, IL) to link formalin fixed whole
cells of E. coli or P. gingivalis to the coupling gel. This kit immobilizes proteins and
other ligands through primary amines (–NH2).
10 ml of formalin fixed E. coli or P. gingivalis cell suspensions were washed 3
times in 0.1 M sodium citrate, 0.05 M sodium carbonate buffer, pH 10 and pelleted by
centrifugation at 4,629 x g for 10 minutes each time. The final suspension was adjusted
to contain 1 X 109 CFU/ml. The AminoLink® Plus coupling gel was equilibrated with 5
ml of 0.1 M sodium citrate, 0.05 M sodium carbonate buffer, pH 10. Three ml citratecarbonate buffer, pH 10 containing 3 X 109 CFU of E. coli or P. gingivalis was added and
mixed with the gel for 4 hours (by end-over-end rocking). The coupling gel was then
washed with 5 ml of 0.1 M phosphate, 0.15 M NaCl, pH 7.2 buffer. Two ml of
phosphate buffered saline, pH 7.2 containing 40 µl of 5 M sodium cyanoborohydride in
0.01 M NaOH was added and mixed with the gel overnight (by end-over-end rocking).
The gel was washed with 4 ml of 1.0 M Tris HCl 0.05% NaN3, pH 7.4 (quenching
buffer). Two ml quenching buffer containing 40 µl of sodium cyanoborohydride was
added and the gel was mixed for 30 minutes (by end-over-end rocking). The gel was
allowed to settle in the column and washed first with 15 ml of 1 M NaCl containing
14
0.05% NaN3 and then with 5 ml of phosphate buffered saline, pH 7.2. The optical
density of the starting bacterial suspensions (600 nm) and the finished suspensions were
used to estimate the coupling efficiencies of 57.4% for E. coli whole cells and 33.9% for
P. gingivalis whole cells to the AminoLink® Plus coupling gel.
1.5 ml of antiserum was added to the column and allowed to completely enter the
gel bed. 0.2 ml of 0.1 M phosphate, 0.15 M NaCl, pH 7.2 was added and allowed to
enter the gel bed. 0.5 ml of 0.1 M phosphate, 0.15 M NaCl, pH 7.2 was added and the
column was incubated for 1 hour at 26o C. The column gel was washed with 12 ml of 0.1
M phosphate, 0.15 M NaCl, pH 7.2. The E. coli (Figure A5) or P. gingivalis (Figure
A6)-specific IgG antibodies were eluted with 8 ml of 0.1 M glycine HCl, pH 2.5. 1 ml
fractions were collected. The pH of each fraction was neutralized by adding 50 µl of 1 M
phosphate buffer/ml of eluted material. Protein content was determined by measuring
absorbance of the eluted material at 280 nm.
E. coli or P. gingivalis-specific IgG antibodies were isolated from the affinity
column eluted fractions and were then concentrated using a HiTrap protein G HP
cartridge (No. 17-0405-01, Pierce, Rockford, IL). Using a syringe attached to the luer
lock, the cartridge gel was equilibrated by passing 10 ml of 20 mM sodium phosphate
buffer, pH 7.0 through it. Fractions were diluted in 20 mM sodium phosphate buffer, pH
7.0 and slowly passed through the cartridge. The filtrate was collected and passed slowly
through the cartridge a second time. The cartridge gel was washed by passing 10 ml of
20 mM sodium phosphate buffer, pH 7.0 through it. 500 µl of 1.0 M Tris-HCl buffer, pH
9.0 was put into a collection tube. The bound IgG antibody was eluted from the
immobilized whole cells of E. coli and P. gingivalis by passing 5 ml 0.1 M glycine-HCl
buffer, pH 2.7 through the column and into the collection tube. The pH was adjusted to
7.0 and the concentration of protein was determined (Figures A5 and A6).
15
Whole Cell ELISA Analysis
For preparation of a whole-cell bacterial antigen, E. coli, P. aeruginosa, S.
marcescens, and S. aureus were grown aerobically in TSB for 24 hours. P. gingivalis
was grown anaerobically in TSB supplemented with hemin and vitamin K for 48 hours as
described above. Bacterial cells were pelleted from the broth at 6,000 x g for 10 minutes
at 4o C. The bacterial cell pellets were washed twice in 140 mM NaCl with 0.3%
formalin each time pelleting the cells at 6,000 x g for 10 minutes at 4o C. After the
second wash, the bacterial cells were suspended in 25 ml of 140 mM NaCl with 0.3%
formalin and incubated overnight at room temperature.
The bacterial suspension was adjusted to 0.108 OD at 600 nm by adding aliquots
of each bacterial suspension to individual tubes of 0.3% formalin (in distilled water) in
the spectrophotometer (Spectronic 20D+, Thermo Fisher Scientific, Inc., Waltham, MA).
This suspension contained 1.0 X 108 CFU/ml. 100 µl of the cell suspension was put into
Immulon 1 microtiter wells (this is equal to 1.0 x 107 CFU/well) and dried overnight at
26o C.
The whole-cell bacterial antigen was incubated with 0.01 M Tris buffer
containing 0.145 M NaCl, 1.0% fish gelatin, and 0.05% (blocking buffer) for 30 minutes
at 26o C. The buffer was aspirated from the wells by vacuum. Blocking buffer was
again added and incubated an additional 30 minutes at 26o C. The blocking buffer was
aspirated from the wells by vacuum. 100 µl of blocking buffer was added to wells in
columns 2-12. 20 µl of rabbit preimmune and antiserum was added to 180 µl blocking
buffer in wells of column 1 and diluted 2-fold. Control wells contained only 100 µl of
blocking buffer. After incubation for 1 hour at 26o C, all wells were washed twice with
blocking buffer. 100 µl/well of peroxidase labeled goat anti-Rabbit IgG antibody (1
µg/ml, KPL, Inc., Gaithersburg, MO No. 074-1506) was added. After incubation for 1
hour at 26o C, all wells were washed twice with blocking buffer. 100 µl/well peroxidase
developing reagent (TMB microwell peroxidase substrate, 1 component, No. 53-00-02,
16
KPL, Inc., Gaithersburg, MD) was added and incubated for 5 minutes at 26o C (or until
color appeared). Then 100 µl stop reagent (TMB stop, 1 component, KPL No. 50-85-05)
was added. The optical density of the wells in the plate were determined in the
spectrophotometer (PowerWavex, BioTek Instruments, Inc., Winooski, VT) at 450 nm.
The values of the blank were subtracted from the test values by the
spectrophotometer. Linear regression of the log2 of dilution (x axis) was graphed against
the optical density of the dilution (y axis) to determine the slope of line. Antibody titer
was calculated as the log2 dilution value at the 0.1 optical density intercept 68.
SMAP28 and IgG Conjugation Procedure
SMAP28 was conjugated to affinity purified IgG antibody by linking the amine
group (-NH2) of SMAP28 to the sulfhydryl group (-SH) of IgG antibody with the linker
maleimide using the Controlled Protein-Protein Cross-Linking Kit (No. 23456, Pierce,
Rockford, IL)(Figure A7).
SMAP28 contains 11 free amine groups, of which 4 are available for linker
attachment, one at each lysine or arginine residue. To prepare the maleimide activated
SMAP28 (SMAP28+maleimide linker), a 1.0 mg/ml suspension of SMAP28 was made in
PBS, pH 7.2. A 5-fold molar excess of Sulfo-SMCC Cross-linking agent was added and
the suspension was incubated for 30 minutes at 26o C. Un-reacted maleimide reagents
were removed by passing the SMAP28+maleimide linker over a dextran desalting
column and collecting the 1 ml fractions. Peptide content was determined and fractions
containing most of the SMAP28+maleimide linker were pooled. The number of linkers
and mass of SMAP28+maleimide linker were determined by Matrix-Assisted Laser
Desorption Time-of-Flight (MALDI-TOF) Mass Spectrometry (High Resolution Mass
Spectrometry Facility, The University of Iowa, Iowa City, IA) and the antimicrobial
activity of SMAP28+maleimide linker was determined in the radial diffusion assay
described below.
17
Two options (IIA and IIB) were performed to prepare sulfhydryl modified IgG
antibody. In option IIA, the IgG antibody was suspended in PBS, pH 7.2, mixed with the
Kit Conjugation/Activation buffer and activated Kit Immobilized–Reductant, and
incubated at 60 minutes at 37o C. The mixture was centrifuged to pellet the Kit
Immobilized–Reductant. The supernatant contained the sulfhydryl-modified IgG
antibody for the protein-protein cross linking procedure with SMAP28+maleimide linker.
In option IIB, sulfhydryl groups were added by reaction with SATA. The IgG
antibody was suspended in PBS, pH 7.2 (1 ml) and a 10 molar excess of SATA solution
(SATA/dimethylformamide) was added and incubated for 20 minutes at 26o C. 100 µl of
hydroxylamine HCl solution was added to the SATA-modified IgG antibody and the
mixture was incubated for 2 hours at 26o C. Unbound reagents were removed by passing
the de-protected sulfhydryl-IgG antibody solution over a desalting column. PBS-EDTA
was used to elute sulfhydryl-IgG antibody and 1 ml fractions were collected. Protein
concentrations were determined at A280 and fractions containing most of the sulfhydrylIgG antibody were pooled.
SMAP28+maleimide linker and sulfhydryl-IgG antibody were mixed in
approximately equal molar amounts and incubated for 60 minutes at 26o C. This was
called the IgG-SMAP28 conjugate (Figure A7).
Mass Spectrometry
Matrix-Assisted Laser Desorption Time-of-Flight (MALDI-TOF) Mass
Spectrometry was performed at the High Resolution Mass Spectrometry Facility (The
University of Iowa, Iowa city, IA). Proteins with masses of 2,867.81 Da and 5,734.61 Da
were used as internal calibration standards.
HPLC
SMAP28 and SMAP28+maleimide linker were separated on an Acclaim 300,
C18, 3 µm analytical column (Dionex Corp., San Francisco, CA) using a Summit HPLC
system (Dionex) with Chromeleon software and eluted with a gradient of acetonitrile (0-
18
100%) in 0.1% TFA. Fractions were collected in a FC 144 fraction collector, pooled with
similar fractions of previous runs, and dried overnight by rotary evaporation under
vacuum at room temperature.
Spot Blot
Spot blots (Figure A8) were used to confirm the presence of SMAP28 and the
presence of IgG antibody in the SMAP28-IgG antibody conjugates. Strips of ImmobilonP transfer membranes were rinsed briefly in absolute methanol, rinsed in distilled water,
and soaked in buffer. 1 µl of the E. coli IgG-SMAP28 conjugate, E. coli rabbit IgG, P.
gingivalis IgG-SMAP28 conjugate, P. gingivalis IgG, ovalbumin+SMAP28 complex,
and ovalbumin were placed on each strip, allowed to dry and soaked in 0.01 M Tris
buffer containing 0.145 M NaCl, 1.0% fish gelatin, and 0.05% (blocking buffer)
overnight at 26o C.
To detect rabbit IgG antibody in the conjugates, the strips were incubated with
peroxidase labeled goat anti-rabbit antibody (KPL No. 074-1506, 1.0 ug/ml) for 1 hour at
37o C on a rocking platform, washed with blocking buffer (3 rinses), and incubated with
developing reagent (KPL No. 50-73-014, CN Peroxidase Substrate) for 5 minutes (or
until color appeared).
To detect SMAP28 in the conjugates, goat anti-SMAP28 antibody 68(diluted 1:20
in blocking buffer) was added and incubated for 1 hour at 37o C, washed with blocking
buffer (3 rinses), incubated with peroxidase labeled rabbit anti-goat antibody (KPL No.
14-13-06, 1 ug/ml) for 1 hour at 37o C on a rocking platform, washed with blocking
buffer (3 rinses), and incubated with developing reagent (KPL No. 50-73-01, 4 CN
Peroxidase Substrate) for 5 minutes (or until color appeared).
Radial Diffusion Antimicrobial Assay
The minimal inhibitory concentrations (MIC) of SMAP28 and
SMAP28+maleimide linker were determined with the radial diffusion assay 88, 89. Cells
were grown to mid-log phase in their appropriate media overnight, centrifuged at 6,000 x
19
g for 10 minutes, rinsed with fresh medium, and suspended in 10 mM sodium phosphate,
pH 7.4. An underlay gel was prepared, which consisted of a mixture of 1% agarose in 10
mM sodium phosphate (pH 7.4) and contained 4 x 106 bacteria. The mixture was
immediately poured into a square Petri dish (100 X 100 X 15 mm, Fisher scientific) and
allowed to solidify. 3 mm diameter wells were punched in the agar and solutions of
SMAP28 or SMAP28+maleimide linker were diluted in 0.01% acetic acid from 112 to
0.8 µg/ml and 5 µl were added to the 3 mm holes punched in the agar. Control wells
contained only 10 mM sodium phosphate buffer and sterile media. The plates were then
incubated under the appropriate aerobic or anaerobic conditions at 37° C for 3 hours to
allow for peptide diffusion. Ten milliliters of a 1% agar overlay gel containing medium
specific for the organism tested was then poured over the first agar layer to provide
nutrients for the cells. The plates were incubated again for 24 to 48 hours. Antimicrobial
activities were expressed as minimal inhibitory concentration, which was calculated as
previously described 88. Zones of inhibition were recorded with a Boley gauge as radial
diffusion units (zone of inhibition - well diameter x 10). The x intercept was obtained
from the relationship between radial diffusion units versus log10 peptide concentration as
determined after regression 68.
Conjugate Specific Killing
in a Mixed Culture Suspension
The specificity and activity of the E. coli IgG-SMAP28 conjugate was arbitrarily
determined in a mixed culture containing E. coli, P. aeruginosa, S. marcescens, and S.
aureus (Figure A9). These organisms were grown for 3 hours in Mueller Hinton broth at
37o C and diluted in saline to a density containing approximately 1 X 108 CFU/ml (0.108
O.D., 600 nm, Spectronic 20D+, Thermo Fisher Scientific, Inc., Waltham, MA). The
culture was diluted 10-fold to contain 104 (CFU/ml). One ml of P. aeruginosa, 1 ml of S.
marcescens, 4 ml of S. aureus, and 2 ml of E. coli were added to 2 ml of saline. The
variable concentrations were done to provide equal numbers of CFUs for counting. 0.6
20
ml of this mixed culture bacterial suspension was mixed with 0.6 ml of conjugate. At
0.0, 0.5, 1.0, 2.0 and 4.0 hours, 50 µl was removed and plated onto agar plates and
incubated at 37o C. At 24 hours, bacteria colonies were counted. E. coli grows as a
grayish colony around 2 mm in diameter, S. aureus grows as a yellow colony 1 mm in
diameter, P. aeruginosa grows as a pinpoint gray colony, and S. marcescens is an easily
visible red colony.
The specificity and activity of the P. gingivalis IgG-SMAP28 conjugate was
determined in a mixed culture containing P. micros, A. actinomycetemcomitans, and P.
gingivalis (Figure A10). These organisms were grown for 48 hours in their respective
broths at 37o C under anaerobic conditions and diluted in reduced saline to a density
containing approximately 1 X 108 CFU/ml (0.108 O.D., 600 nm, Spectronic 20D+,
Thermo Fisher Scientific, Inc., Waltham, MA). Each culture was diluted 10-fold to
contain 104 (CFU/ml). 1 ml each of the suspensions was added to 7 ml of saline. 0.6 ml
of this mixed culture bacterial suspension was mixed with 0.6 ml of conjugate. At 0, 10,
20, and 30 minutes 50 µl was removed and plated onto anaerobic blood agar (CDC
formulation, Remel, Lenexa, KS) and incubated at 37o C. After 9 days, bacterial colonies
of differing morphologies, sizes, and color were counted by 3 different observers.
Counting was done by hand using a differential counting device and illuminating the
plates from underneath with a standard dental radiograph reader.
Statistical Analyses
The experiment was divided into four treatments at different timepoints:
Conjugate IIA, Conjugate IIB, P. gingivalis antibody control, and pre-immune IgG
antibody control, all at 0, 10, 20, and 30 minutes. Sixteen total treatments were defined
for the study. Each treatment had three plates, and the experiments were performed on
two different days. Since day to day differences could affect the assays, each day was
considered a block. A multi-factorial analysis of variance model was used to analyze the
effect of treatments, time and day on the number of CFUs, as well as to assess two- and
21
three- way interactions. Logarithm transformations of the form Log10(CFU+1) were
used as needed to conform to model assumptions such as normality and
homoscedasticity.
Intraclass correlation coefficients were used to assess rater reliability. Perfect
agreement corresponded to a correlation coefficient of 1. For this analysis both CFU and
base 10 log transformations were used. The general linear model procedure was
performed using SAS statistical software to assess time, treatment, and day effects and
interactions. For multiple comparisons of the four treatments, Tukey’s studentized range
test was used in conjunction with an overall Type I error level of 0.05. A simple linear
regression model and Spearman Correlation was used to assess the relationship between
the number of CFUs and time for a given treatment and microorganism.
22
CHAPTER V
RESULTS
E. coli and P. gingivalis IgG Antibodies
Were Isolated and Purified
1 X 108 CFU whole cells of E. coli or P. gingivalis with 25 µg muramyl
dipeptide/ml 140 mM NaCl with 0.3% formalin in a 50% emulsion of Freund’s complete
adjuvant induced the production of E. coli and P. gingivalis specific antibodies in rabbits.
ELISA determination of antibody titers showed that there were preimmune antibody
titers to E. coli (mean log2 titer 6.5), P. aeruginosa (mean log2 titer 6.2), S. marcescens
(mean log2 titer 7.3), and S. aureus (mean log2 titer 9.9). Nine weeks later, immune
antiserums contained elevated antibody titers to E. coli (mean log2 titer 11.4) and natural
titers to P. aeruginosa (mean log2 titer 8.8), S. marcescens (mean log2 titer 8.6), and S.
aureus (mean log2 titer 9.9). Imgenex Corporation likely uses conventionally reared
rabbits for their custom polyclonal antibody production services and titers to commensals
like P. aeruginosa, S. marcescens, and S. aureus are not unusual.
Specific antibodies directed to the outer surface of E. coli or P. gingivalis are
required for directed targeting of antimicrobial peptides in conjugates. Antibodies to P.
aeruginosa, S. marcescens, and S. aureus would interfere with the concepts of the
hypothesis. Therefore, specific cell surface antibodies to E. coli or P. gingivalis were
isolated by affinity chromatography using columns containing immobilized whole cells
of E. coli or P. gingivalis as the ligand (Figures A5 and A6). Fractions were collected
and assessed for antibody by ELISA. Fraction 2 of the column wash contained serum
proteins and antibodies to E. coli, P. aeruginosa, S. marcescens, and S. aureus. Fractions
10, 11, and 12 of the eluted affinity bound protein contained only antibodies to E. coli
(Figure A5). These fractions were combined and contained 0.12 mg protein/ml of E. coli
affinity purified IgG antibody and 0.30 mg protein/ml P. gingivalis affinity purified IgG
23
antibody. The pH was adjusted to pH 7.2 and the IgG antibody in these fractions were
concentrated and filter sterilized. Five ml of 0.24 mg/ml (total protein 1.2 mg) of affinity
purified E. coli IgG antibody and 7 ml of 0.10 mg/ml (total protein 0.7 mg) of affinity
purified P. gingivalis IgG antibody were isolated.
The Antimicrobial Activity of SMAP28
Conjugated to Antibody Reagents was Determined
A primary amine group (e.g., an N-terminus amine or lysine residue) must be
present on SMAP28 for attachment of the Sulfo-SMCC reagent containing the maleimide
linker. SMAP28 has a mass of 3,198.95 Da peptide and a net 11+ charge. It has one Nterminus amine and 3 internal lysine residues, constituting 4 possible maleimide linker
binding sites.
After attaching maleimide, the SMAP28+maleimide linker was assessed by
MALFI-TOF analysis to determine if and how many maleimide linkers were attached.
The linker was successfully attached to SMAP28 and four distinct peaks were seen
(Figure A11). Each peak corresponded to the molecular mass of SMAP28 (i.e., 3,198.95
Da) and number of maleimide linkers attached (i.e., 219.24 Da). Predicted masses were
3,418.19 Da (SMAP28+1 maleimide linker), 3,637.43 Da (SMAP28+2 maleimide
linkers), 3,856.67 Da (SMAP28+3 maleimide linkers), and 4,075.91 Da (SMAP28+4
maleimide linkers). Distinct peaks were seen at 3,419.54 m/z, 3638.75 m/z, 3,857.80
m/z, and 4,077.32 m/z. Whether the maleimide linker attached consistently to specific
residue amines in the peptide or attached to every conceivable amine in every
conceivable combination is not known and was not determined in this study.
The antimicrobial activities of SMAP28 and SMAP28+maleimide linker were
nearly identical suggesting that the presence of the linkers does not affect antimicrobial
activity. The MIC for native SMAP28 was 3.9, 5.9, and 5.0 ug/ml for E. coli, P.
aeruginosa, and S. aureus, respectively. The MIC for SMAP28+maleimide linker
24
solution was 4.7, 4.7, and 4.4 ug/ml, for E. coli, P. aeruginosa, and S. aureus,
respectively.
To assess whether the number of attached maleimide linkers altered the
antimicrobial activity of SMAP28, the SMAP28+maleimide linker solution was
separated into fractions by HPLC, dried, assessed by MALDI-TOF for content, and
assayed for antimicrobial activity. Fractions contained SMAP28+1 maleimide linker,
SMAP28+2 maleimide linkers, SMAP28+3 maleimide linkers, and SMAP28+4
maleimide linkers. SMAP28 alone eluted at 13.78 minutes in ~45.9% acetonitrile
(fraction 14) (Figure A12). The SMAP28+maleimide linker solution contained four
broad peaks, which eluted at 13.51 minutes (fraction 14), 14.31 minutes (fraction 15),
14.82 minutes (fraction 15), and 15.63 minutes (fraction 16). The first peak in fraction
14 was likely SMAP28. Fractions 15, 16, 17 had antimicrobial activity against E. coli, P.
aeruginosa, and S. aureus. Subsequent fraction 18 had antimicrobial activity against S.
aureus and fraction 19 did not have antimicrobial activity against any microorganism.
Antimicrobial activity in fractions 15, 16, 17 suggests that SMAP28+maleimide linker
retained their antimicrobial activity.
These peak fractions were also assessed for the mass of the contents by MALDITOF. Fraction 15 contained masses 3,199.64 m/z, 3,418.32 m/z, and 3,637.90 m/z
corresponding to SMAP 28, SMAP28+1 maleimide linker, and SMAP28+2 maleimide
linkers, respectively. Fraction 16 contained masses 3,419.65 m/z, 3,638.87 m/zDa, and
3,860.11 m/z corresponding to SMAP28+1 maleimide linker, SMAP28+2 maleimide
linkers, and SMAP28+3 maleimide linkers, respectively. Fraction 17 contained masses
3,419.35 m/z, 3,637.90 m/z, and 3,860.03 m/z corresponding to SMAP28+1 maleimide
linker, SMAP28+2 maleimide linkers, and SMAP28+3 maleimide linkers, respectively.
Although easily separated by HPLC, the fractions 15, 16, and 17 contained SMAP28 with
multiple maleimide linkers. This suggests that the attachment does not dramatically alter
25
the chemical properties of SMAP28 and also suggests that they cannot be easily
separated by this method.
The Antimicrobial Specificity of SMAP28
Conjugated to Organism Specific Antibodies
in a Community of Microorganisms was Determined
Activated SMAP28+maleimide linker was attached to the affinity purified IgG
antibody (prepared as described above) via a free cysteine residue on the IgG antibody.
Since it was not known if the affinity purified IgG antibody had a free –SH on a cysteine
or an available disulfide (cysteine) residue, 2 procedures were used.
In first procedure (option IIA) disulfide bonds on the affinity purified IgG
antibody, if present, were reduced to free sulfhydryls groups. In the second procedure,
(option IIB) free sulfhydryls were added by adding SATA to a primary amine on the IgG
antibody. Preliminary work indicated that option IIB was the optimum choice.
A dot blot assay was used to verify the coupling between SMAP28 and the
affinity purified IgG antibody. The presence of the affinity purified IgG rabbit antibody
in the IgG-SMAP28 conjugate was confirmed using peroxidase labeled goat anti-rabbit
antibody (Figure A8). The presence of the SMAP28 peptide in the IgG-SMAP28
conjugate was confirmed first using goat-anti-SMAP28 and then peroxidase labeled
rabbit anti-goat antibody (Figure A8). Although the dot blot assay confirmed the
presence of both the affinity purified rabbit IgG antibody and SMAP28, it did not give
any information on the stoichiometry of the binding, the number of SMAP28 molecules
bound to the affinity purified rabbit IgG antibody, nor the location of the bound SMAP28
on the affinity purified rabbit IgG antibody. The specificity of the IgG-SMAP28
conjugate does suggest, however, that SMAP28 did not bind in the Fab portion of the
affinity purified IgG antibody.
26
The Antimicrobial Activity of the SMAP28-E. coli IgG
Antibody Conjugate was Determined
The potency of the SMAP28-E. coli IgG antibody conjugate was unknown and its
antimicrobial activity was first assessed independently against E. coli and S. aureus.
Initial data showed the undiluted conjugate was too concentrated and killed both
organisms. A diluted conjugate showed differential, specific killing (Figure A13). It was
realized that this solution may contain unbound SMAP28+maliamide linker. In a pilot
study the SMAP28-E. coli IgG antibody conjugate was dialyzed (12-14,500 NMWCO)
against 0.01 M PBS, pH 7.2 and then diluted 2-fold in microtiter plates in the broth
microdilution assay. Suspensions of Mueller Hinton broth containing E. coli or S. aureus
were added, incubated overnight at 37o C, and the optical density of the turbidity of broth
in the microtiter plates was read at 600 nm. The microorganisms were plated in the
presence of the SMAP28-IgG conjugate, and E. coli was more susceptible than S. aureus
which strongly supported the hypothesis that E. coli would be selectively targeted (Figure
A14). 80 ug/ml of SMAP28-E. coli IgG antibody conjugate (total protein) was chosen as
the concentration to use.
Before assessing the specificity and activity of the SMAP28-E. coli IgG antibody
conjugate, the concentrations of E. coli, P. aeruginosa, S. marcescens, and S. aureus
were first titrated with respect to each other. Briefly, each culture was adjusted to an
optical density of 0.108 and then diluted 10-4 fold in saline. 2.0 ml, 1.0 ml, 1.0 ml, and
4.0 ml of E. coli, P. aeruginosa, S. marcescens, and S. aureus, respectively, were added
to 2 ml of saline (e.g., constituting the 10-5 dilution of each). 800 µl of this suspension
was mixed with 800 µl of hypothetical test or control solution. These dilutions resulted
in approximately 0.75-1.2 x 103 CFU/ml of each organism per plate (Figure A15) which
remained constant up to 240 minutes.
The SMAP28-E. coli IgG antibody conjugate was antimicrobial for E. coli in the
presence of a microbial community containing E. coli, P. aeruginosa, S. marcescens, and
27
S. aureus (Figure A14). The concentrations of E. coli, P. aeruginosa, S. marcescens, and
S. aureus remained constant in the control saline and the control antibody solutions. In
these control solutions, colony forming units ranged from 3.3-5.8 x 102 CFU/ml for E.
coli, 3.4-6.9 x 102 CFU/ml for P. aeruginosa, 0.92-1.9 x 103 CFU/ml for S. marcescens,
and 2.8-5.2 x 102 CFU/ml for S. aureus throughout the 240 minute incubation period.
The concentrations of E. coli, P. aeruginosa, and S. aureus declined in the presence of
the SMAP28-E. coli IgG antibody conjugate. In this conjugate, S. aureus was very
susceptible and was killed almost instantly; there was a mean of 1.3 CFU at time 0. In
this conjugate, E. coli was also very susceptible and was killed within 30 minutes. There
was a mean of 4.6 CFU E. coli at 30 minutes, which was down from a mean of 54.7 CFU
at 0 minutes. In this conjugate, P. aeruginosa was susceptible and was killed within 120
minutes. There was a mean of 1 CFU P. aeruginosa at 120 minutes, which was down
from a mean of 48.7 CFU at 0 minutes. Finally, in this conjugate, S. marcescens was
resistant (Figure A14 and A15). Colony forming units ranged from a mean of 68.7 to 97
CFU for S. marcescens in the SMAP28-E. coli IgG antibody conjugate throughout the
240 minute incubation period.
The Antimicrobial Activity of the SMAP28-P. gingivalis IgG
Antibody Conjugate was Determined
Before assessing the specificity and activity of the SMAP28-P. gingivalis IgG
antibody conjugate, the concentrations of P. gingivalis, A. actinomycetemcomitans, and
P. micros were first titrated with respect to each other. Briefly, each culture was adjusted
to an optical density of 0.108 and then diluted 10-4 fold in reduced, anaerobic saline. 2.0
ml, 0.25 ml, and 2.0 ml of P. gingivalis, A. actinomycetemcomitans, and P. micros,
respectively, were added to 5.75 ml of reduced, anaerobic saline (e.g., constituting the 105
dilution of each) and diluted 2-fold with the SMAP28-P. gingivalis IgG antibody
conjugate. These dilutions resulted in approximately 50-75 CFU of each organism per
plate. Preliminary results showed that antimicrobial activity was rapid and occurred
28
within 30 minutes of incubation with the SMAP28-P. gingivalis IgG antibody conjugate.
Therefore, the activity and specificity of the SMAP28-P. gingivalis IgG antibody
conjugate to P. gingivalis, A. actinomycetemcomitans, and P. micros were determined at
0, 10, 20, and 30 minute intervals.
The specificity and activity of the SMAP28-P. gingivalis IgG antibody conjugate,
coupled by option IIA and IIB and containing 100 µg total protein/ml (50 ug total
protein/ml) was determined (Tables A4-A7, Figures A16-A17). At this concentration,
there was no difference between the antimicrobial activity of IIA and IIB, as both were
antimicrobial (Figures A16 and A17). At high concentrations, the conjugate lacked
specificity and killed P. gingivalis, A. actinomycetemcomitans, and P. micros. In the
controls containing P. gingivalis IgG antibody and Pre-Immune Control IgG antibody,
the concentration of P. micros fell off rapidly. In a separate experiment, the viability of
P. micros held in reduced, anaerobic saline fell off dramatically (Figure A18).
The specificity and activity of the SMAP28-P. gingivalis IgG antibody conjugate,
coupled by option IIA and IIB and containing 40 µg total protein/ml (20 µg total
protein/ml total concentration) was then determined. In these studies, there were large
differences in the average and standard deviations of the CFU on each of the two days
(Table A7). Since the conjugates were prepared separately on these days, there may be
differences in binding efficiencies of SMAP28 to IgG antibody that would account for
some variation in the results. Also, since the mixed community cultures were prepared
on different days, there were obvious differences in the number of organisms in the test
mixtures; the CFU in the mixture on September 22 were considerably less than the CFU
in the mixture on November 3.
There was strong and highly significant agreement among the three raters (Tables
A4 and A5). For P. gingivalis, option IIB demonstrated a significant negative correlation
with time for both treatment days (p=0.0001) (Figure A20). The pre-immune IgG
antibody control also demonstrated a significant negative correlation with time (p=0.04)
29
with regards to P. gingivalis (Figure A20). Option IIA and P. gingivalis antibody control
did not show significant correlation. A significant decline of P. gingivalis CFUs over
time for option IIB was seen (43 over 10 minutes). For A. actinomycetemcomitans, no
significant effects were noted for any treatment at any time. At this concentration, there
was no difference between the antimicrobial activity of option IIA and IIB conjugation,
both were antimicrobial although option IIB was more effective and specific for P.
gingivalis (Figures A19 and A20). Representative IIB plate samples are seen at time 0
minutes and 30 minutes (Figure A25). In the controls containing P. gingivalis IgG
antibody and Pre-Immune Control IgG antibody, the concentration of P. micros fell off
rapidly.
For P. gingivalis, there was a significant interaction between treatments and time
(P< 0.0001). The treatment mean curves (Figure A22) for the four time levels support
this conclusion. The number of P. gingivalis CFU declined over time under all four
treatments, but this relationship with time was significant under some treatments and not
significant under others. Table A6 lists the Spearman Correlation for this microorganism
by treatment. The PgAb, IIB treatment showed a significant negative correlation with
time (Spearman Correlation -0.705, P = 0.0001) and similarly, IgGAb control resulted in
a significant negative correlation with time (Spearman Correlation -0.420, p =0.04). The
PgAb, IIA treatment (Spearman Correlation = -.137, p = 0.5) and PgAb control
(Spearman Correlation = -.076, p =0.81) showed a non-significant correlation with time.
Using a simple linear regression line, the average rate of decline over time was estimated
and listed in Table A6. The average decline in the number of CFUs for every 10 minutes,
for this microorganism is 9.7 (PgAb, IIA), 43 (PgAb, IIB), 1.5 (PgAb control), and 21.8
(IgGAb control).
For P. micros the interaction between time and treatment was statistically
significant if the data for each day of experiment was analyzed separately (P = 0.01 for
September 9 and p = 0.0005 for November 9). The treatment mean curves for P. micros
30
are shown in Figure A23. When the data for the two days were combined and analyzed,
the interaction between time and treatment become non-significant (P = 0.2), while both
time and treatment were significant factors in the CFUs. This implies that there was a
significant interaction (P = 0.0046) between the treatment, time and the day the
experiment was performed. Tukey’s studentized range test shows a significant difference
(at 5% level) between treatment 1 ( PgAb, IIA) and treatment 2 (PgAb, IIB) and
treatment 3 (PgAb control). P. micros showed decline in bacterial counts over time for
all four treatments (Figure A23). The Spearman Correlation for this organism is listed in
table A6. The P. micros count in PgAb, IIA treatment (Spearman Correlation = -0.45, P
= 0.027), IgGAb control (Spearman Correlation -0.42, p =0.04) and PgAb control
(Spearman Correlation = -0.89, p =0.0001) showed a significant negative correlation
with time but this negative correlation was not significant for P. micros count in PgAb,
IIB treatment (Spearman Correlation = -.347, p = 0.095). Using a simple linear
regression line, the estimate of average rate of decline in CFUs over time was evaluated
and listed in Table A6. This rate of decline for every 10 minutes is 41 (PgAb, IIA), 10.2
(PgAb, IIB), 8.3 (PgAb control), and 47 (IgGAb control).
For A. actinomycetemcomitans, the interaction between treatment and time was
not significant (p = 0.48) for the September 9 experiment and it was significant (p =
0.0012) for November 9. The data for September 9 shows that time is not a significant
factor (p =0.23) while treatment is significant (.02). Tukey’s studentized range test
shows that treatment 2 and 4 are significantly different. When the data is combined for
the two days, the interaction between treatment and time becomes insignificant (p =
0.42). The Tukey’s studentized range test for the combined data indicated that except for
treatment 4 and 1 all the pair wise comparisons between treatments are significant at 5%
level. There was interaction between the treatment, time and the day of the experiment
for this microorganism. Figure A24 showed the treatment mean curves for this
microorganism.
31
The rate of decline in CFUs over time is small compared to the other two
microorganisms and varied from September 9 to November 9. The Spearman Correlation
for this organism is listed in Table A6. The negative Correlation between the number of
bacterial counts over time was not statistically significant for any of the four treatments
and controls (PgAb, IIA, Spearman Correlation = -0.21, P = 0.32; PgAb, IIB, Spearman
Correlation = -0.33, P = 0.11; PgAb control, Spearman Correlation = -.11, p =0.73;
IgGAb control, Spearman Correlation = -.13, p = 0.5). Using a simple linear regression
line, the average rate of decline in CFUs over time was evaluated and listed in Table A6.
This rate of decline, for every 10 minutes was 2.85 (PgAb, IIA), 8.2 (PgAb, IIB), 1
(PgAb control), and 1.9 (IgGAb control).
In summary, the descriptive statistics (Table A7) show that the mean and standard
deviation for the number of colony forming units (CFUs) for the three organisms, by
treatment and day of experiment, are very different for the two days of the experiment.
This is also evident in the Figures (A22 –A24). The reason for this is not known. The
two factor analysis of variance showed a strong interaction between treatment, time and
day for all three microorganisms. Three factor interactions are difficult to understand and
interpret. This disparity in experiment results for the two days where the experiment
were performed was more profound for A. actinomycetemcomitans and P. micros where
two way interaction between time and treatment was significant for one day and not
significant for the other and the combined data showed non-significant interaction. Base
10 log transformations of the CFUs were used to stabilize the error variance and to make
error distribution more normal in analysis of variance procedure. Significant two way
interaction between time and treatment implies that the difference among levels of one
factor (time) is not constant at all levels of the second factor (treatment). Therefore, it is
generally not meaningful to speak of factor effect (even if the factor is statistically
significant) if there is a significant interaction effect. Additional work is needed to
32
identify the underlying factors resulting in variability associated with the time, treatment,
and day effects and their interactions. Additional replications should provide some clues.
33
CHAPTER VI
DISCUSSION
Periodontitis consists of both acute and chronic inflammation of the tissues
supporting the teeth. Periodontitis is likely the result of the presence of polymicrobial
communities 75. These communities may contain any number or combination of over 700
species of microorganisms. Paster, et al. assessed the diversity of all cultivable and notyet-cultivated species of human oral bacteria by sequencing full 16S ribosomal DNA
(rDNA) bacterial genes from DNA isolated from subgingival plaque samples from 31
human volunteers 90. 16S rDNA was PCR amplified and cloned into E. coli. The
sequences of cloned 16S rDNA inserts were used to determine species identity. From
2,522 clones, approximately 60% represented 132 known species and approximately 40%
were novel phylotypes. Hutter, et al. assessed the diversity of microbial species in
subgingival plaque samples from 26 human volunteers 91. 16S rDNA was amplified
using two different primer sets. From 578 sequences, about 70% showed a similarity to
at least 99% of the sequences deposited in public databases. These included
Actinobacteria spp., Bacillus spp., Bacteroidetes spp., Clostridia spp., Deferribacteres
spp., Flavobacteria spp., Fusobacteria spp., Mollicutes spp., Spirochaetes spp., and
Proteobacteria spp. The remaining 30% were novel phylotypes. Aas, et al. assessed the
diversity of human oral bacteria from nine sites from five clinically healthy volunteers.
These sites included: tongue dorsum, lateral sides of tongue, buccal epithelium, hard
palate, soft palate, supragingival plaque of tooth surfaces, subgingival plaque, maxillary
anterior vestibule, and tonsils 72. 16S rRNA genes from sample DNA were amplified,
cloned, and transformed into E. coli. Sequences of 16S rRNA genes were used to
determine species identity or closest relatives. From 2,589 clones, about 40% were 141
known species and about 60% were novel phylotypes. In all, more than 700 bacterial
species or phylotypes were detected and thirteen new phylotypes were identified.
34
Ledder, et al. assessed the diversity of human oral bacteria with PCR-denaturing gradient
gel electrophoresis with primers specific for the V2-V3 region of the eubacterial 16S
rRNA gene from DNA isolated from subgingival samples from healthy gingiva or
clinically diagnosed chronic periodontitis from 47 human volunteers 92. From 52
microbial species, about 98% were 51 known species and about 2% was one novel
phylotype.
Among these extensive communities are pathogens that are characteristically
associated with periodontitis. These include, but were not limited to, the putative
periodontal pathogens A. actinomycetemcomitans 92, P. gingivalis 90-92, Tannerella
forsythensis 92, Tannerella forsythus 90, and T. denticola 90. Since conventional antibiotic
therapy is often broad-spectrum, a strategy was conceived in this thesis project to
specifically target antimicrobial activity to select pathogens without harming normal
commensals or favoring the growth of opportunistic organisms like C. albicans. Here, a
potent cathelicidin, SMAP28, was conjugated to affinity purified E. coli or P. gingivalisspecific IgG antibodies and hypothesized to selectively kill E. coli or P. gingivalis, in
mixed aerobic and anaerobic microbial cultures, respectively.
The concept was initially developed using SMAP28-E. coli IgG antibody
conjugate for the following reasons. First, the conditions could be determined for the
preparation of the specific antiserum and affinity isolation and concentration of E. coli
specific IgG antibody. Second, the conditions could be determined for the linking of
maleimide to SMAP28 and determination of its antimicrobial activity. Third, the
conditions could be determined for conjugating the SMAP28+maleimide linker moiety to
the affinity purified E. coli specific IgG antibody. Finally, the conditions could be
determined for antimicrobial activity and specificity of the SMAP28-E. coli antibody
conjugate for E. coli in the presence of a microbial community containing E. coli, P.
aeruginosa, S. marcescens, and S. aureus. This preliminary work was necessary in
establishing proof of the concept, delineating the details of the conjugation procedure,
35
and assessing the specificity and activity of the SMAP28-E. coli antibody conjugate
under aerobic conditions. An optimized procedure was used to prepare a SMAP28-P.
gingivalis IgG antibody conjugate. The specificity and antimicrobial activity of this
conjugate was determined in an artificially generated microbial community containing
equal concentrations of three bacteria associated with periodontal disease. These are
arbitrary test conditions and I recognize that these organisms likely: a) occur in differing
concentrations in periodontal lesions, b) occur as members of a mature biofilm, and c)
grow in an environment that contains a plethora of electrolytes, host proteins, antibodies,
and defense molecules in gingival crevicular and serous fluids. These anaerobic
periodontal pathogens were selected for this project because of their ease of identification
when grown on blood agar plates. A. actinomycetemcomitans grows as a white colony
surrounded by a halo, P. micros grows as a small gray colony, and P. gingivalis grows as
a black colony.
The SMAP28-E. coli IgG antibody conjugate had both specific antimicrobial
activity for E. coli and non-specific antimicrobial activity for S. aureus and P. aeruginosa
when added to an artificial community containing E. coli, S. aureus, P. aeruginosa, and
S. marcescens. S. aureus was very susceptible to the conjugate and was killed almost
instantly; there was a mean of 13 CFU/ml at time 0. This is likely due to the Fc portion
of the IgG in the SMAP28-E. coli IgG antibody conjugate binding to protein A on the
Staphylococcus surface. However, this is not known. As hypothesized, E. coli was very
susceptible and was killed within 30 minutes. There was a mean of 46 CFU/ml at 30
minutes, which was down from a mean of 547 CFU/ml at 0 minutes. Also, P. aeruginosa
was susceptible and was killed within 120 minutes. This is not unusual, P. aeruginosa is
very susceptible to SMAP28 (KALFA). In retrospect, S. aureus and P. aeruginosa
should have been replaced with other Enterobacteriaceae. S. marcescens was resistant.
The specificity and activity of the SMAP28-P. gingivalis IgG antibody conjugate
for P. gingivalis was found to be dependent upon the conjugation procedure and the
36
concentration of conjugate. Conjugation by option IIB was more effective and a
SMAP28-P. gingivalis antibody conjugate developed this way was more specific for P.
gingivalis than option IIA. Option IIB generates a free sulfhydryl by adding SATA to a
primary amine on the P. gingivalis IgG antibody, which would be more accessible to
reactivity with the SAMP28-maleimide linker. The concentration of the SMAP28-P.
gingivalis IgG antibody conjugate was also important. The conjugate, containing 100 µg
total protein/ml (50 µg total protein/ml) lacked specificity and killed P. gingivalis, A.
actinomycetemcomitans, and P. micros. However, the specificity and activity of the
SMAP28-P. gingivalis IgG antibody conjugate containing 40 µg total protein/ml (20 µg
total protein/ml, final concentration) when diluted with cultures was more effective and
specific for P. gingivalis. Overall, the numbers of P. gingivalis in the presence of the
SMAP28-P. gingivalis IgG antibody conjugate declined with respect to a) the numbers of
the P. gingivalis incubated similarly in affinity purified IgG antibody alone, b) the
numbers of the P. gingivalis incubated similarly in IgG isolated from pre-immune serum,
or c) the numbers of A. actinomycetemcomitans, and P. micros in the presence of the
SMAP28-P. gingivalis IgG antibody conjugate. The specificity of the antimicrobial
activity was related to the concentration of the SMAP28-P. gingivalis IgG antibody
conjugate; 40 µg/ml was more specific killing P. gingivalis whereas 100 µg/ml was not
specific killing P. gingivalis, A. actinomycetemcomitans, and P. micros. In this
experiment, saline was used to control the growth of the microorganisms during the
assay. If a protein-rich medium had been used, the CFUs for each microorganism would
have increased over time and the dilution procedure designed to provide countable CFU
numbers would have been ineffective.
Antimicrobial Activity of SMAP28 with
Attached Maleimide and IgG Antibody
37
The deduced peptide SMAP29 63 and the isolated peptide SMAP28 are potent
antimicrobial peptides 60. SMAP28 and SMAP29 have MICs of 0.04 – 16.0 µM for
Gram-negative bacteria 57, 64, 68, 93; 0.08 – 1.0 µM for Gram-positive bacteria 57, 64, 93; 0.3 > 32.0 µM for fungi 57, 64, 93; and 3.1 – 30.7 µM for Cryptosporidium parvum 94.
Spirochaetes are susceptible but at much higher levels of peptide 95. Congeners of
SMAP29 are also antimicrobial against bacteria 67, 68 and fungi 96.
The results of this study dispute current dogma concerning the mechanisms of
antimicrobial peptide activity. To be effective, peptides like SMAP28 must first be
attracted to bacterial surfaces and one obvious mechanism is electrostatic bonding
between anionic or cationic peptides and structures on the bacterial surface 97. Once
close to the microbial surface, peptides must traverse capsular polysaccharides before
they can interact with the outer membrane containing lipopolysaccharide in Gramnegative bacteria and capsular polysaccharides, teichoic acids, and lipoteichoic acids
before they can interact with the cytoplasmic membrane in Gram-positive bacteria. Once
at the cytoplasmic membrane, peptides are free to interact with lipid bilayers. In vitro
studies of antimicrobial peptides incubated with single or mixed lipids in membranes or
vesicles show that peptides bind in 2 physically distinct states 98. At low peptide-to-lipid
ratios, peptides are bound parallel to a lipid bilayer 99. As the peptide-to-lipid ratio
increases, peptides begin to orientate perpendicular to the membrane. At high peptide-tolipid ratios, peptide molecules are orientated perpendicularly and insert into the bilayer
forming transmembrane pores. In the “barrel-stave model,” peptide helices form a
bundle in the membrane with a central lumen, much like a barrel composed of helical
peptides as the staves 99, 100. This type of transmembrane pore is unique and induced with
alamethicin. In the “toroidal pore model,” antimicrobial peptide helices insert into the
membrane and induce the lipid monolayers to bend continuously through the pore so that
the water core is lined by both the inserted peptides and the lipid head groups 101. This
type of transmembrane pore is induced by magainins, protegrins, and melittin 99, 101, 102.
38
In the “carpet model,” peptides accumulate on the bilayer surface 103. This model can
explain the activity of antimicrobial peptides, like ovispirin, a congener of SMAP28 104,
that orientate parallel, “in-plane,” to the membrane surface 105. Peptides are
electrostatically attracted to the anionic phospholipid head groups at numerous sites
covering the surface of the membrane in a ‘carpet-like’ manner. At high peptide
concentrations, surface oriented peptides are thought to disrupt the bilayer in a detergentlike manner eventually leading to the formation of micelles 106, 107. At a critical, threshold
concentration, the peptides form toroidal transient holes in the membrane allowing
additional peptides to access the membrane. Finally, the membrane disintegrates and
forms micelles after disruption of the bilayer curvature 105, 108.
SMAP28 is amphipathic and forms an alpha helix in an organic environment. It
was not known if attachment of linkers or proteins would alter its helical structure and
impair its ability to insert into microbial membranes. In this study, SMAP28 was
observed to be bactericidal with attached maleimide linkers and with an attached affinity
purified IgG antibody. The exact mechanism of this antimicrobial activity is not known
but it is likely that the maleimide linkers are attached to lysine or arginine residues on the
hydrophilic face of the SMAP28 alpha helix. Maleimide, linked to the hydrophilic face
would allow the peptide to form an alpha helix and insert into the bacterial membrane.
However, there is evidence that other peptides attached to carrier proteins, surfaces, or
incorporated into films are also bacteriostatic or bactericidal 109. For example, nisin, a
24-amino acid lantibiotic produced by certain strains of Lactococcus lactis subsp., causes
pore formation in the membranes of sensitive bacteria. However, when absorbed to food
preparation surfaces, it still reduces attachment and kills Enterococcus spp. and Listeria
spp. 110. Variables include the composition of the surface, the temperature, the bacterial
strain, and the concentration of the bacterial suspension. Nisin can also be incorporated
into polyethylene or polyethylene oxide polymer films 111. Here, nisin reduces
Brochothrix thermosphacta, a meat spoilage organism, on beef surfaces up to 21 days to
39
a greater extent than the control plastics 112. The mechanisms of defensins are not as well
defined 113 but they are thought to permeabilize membrane bilayers containing negatively
charged phospholipids 113, 114. In planar lipid bilayers, HNP-1 and rabbit NP-1 form
transmembrane pores when a physiologic relevant negative potential was applied to the
membrane side opposite to the defensin-containing diluent 115. In large unilamellar
vesicles, NP-1 creates large, transient defects in phospholipid bilayers 116 and NHP-2
forms 2.5 nm pores 117. Defensins from Anopheles gambiae mosquitoes inserted into
polyelectrolyte multilayer films built by the alternate deposition of polyanions and
polycations are also antimicrobial 118. The growth of E. coli D22 was inhibited at the
surface of defensin-functionalized films; inhibition was found to be 98% when 10
antimicrobial peptide layers were inserted in the film architecture. By confocal or
electron microscopy, there was a close interaction of the bacteria with the positively
charged ends of the films, which allows defensin to interact with the bacterial membrane
structure. Thus it appears that the lytic models proposed for attraction, attachment, and
insertion of antimicrobial peptides needs to be modified to include antimicrobial peptides
that are tethered to inert surfaces or attached to larger proteins.
Alternate Carriers
A unique and specific carrier is needed to carry the lytic antimicrobial peptide to
the targeted microbial surface. Peschen, et al. successfully used a Fusarium spp.-specific
antibody linked to antifungal peptides to protect plants against a fungal pathogen 86. Qiu,
et al used staphylococcal AgrD1 pheromone fused with the channel forming domain of
colicin Ia to kill methicillin-sensitive and methicillin-resistant S. aureus, but not
Staphylococcus epidermidis or Streptococcus pneumoniae 1. Qiu, et al also used
enterococcal cCF109 pheromone fused with the channel forming domain of colicin Ia to
kill vancomycin-resistant E. faecalis 82. Interestingly, mice made bacteremic with
vancomycin-resistant E. faecalis survived when they were treated with enterococcal
cCF109 pheromone fused with the channel forming domain of colicin Ia, while all of the
40
untreated controls died 82. Other attractants are also feasible and include lectins to
capsular polysaccharides, lytic phage peptides, siderophores, and other ligands for
surface receptors. Since many of these are not known for P. gingivalis, an affinity
purified IgG antibody, specific to the microbial cell surface, was used. However, other
strategies may be considered to further increase the specificity and activity of
antimicrobial peptide targeted activity. These may include ligands to surface proteins on
P. gingivalis, lectins to surface carbohydrates on P. gingivalis, or specific proteins from
bacteriophages to P. gingivalis receptors.
Applicability
The results shown here are important, initial steps towards the development of a
selective, targeted antimicrobial agent. Characterizing the peptide antimicrobial activity
with and without the linked E. coli and P. gingivalis-specific IgG antibodies will give
insight on how to reduce the broad-spectrum antimicrobial activity of this conjugate and
increase the specific targeted pathogen antimicrobial activity in a mixed microbial
culture. Such reagents may be useful to eliminate specific periodontal pathogens (e.g., P.
gingivalis) from patients with periodontal disease without harming their normal
commensal population.
Future Directions
Further efforts are needed to evaluate the clinical possibilities of this conjugate
model. Continued in vitro studies are needed to refine the appropriate concentrations of
peptide conjugate, as well as add more species of oral bacteria, both beneficial and
pathological species. An animal model would be the next step to determine actual
clinical efficacy. The delivery mechanism for the conjugate will likely be oral rather than
parenteral, and topical rather than systemic. A systemic delivery would not be possible
due to enzymatic inactivation of the conjugate’s activity. Topical delivery would be
favored for this reason and for the ease of use for dental providers. The delivery
mechanism and antimicrobial activity would need to have substantivity in the mouth,
41
similar to chlorhexidine, to ensure continued delivery of the antimicrobial. A likely
mechanism for delivery would mimic the delivery of local antibiotics in subgingival
pockets. Local antibiotic therapy has shown some efficacy in reducing probing depths in
treatment of periodontal disease, and many forms are now available as sustained delivery,
commercially-produced products. These include: a chlorhexidine chip, minocyclineHCl microspheres, tetracycline-HCl in an ethylene/vinyl acetate copolymer fiber, and
doxycycline hyclate in a gel delivery system. 119 It is reasonable to speculate that this
conjugate could be incorporated into a formulation for use in the subgingival periodontal
pocket, under the supervision of a dental health provider.
42
CHAPTER VI
SUMMARY AND CONCLUSIONS
In this study, E. coli and P. gingivalis IgG antibody conjugate-mediated targeted
killing was demonstrated without drastically affecting the growth of other
microorganisms in a mixed culture. Such reagents may be useful to eliminate specific
periodontal pathogens (e.g. P. gingivalis) from patients suffering from periodontal
disease without harming the normal commensal flora. Future efforts will focus on a
delivery mechanism for clinical application.
43
APPENDIX A
Table A1. Antimicrobial Proteins and Peptides Found in Human Mucosa and Mucosal
Secretions
System
Protein
Relative Mass (kDa)
Reference
Respiratory tract
Anionic peptides
<1
120
-defensins
4
121
-defensins
4
122
Surfactant proteins
8
123
Lysozyme
12
124
Secretory
12
125
80
126
leukoprotease
inhibitor
Lactoferrin
36
salivary mucin
glycoprotein MG2
Intestinal tract
Salivary histatin 5
37
cathelicidins
127
Secretory
12
128
12
128
leukoprotease
inhibitor
Lysozyme
44
Table A1. Continued
Urogenital tract
-defensins
4
129
-defensins
4
130
Cryptdin
4
131
Secretory
12
128
12
128
-defensins
4
132
-defensins
4
133
4
134
leukoprotease
inhibitor
Lysozyme
Cryptdin
hCAP18/LL-37
45
RK-1 defensin-like
135
peptide
45
Table A2. Antimicrobial Proteins and Peptides Found in Human Oral Mucosa and Oral
Secretions
Peptide
Source
Reference
Lysozyme
Saliva, gingival crevicular
29
fluid
Lactoferrin
Saliva, gingival crevicular
30
fluid
Acidic proline-rich proteins
Saliva
29, 32
Plunc
Saliva
33
Salivary mucin glycoprotein Saliva
30, 36
Histatin
Saliva
29, 32, 37
Defensins
Oral mucosa and salivary
38, 40, 42, 136
glands
Cathelicidins (LL37)
Saliva
45, 137
46
Table A3. Characteristics of SMAP28, a cathelicidin from sheep with potent
antimicrobial activity
Name:
SMAP-28
Source:
Sheep
Sequence:
RGLRRLGRKIAHGVKKYGPTVLRIIRIA-(AMID)
Length:
28 amino acid residues
Structure:
Rich
Average mass:
3198.95 Da
Monoisotopic mass:
3197.01Da
Theoretical pI:
12.31
Activity:
SMAP28 and SMAP29 have MICs of 0.04 – 16.0 µM for
Gram-negative bacteria 57, 64, 68, 93; 0.08 – 1.0 µM for Grampositive bacteria 57, 64, 93; 0.3 - > 32.0 µM for fungi 57, 64, 93; and
3.1 – 30.7 µM for C. parvum 94, 95. Spirochaetes are
susceptible oral microorganisms at higher levels of peptide 67
Net charge:
11
Hydrophobic
37%
percentage:
References:
58, 61-64, 138
47
Table A4. Intraclass correlation coefficients (ICC) were used to assess
rater reliability in counting colony forming units (CFU). ICC are alternative
statistics for measuring homogeneity for sets of measurements. For this analysis,
the number of CFU were used. Note that the tables list pair wise and
overall concordance among the three raters as well as Intra-rater agreement
and statistical significance.
Rater
Rater
Rater
Organism
All
1-2
1-3
2-3
0.9927
0.9944
0.9902
0.9939
(<0.0001)
(<0.0001)
(<0.0001)
(<0.0001)
0.9818
0.9689
0.9803
0.9791
(<0.0001)
(<0.0001)
(<0.0001)
(<0.0001)
0.9956
0.9961
0.9942
0.9958
(<0.0001)
(<0.0001)
(<0.0001)
(<0.0001)
Pga
Aa
b
c
Pm
a
P. gingivalis
b
c
Aggregatibacter actinomycetemcomitans
Peptostreptococcus micros
48
Table A5. Intraclass correlation coefficients (ICC) were used to assess
rater reliability of base 10 log transformations of colony forming units (CFU).
ICC are alternative statistics for measuring homogeneity for sets of measurements.
For this analysis, the base 10 log transformations of CFU were used.
Note that the tables list pair wise and overall concordance among the
three raters as well as the intra-rater agreement using base 10 log transformations
of CFU and statistical significance.
Rater
Rater
Rater
1-2
1-3
2-3
0.9961
0.9956
0.9935
0.9959
(<0.0001)
(<0.0001)
(<0.0001)
(<0.0001)
0.9443
0.9482
0.9812
0.9791
(<0.0001)
(<0.0001)
(<0.0001)
(<0.0001)
0.9982
0.9985
0.9963
0.9981
(<0.0001)
(<0.0001)
(<0.0001)
(<0.0001)
Organism
All
a
Pg
Aab
Pmc
a
P. gingivalis
b
c
Aggregatibacter actinomycetemcomitans
Peptostreptococcus micros
49
Table A6. Estimate of the average rate of change in the number of CFUs over time.
a
PgAb, IIA
PgAb, IIB
PgAb control
IgGAb control
a
c
b
Pm
c
Aa
Spearman Correlation
-0.137
-0.45
-0.21
(p-value)
(0.5)
(0.027)
(0.32)
Average Rate/10 min
9.7
41
2.85
Spearman Correlation
-0.72
-0.35
-0.33
(p-value)
(.0001)
(0.095)
(0.11)
Average Rate/10 min
43
10.2
8.2
Spearman Correlation
-.076
-.089
-0.11
(p-value)
(0.81)
(0.0001)
(0.73)
Average Rate/10 min
1.5
8.3
1
Spearman Correlation
-0.42
-0.42
-0.13
(p-value)
(0.04)
(0.04)
(0.5)
Average Rate/10 min
21.8
47
1.9
P. gingivalis
b
Pg
Peptostreptococcus micros
Aggregatibacter actinomycetemcomitans
50
Table A7. Descriptive statistics of the mean and standard deviation for the
number of colony forming units (CFUs) for the three organisms by treatment
and day of experiment. The mean and standard deviation are very different
for the two days of the experiment.
P. gingivalis
0 minutes
10 minutes
20 minutes
30 minutes
September 22
Conjugate IIA
x (s )
54 (2.65)
66.3(7.6)
64(4.6)
50(10.4)
Conjugate IIB
x (s )
74.7(10.4)
63.7(2.5)
18(4)
4(1.7)
PgAb Control
x (s )
78.3(17.2)
71.7(14.5)
84(15.1)
79.3(3.1)
IgGAb Control
x (s )
81.7(5.7)
79.7(8.9)
38.7(6.1)
22.3(4.7)
Conjugate IIA
x (s )
206.3(28.7) 220.3(21.5) 214.3(20.3) 148.7(18.3)
Conjugate IIB
x (s )
223.3(27.5) 179.6(27.8) 136.3(18.3) 36.7(7.4)
PgAb Control
x (s )
NA
IgGAb Control
x (s )
194.5(12.8) 195.2(3.9)
166.8(17.3) 131.3(20.8)
0 minutes
10 minutes
20 minutes
30 minutes
November 3
A.
NA
NA
NA
actinomycetemcomitans
September 22
Conjugate IIA
x (s )
40.7(6.6)
31(2.6)
27.3(4.04)
30.7(2.7)
Conjugate IIB
x (s )
32.3(2.3)
25(6.2)
30(6.6)
28(1)
PgAb Control
x (s )
34.7(12.7)
8.7(4)
33(7.2)
33(6)
IgGAb Control
x (s )
37.3(2.5)
4.7(6.4)
33.3(10.2)
37.3(1.2)
51
Table A7. Continued
November 3
Conjugate IIA
x (s )
99.7(18.6)
107.7(8.4)
107.3(6.1)
92(12.3)
Conjugate IIB
x (s )
89.3(2.1)
73.3(8.5)
61(11.3)
41.7(3.2)
PgAb Control
x (s )
NA
NA
NA
NA
IgGAb Control
x (s )
101.2(4.6)
104.7(12.6) 102.3(22.8) 91.5(5.6)
0 minutes
10 minutes
20 minutes
30 minutes
P. micros
September 22
Conjugate IIA
x (s )
57.7(11)
4(1)
0
0
Conjugate IIB
x (s )
10.7(2.1)
0
0
0
PgAb Control
x (s )
26.3(5.9)
0
1.6(2.1)
1(1)
IgGAb Control
x (s )
8.3(3.2)
0
1(1)
.33(0.6)
Conjugate IIA
x (s )
415(24.2)
Conjugate IIB
x (s )
79.3(9.1)
32.7(2.1)
2.5(2.5)
25.2(8.1)
PgAb Control
x (s )
NA
NA
NA
NA
IgGAb Control
x (s )
377.2(19.)
303(23.1)
173.2(30.4)
118(16.1)
November 3
345.7(51.6) 280.3(72.1)
222(27.5)
52
Figure A1. An overview of the procedure used to construct the SAM28-IgG antibody
conjugate. SMAP28 was first synthesized. The maleimide linker was then attached and
the mixture was characterized by mass spectrometry. Antiserum to whole cells of E. coli
or P. gingivalis were prepared in rabbits. Specific cell surface antibodies were then
isolated with an immunoaffinity column and specific IgG was isolated using a Protein G
column. SMAP28+maleimide linker was then attached to the specific IgG, dialyzed, and
the SAM28-IgG antibody conjugate was tested for specific antimicrobial activity.
53
Figure A2. The NMR solution structure for the SMAP28 peptide containing
RGLRRLGRKIAHGVKKYGPTVLRIIRIA-(AMID). High resolution images were
obtained from PDBsum and generated using Molscript and Raster3D with permission
from Dr. Roman Laskowski, European Bioinformatics Institute, Wellcome Trust Genome
Campus, Hinxton, Cambridge, CB10 1SD, United Kingdom.
Source: SwissProt ID: PDB ID: 1FRY and Brogden97.
54
Figure A3. MALDI-TOF assessment of the purity of the SMAP28 sample showing the
major peak at 3200.78 m/z and a number of smaller peaks at 2718.54, 3226.16, 3410.92,
and 3620.39 m/z.
Source: High Resolution Mass Spectrometry Facility, University of Iowa, Iowa City, IA
55
Figure A4. Reversed Phase-High Performance Liquid Chromatography (HPLC)
assessment of the purity of the SMAP28 sample. A single peak eluted from the column
at 13.5 minutes.
56
Figure A5. A chromatogram showing the profile of proteins eluted after rabbit antiserum
was passed over the immunoaffinity column containing bound whole cells of E. coli.
Fractions were tested by ELISA for antibodies to E. coli, S. aureus, S. marcescens, and P.
aeruginosa. Fractions 1 through 4 contained the majority of serum proteins as well as
antibodies to E. coli, S. aureus, S. marcescens, and P. aeruginosa (fraction 2) After the
unbound material was washed from the column (fractions 5-9), specifically bound protein
was eluted (fractions 10-12), and contained only antibody to E. coli. Fractions 11-12
were then pooled.
57
Antibody titer
1
P.
2
3
4
5
6
7
8
9
10
11
12
13
14
15
+
gingivalis
Figure A6. A chromatogram showing the profile of proteins eluted after rabbit antiserum
was passed over the immunoaffinity column containing bound whole cells of P.
gingivalis. Fractions were tested by ELISA for antibodies to P. gingivalis. Fractions 1
through 5 contained the majority of serum proteins. After the unbound material was
washed from the column (fractions 5-9), specifically bound protein was eluted (fraction
10) contained only antibody to P. gingivalis.
58
Antibody
Maleimide
Linker
SMAP28
Figure A7. A schematic diagram of the conjugate that shows the concept of the linkage
between SMAP28 and the specific antibody. Hypothetically, more than one SMAP28
may link to the IgG and this will be dependent on the number of available binding sites.
59
Figure A8. A dot blot showing the constituents present in the SMAP28-IgG antibody
conjugate. A SMAP28-ovalbumin conjugate was prepared as a control. In the left
figure, rabbit IgG antibody was detected in the SMAP28-E. coli IgG antibody conjugate
(1), the E. coli IgG antibody control (2), the SMAP28-P.gingivalis IgG antibody
conjugate (3), and the P. gingivalis IgG antibody control (4) but not in the SMAP28–
ovalbumin conjugate (5) nor the ovalbumin control (6). In the right figure, SMAP28 was
detected in the SMAP28-E. coli IgG antibody conjugate (1) and the SMAP28P.gingivalis IgG antibody conjugate (3) but not the E. coli IgG antibody control (2), the
P. gingivalis IgG antibody control (4) the SMAP28–ovalbumin conjugate (5) nor the
ovalbumin control (6).
60
Figure A9. A diagram showing the method used to prepare the mixed microbial culture
of P. aeruginosa, S. marcescens, S. aureus, and E. coli. 0.8 ml of this mixed culture was
added to 0.8 ml of the SMAP28-E. coli IgG antibody conjugate, the antibody control, or
the saline control. At 0, 30, 60, 120, and 240 minutes, 0.1 ml was removed and plated on
Trypticase Soy Agar containing 5% defibrinated sheep blood. After n24 hours, colonies
were then counted.
61
Figure A10. A diagram showing the method used to prepare the mixed microbial culture
of P. gingivalis, P. micros, and A. actinomycetemcomitans. Similarly, 0.8 ml of this
mixed culture was added to 0.8 ml of the SMAP28-P. gingivalis IgG antibody conjugate
IIA, the SMAP28-P. gingivalis IgG antibody conjugate IIB, the P. gingivalis IgG
antibody control, or the IgG antibody control. At 0, 10, 20, and 30 minutes, 0.1 ml was
removed and plated onto pre-reduced, Anaerobic Blood Agar (CDC formulation, Remel,
Lenexa, KS) and incubated anaerobically at 37oC. After 9 days, bacterial colonies of
differing morphologies, sizes, and colors were counted by 3 different observers.
62
Figure A11. After attaching maleimide, the SMAP28+maleimide linker was assessed by
MALFI-TOF analysis to determine if and how many maleimide linkers were attached.
The linker was successfully attached to SMAP28 and four distinct peaks were seen. Each
peak corresponded to the molecular mass of SMAP28 (i.e., 3,198.95 Da) and number of
maleimide linkers attached (i.e., 219.24 Da). Predicted masses were 3,418.19 Da
(SMAP28+1 maleimide linker), 3,637.43 Da (SMAP28+2 maleimide linkers), 3,856.67
Da (SMAP28+3 maleimide linkers), and 4,075.91 Da (SMAP28+4 maleimide linkers).
Distinct peaks were seen at 3,419.54 m/z, 3638.75 m/z, 3,857.80 m/z, and 4,077.32 m/z.
Source: High Resolution Mass Spectrometry Facility, University of Iowa, Iowa City, IA
63
Figure A12. The SMAP28+maleimide linker solution was separated into fractions by
HPLC, dried, and assessed by MALDI-TOF for content. The SMAP28+maleimide linker
solution contained four broad peaks, which eluted at 13.514 minutes (fraction 14), 14.306
minutes (fraction 15), 14.815 minutes (fraction 15), and 15.630 minutes (fraction 16).
Fraction 15 contained masses 3,199.64 m/z, 3,418.32 m/z, and 3,637.90 m/z
corresponding to SMAP 28, SMAP28+1 maleimide linker, and SMAP28+2 maleimide
linkers, respectively. Fraction 16 contained masses 3,419.65 m/z, 3,638.87 m/zDa, and
3,860.11 m/z corresponding to SMAP28+1 maleimide linker, SMAP28+2 maleimide
linkers, and SMAP28+3 maleimide linkers, respectively. In other runs, a Fraction 17
contained masses 3,419.35 m/z, 3,637.90 m/z, and 3,860.03 m/z corresponding to
SMAP28+1 maleimide linker, SMAP28+2 maleimide linkers, and SMAP28+3
maleimide linkers, respectively (not shown here)
64
Figure A13. The E. coli IgG conjugate was too concentrated and showed non-specific
killing for both E. coli and S. aureus and needed to be diluted to show differential,
specific killing. The minimal dilution for killing E. coli was 1:8 (0.125) and the minimal
dilution for killing S. aureus was 1:4 (0.25).
65
A.
C.
B.
D.
Figure A14. The SMAP28-E. coli IgG antibody conjugate was antimicrobial for E. coli
in the presence of a microbial community containing E. coli, P. aeruginosa, S.
marcescens, and S. aureus. The concentrations of E. coli (A), P. aeruginosa (B), and S.
aureus (C) declined in the presence of the SMAP28-E. coli IgG antibody conjugate. In
this conjugate, S. marcescens was resistant (D).
66
Figure A15. In the mixed microbial culture at 0 minutes, E. coli grows as a grayish
colony around 2 mm in diameter, S. aureus grows as a yellow colony 1 mm in diameter,
P. aeruginosa grows as a pinpoint gray colony, and S. marcescens is an easily visible red
colony. In the SMAP28-E. coli IgG antibody conjugate, S. aureus was very susceptible
and was killed almost instantly; E. coli was also very susceptible and was killed within 30
minutes; and P. aeruginosa was susceptible and was killed within 120 minutes. S.
marcescens was resistant and Colony forming units ranged from a mean of 68.7 to 97
CFU throughout the 240 minute incubation period.
67
Figure A16. The specificity and activity of the SMAP28-P. gingivalis IgG antibody
conjugate, coupled by option IIA containing 100 µg total protein/ml (50 ug total
protein/ml, final concentration) was determined. At this concentration, there was no
difference in the antimicrobial activity of IIA for different microorganisms as it was
highly antimicrobial.
68
Figure A17. The specificity and activity of the SMAP28-P. gingivalis IgG antibody
conjugate, coupled by option IIB and containing 100 µg total protein/ml (50 ug total
protein/ml, fial concentration) was determined. At this concentration, there was no
difference in the antimicrobial activity of IIB for different microorganisms as it was
highly antimicrobial.
69
Figure A18. A graph showing the rapid drop of viability of P. micros incubated in
reduced, anaerobic saline. This drop in viability complicated the assessment of the
SMAP28-P. gingivalis IgG antibody conjugate for P. micros in the mixed microbial
community.
70
CONJ_IIA Sep-09-06
70
60
CFU
50
40
30
PG
20
10
AA
PM
0
0
10
20
30
Time
CONJ_IIA Nov-09-06
450
PG
400
AA
350
PM
CFU
300
250
200
150
100
50
0
0
10
20
30
Time
Figure A19. SMAP28-IgG Pg Antibody Conjugate Mediated Killing in Mixed Microbial
Culture (40 µg/ml) Method IIA.
71
CONJ_IIB Sep-09-06
80
PG
70
AA
60
PM
CFU
50
40
30
20
10
0
0
10
20
30
Time
CONJ_IIB Nov-09-06
250
200
PG
AA
150
CFU
PM
100
50
0
0
10
20
30
Time
Figure A20. SMAP28-IgG Pg Antibody Conjugate Mediated Killing in Mixed Microbial
Culture (40 µg/ml) Method IIB.
72
IGGAB_CTL Sep-09-06
90
80
PG
70
AA
CFU
60
PM
50
40
30
20
10
0
0
10
20
30
Time
IGGAB_CTL Nov-09-06
400
PG
350
AA
300
PM
CFU
250
200
150
100
50
0
0
10
20
30
Time
Figure A21. Pre-Immune IgG Antibody Control Mediated Killing in Mixed Microbial
Culture (40 µg/ml).
73
PG Sep-09-06
90
80
70
CFU
60
50
40
CONJ_IIA
30
CONJ_IIB
20
IGGAB_CTL
10
PGAB_CTL
0
0
10
20
30
20
30
Time
PG Nov-03-06
250
200
CFU
150
100
CONJ_IIA
CONJ_IIB
50
IGGAB_CTL
0
0
10
Time
Figure A22. The increased antimicrobial activity of the SMAP28-P. gingivalis IgG
antibody conjugate option IIB (CONJ_IIB) for P. gingivalis compared to SMAP28-P.
gingivalis IgG antibody conjugate option IIA (CONJ_IIA), P. gingivalis IgG antibody
(PGAB_CTL), or control antibody (IGGAB_CTL) on two separate days.
74
PM Sep-09-06
70
CONJ_IIA
60
CONJ_IIB
CFU
50
IGGAB_CTL
PGAB_CTL
40
30
20
10
0
0
10
20
30
20
30
Time
PM Nov-03-06
450
400
350
CFU
300
250
CONJ_IIA
200
CONJ_IIB
150
IGGAB_CTL
100
50
0
0
10
Time
Figure A23. The rapid drop in viability of P. micros in the SMAP28-P. gingivalis IgG
antibody conjugate option IIB (CONJ_IIB), SMAP28-P. gingivalis IgG antibody
conjugate option IIA (CONJ_IIA), P. gingivalis IgG antibody (PGAB_CTL), and control
antibody (IGGAB_CTL) on two separate days. This rapid drop in viability is similar to
that seen Figure A18.
75
AA Sep-09-06
45
40
35
CFU
30
25
20
CONJ_IIA
15
CONJ_IIB
10
IGGAB_CTL
5
PGAB_CTL
0
0
10
20
30
20
30
Time
AA Nov-03-06
120
100
CFU
80
60
40
CONJ_IIA
CONJ_IIB
20
IGGAB_CTL
0
0
10
Time
Figure A24. The decreased antimicrobial activity of the SMAP28-P. gingivalis IgG
antibody conjugate option IIB (CONJ_IIB) for A. actinomycetemcomitans compared to
SMAP28-P. gingivalis IgG antibody conjugate option IIA (CONJ_IIA), P. gingivalis IgG
antibody (PGAB_CTL), or control antibody (IGGAB_CTL) on two separate days.
Antimicrobial activity was no different than other solutions on September 9, 2006 but
slightly more active on November 3, 2006.
76
Figure A25. The decline of P. gingivalis is visible after incubation in the SMAP28-P.
gingivalis IgG antibody conjugate option IIB. After 30 minutes there was a clear
reduction of small black P. gingivalis colonies. The numbers of colonies of A.
actinomycetemcomitans and P. micros remain unchanged.
77
REFERENCES
1.
Qiu, X. Q.; Wang, H.; Lu, X. F.; Zhang, J.; Li, S. F.; Cheng, G.; Wan, L.; Yang,
L.; Zuo, J. Y.; Zhou, Y. Q.; Wang, H. Y.; Cheng, X.; Zhang, S. H.; Ou, Z. R.; Zhong, Z.
C.; Cheng, J. Q.; Li, Y. P.; Wu, G. Y., An engineered multidomain bactericidal peptide as
a model for targeted antibiotics against specific bacteria. Nat Biotechnol 2003, 21, (12),
1480-5.
2.
Socransky, S. S.; Haffajee, A. D., The bacterial etiology of destructive periodontal
disease: current concepts. J Periodontol 1992, 63, (4 Suppl), 322-31.
3.
Brogden, K. A.; Ackermann, M.; Zabner, J.; Welsh, M. J., Antimicrobial peptides
suppress microbial infections and sepsis in animal models. In Mammalian Host Defense
Peptides, Devine, D. A.; Hancock, R. E. W., Eds. Cambridge University Press:
Cambridge, 2004; pp 189-228.
4.
Brown, L. J.; Loe, H., Prevalence, extent, severity and progression of periodontal
disease. Periodontol 2000 1993, 2, 57-71.
5.
Medzhitov, R.; Janeway, C., Jr., The toll receptor family and microbial
recognition. Trends in Microbiology 2000, 8, (10), 452-6.
6.
Germain, R. N., The art of the probable: system control in the adaptive immune
system. Science 2001, 293, (5528), 240-5.
7.
Medzhitov, R.; Janeway, C., Jr., Innate immune recognition: mechanisms and
pathways. Immunol Rev 2000, 173, 89-97.
8.
Holmskov, U. L., Collectins and collectin receptors in innate immunity. APMIS
Suppl 2000, 100, 1-59.
9.
Bendelac, A.; Fearon, D. T., Innate pathways that control acquired immunity.
Curr Opin Immunol 1997, 9, (1), 1-3.
10.
Medzhitov, R.; Janeway, C. A., Jr., An ancient system of host defense. Curr Opin
Immunol 1998, 10, (1), 12-5.
11.
Unanue, E. R., Inter-relationship among macrophages, natural killer cells and
neutrophils in early stages of Listeria resistance. Curr Opin Immunol 1997, 9, (1), 35-43.
12.
Fearon, D. T., Seeking wisdom in innate immunity. Nature 1997, 388, (6640),
323-4.
78
13.
Dempsey, P. W.; Allison, M. E.; Akkaraju, S.; Goodnow, C. C.; Fearon, D. T.,
C3d of complement as a molecular adjuvant: bridging innate and acquired immunity.
Science 1996, 271, (5247), 348-50.
14.
Song, W.; Sarrias, M. R.; Lambris, J. D., Complement and innate immunity.
Immunopharmacology 2000, 49, (1-2), 187-98.
15.
Prodinger, W. M., Complement receptor type two (CR2,CR21): a target for
influencing the humoral immune response and antigen-trapping. Immunol Res 1999, 20,
(3), 187-94.
16.
Jiang, W.; Swiggard, W. J.; Heufler, C.; Peng, M.; Mirza, A.; Steinman, R. M.;
Nussenzweig, M. C., The receptor DEC-205 expressed by dendritic cells and thymic
epithelial cells is involved in antigen processing. Nature 1995, 375, (6527), 151-5.
17.
Vella, A. T.; McCormack, J. E.; Linsley, P. S.; Kappler, J. W.; Marrack, P.,
Lipopolysaccharide interferes with the induction of peripheral T cell death. Immunity
1995, 2, (3), 261-70.
18.
Lenschow, D. J.; Walunas, T. L.; Bluestone, J. A., CD28/B7 system of T cell
costimulation. Annu Rev Immunol 1996, 14, 233-58.
19.
Harris, N. L.; Ronchese, F., The role of B7 costimulation in T-cell immunity.
Immunol Cell Biol 1999, 77, (4), 304-11.
20.
Slavik, J. M.; Hutchcroft, J. E.; Bierer, B. E., CD28/CTLA-4 and CD80/CD86
families: signaling and function. Immunol Res 1999, 19, (1), 1-24.
21.
Greenfield, E. A.; Nguyen, K. A.; Kuchroo, V. K., CD28/B7 costimulation: a
review. Crit Rev Immunol 1998, 18, (5), 389-418.
22.
Fearon, D. T.; Locksley, R. M., The instructive role of innate immunity in the
acquired immune response. Science 1996, 272, (5258), 50-3.
23.
Janeway, C. A., Jr.; Medzhitov, R., Innate immune recognition. Annu Rev
Immunol 2002, 20, 197-216.
24.
Bendelac, A.; Medzhitov, R., Adjuvants of immunity: harnessing innate immunity
to promote adaptive immunity. J Exp Med 2002, 195, (5), F19-23.
25.
Boman, H. G., Peptide antibiotics and their role in innate immunity. Annual
Review of Immunology 1995, 13, 61-92.
26.
Hayday, A.; Viney, J. L., The ins and outs of body surface immunology. Science
2000, 290, (5489), 97-100.
79
27.
Lieberman, N.; Mandelboim, O., The role of NK cells in innate immunity. Adv
Exp Med Biol 2000, 479, 137-45.
28.
Virella, G.; Goudswaard, J., Measurement of salivary lysozyme. J Dent Res 1978,
57, (2), 326-8.
29.
Schenkels, L. C.; Veerman, E. C.; Nieuw Amerongen, A. V., Biochemical
composition of human saliva in relation to other mucosal fluids. Crit Rev Oral Biol Med
1995, 6, (2), 161-75.
30.
Groenink, J.; Walgreen-Weterings, E.; Nazmi, K.; Bolscher, J. G.; Veerman, E.
C.; van Winkelhoff, A. J.; Nieuw Amerongen, A. V., Salivary lactoferrin and low-Mr
mucin MG2 in Actinobacillus actinomycetemcomitans-associated periodontitis. J Clin
Periodontol 1999, 26, (5), 269-75.
31.
Cumberbatch, M.; Dearman, R. J.; Uribe-Luna, S.; Headon, D. R.; Ward, P. P.;
Conneely, O. M.; Kimber, I., Regulation of epidermal Langerhans cell migration by
lactoferrin. Immunology 2000, 100, (1), 21-8.
32.
Lamkin, M. S.; Oppenheim, F. G., Structural features of salivary function. Crit
Rev Oral Biol Med 1993, 4, (3-4), 251-9.
33.
Sung, Y. K.; Moon, C.; Yoo, J. Y.; Pearse, D.; Pevsner, J.; Ronnett, G. V., Plunc,
a member of the secretory gland protein family, is up-regulated in nasal respiratory
epithelium after olfactory bulbectomy. J Biol Chem 2002, 277, (15), 12762-9.
34.
Wheeler, T. T.; Haigh, B. J.; McCracken, J. Y.; Wilkins, R. J.; Morris, C. A.;
Grigor, M. R., The BSP30 salivary proteins from cattle, LUNX/PLUNC and von Ebner's
minor salivary gland protein are members of the PSP/LBP superfamily of proteins.
Biochim Biophys Acta 2002, 1579, (2-3), 92-100.
35.
Bingle, C. D.; Craven, C. J., PLUNC: a novel family of candidate host defence
proteins expressed in the upper airways and nasopharynx. Hum Mol Genet 2002, 11, (8),
937-43.
36.
Antonyraj, K. J.; Karunakaran, T.; Raj, P. A., Bactericidal activity and poly-Lproline II conformation of the tandem repeat sequence of human salivary mucin
glycoprotein (MG2). Arch Biochem Biophys 1998, 356, (2), 197-206.
37.
Raj, P. A.; Marcus, E.; Sukumaran, D. K., Structure of human salivary histatin 5
in aqueous and nonaqueous solutions. Biopolymers 1998, 45, (1), 51-67.
38.
Mathews, M.; Jia, H. P.; Guthmiller, J. M.; Losh, G.; Graham, S.; Johnson, G. K.;
Tack, B. F.; McCray, P. B., Jr., Production of -defensin antimicrobial peptides by the
oral mucosa and salivary glands. Infection and Immunity 1999, 67, (6), 2740-5.
80
39.
Krisanaprakornkit, S.; Weinberg, A.; Perez, C. N.; Dale, B. A., Expression of the
peptide antibiotic human beta-defensin 1 in cultured gingival epithelial cells and gingival
tissue. Infect Immun 1998, 66, (9), 4222-8.
40.
Krisanaprakornkit, S.; Weinberg, A.; Perez, C. N.; Dale, B. A., Expression of the
peptide antibiotic human -defensin 1 in cultured gingival epithelial cells and gingival
tissue. Infection and Immunity 1998, 66, (9), 4222-4228.
41.
Weinberg, A.; Krisanaprakornkit, S.; Dale, B. A., Epithelial antimicrobial
peptides: review and significance for oral applications. Critical Reviews in Oral Biology
& Medicine 1998, 9, (4), 399-414.
42.
Harder, J.; Bartels, J.; Christophers, E.; Schroder, J. M., Isolation and
characterization of human -Defensin-3, a novel human inducible peptide antibiotic.
Journal of Biological Chemistry 2001, 276, 5707-5713.
43.
Jia, H. P.; Schutte, B. C.; Schudy, A.; Linzmeier, R.; Guthmiller, J. M.; Johnson,
G. K.; Tack, B. F.; Mitros, J. P.; Rosenthal, A.; Ganz, T.; McCray, P. B., Jr., Discovery
of new human -defensins using a genomics-based approach. Gene 2001, In press.
44.
Garcia, J. R.; Krause, A.; Schulz, S.; Rodriguez-Jimenez, F. J.; Kluver, E.;
Adermann, K.; Forssmann, U.; Frimpong-Boateng, A.; Bals, R.; Forssmann, W. G.,
Human -defensin 4: a novel inducible peptide with a specific salt- sensitive spectrum of
antimicrobial activity. Federation of American Societies for Experimental Biology
Journal 2001, 15, (10), 1819-21.
45.
Frohm Nilsson, M.; Sandstedt, B.; Sorensen, O.; Weber, G.; Borregaard, N.;
Stahle-Backdahl, M., The human cationic antimicrobial protein (hCAP18), a peptide
antibiotic, is widely expressed in human squamous epithelia and colocalizes with
interleukin-6. Infect Immun 1999, 67, (5), 2561-6.
46.
Chertov, O.; Yang, D.; Howard, O. M.; Oppenheim, J. J., Leukocyte granule
proteins mobilize innate host defenses and adaptive immune responses. Immunol Rev
2000, 177, 68-78.
47.
Yang, D.; Chertov, O.; Oppenheim, J. J., The role of mammalian antimicrobial
peptides and proteins in awakening of innate host defenses and adaptive immunity. Cell
Mol Life Sci 2001, 58, (7), 978-89.
48.
McNeela, E. A.; Mills, K. H., Manipulating the immune system: humoral versus
cell-mediated immunity. Adv Drug Deliv Rev 2001, 51, (1-3), 43-54.
49.
Neurath, M. F.; Finotto, S.; Glimcher, L. H., The role of Th1/Th2 polarization in
mucosal immunity. Nat Med 2002, 8, (6), 567-73.
81
50.
Kidd, P., Th1/Th2 balance: the hypothesis, its limitations, and implications for
health and disease. Altern Med Rev 2003, 8, (3), 223-46.
51.
Flynn, J. L.; Chan, J., Immunology of tuberculosis. Annu Rev Immunol 2001, 19,
93-129.
52.
Yingst, S.; Hoover, D. L., T cell immunity to brucellosis. Crit Rev Microbiol
2003, 29, (4), 313-31.
53.
Penttila, J. M.; Anttila, M.; Puolakkainen, M.; Laurila, A.; Varkila, K.; Sarvas,
M.; Makela, P. H.; Rautonen, N., Local immune responses to Chlamydia pneumoniae in
the lungs of BALB/c mice during primary infection and reinfection. Infect Immun 1998,
66, (11), 5113-8.
54.
Esche, C.; Stellato, C.; Beck, L. A., Chemokines: key players in innate and
adaptive immunity. J Invest Dermatol 2005, 125, (4), 615-28.
55.
Scocchi, M.; Wang, S. L.; Zanetti, M., Structural organization of the bovine
cathelicidin gene family and identification of a novel member. FEBS Letters 1997, 417,
(3), 311-315.
56.
Scocchi, M.; Bontempo, D.; Boscolo, S.; Tomasinsig, L.; Giulotto, E.; Zanetti,
M., Novel cathelicidins in horse leukocytes. FEBS Lett 1999, 457, (3), 459-64.
57.
Gennaro, R.; Zanetti, M., Structural features and biological activities of the
cathelicidin- derived antimicrobial peptides. Biopolymers 2000, 55, (1), 31-49.
58.
Travis, S. M.; Anderson, N. N.; Forsyth, W. R.; Espiritu, C.; Conway, B. D.;
Greenberg, E. P.; McCray, P. B., Jr.; Lehrer, R. I.; Welsh, M. J.; Tack, B. F., Bactericidal
activity of mammalian cathelicidin-derived peptides. Infect Immun 2000, 68, (5), 274855.
59.
Zanetti, M.; Gennaro, R.; Romeo, D., Cathelicidins: a novel protein family with a
common proregion and a variabe C-terminal antimicrobial domain. Federation of
European Biological Sciences Letters 1995, 374, 1-5.
60.
Brogden, K. A.; Ackermann, M.; McCray, P. B.; Tack, B. F., Antimicrobial
peptides in animals and their role in host defences. Int J Antimicrob Agents 2003, 22, (5),
465-78.
61.
Brogden, K. A.; Kalfa, V. C.; Ackermann, M. R.; Palmquist, D. E.; McCray, P.
B., Jr.; Tack, B. F., The ovine cathelicidin SMAP29 kills ovine respiratory pathogens in
vitro and in an ovine model of pulmonary infection. Antimicrobial Agents and
Chemotherapy 2001, 45, (1), 331-4.
82
62.
Bagella, L.; Scocchi, M.; Zanetti, M., cDNA sequences of three sheep myeloid
cathelicidins. Federation of European Biological Sciences Letters 1995, 376, (3), 225228.
63.
Mahoney, M. M.; Lee, A. Y.; Brezinski-Caliguri, D. J.; Huttner, K. M., Molecular
analysis of the sheep cathelin family reveals a novel antimicrobial peptide. Federation of
European Biological Sciences Letters 1995, 377, 519-522.
64.
Skerlavaj, B.; Benincasa, M.; Risso, A.; Zanetti, M.; Gennaro, R., SMAP-29: a
potent antibacterial and antifungal peptide from sheep leukocytes. Federation of
European Biological Sciences Letters 1999, 463, (1-2), 58-62.
65.
Saiman, L.; Tabibi, S.; Starner, T. D.; San Gabriel, P.; Winokur, P. L.; Jia, H. P.;
McCray, P. B., Jr.; Tack, B. F., Cathelicidin peptides inhibit multiply antibiotic-resistant
pathogens from patients with cystic fibrosis. Antimicrob Agents Chemother 2001, 45,
(10), 2838-44.
66.
Guthmiller, J. M.; Vargas, K. G.; Srikantha, R.; Schomberg, L. L.; Weistroffer, P.
L.; McCray, P. B., Jr.; Tack, B. F., Susceptibilities of oral bacteria and yeast to
mammalian cathelicidins. Antimicrob Agents Chemother 2001, 45, (11), 3216-9.
67.
Weistroffer, P. L., S. Joly, R. Srikantha, B. Tack, K. Brogden, and J. Guthmiller,
SMAP29 congeners demonstrate activity against oral bacteria and reduced toxicity
against oral keratinocytes. OMI - Oral Microbiology and Immunology 2007, in press.
68.
Kalfa, V. C.; Jia, H. P.; Kunkle, R. A.; McCray, P. B., Jr.; Tack, B. F.; Brogden,
K. A., Congeners of SMAP29 kill ovine pathogens and induce ultrastructural damage in
bacterial cells. Antimicrobial Agents and Chemotherapy 2001, 45, (11), 3256-61.
69.
AAM In Microbial Communities: From life apart to life together, Microbial
Communities: From life apart to life together, Tucson, AZ, May 3-5, 2002, 2003;
American Academy of Microbiology: Tucson, AZ, 2003.
70.
Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P., Bacterial biofilms: from the
natural environment to infectious diseases. Nat Rev Microbiol 2004, 2, (2), 95-108.
71.
Tannock, G., Normal Microflora. Chapman & Hall: 1995.
72.
Aas, J. A.; Paster, B. J.; Stokes, L. N.; Olsen, I.; Dewhirst, F. E., Defining the
normal bacterial flora of the oral cavity. J Clin Microbiol 2005, 43, (11), 5721-32.
73.
Tannock, G., Medical Importance of the Normal Microflora. Kluwer Academic
Publishers: 1999.
83
74.
Tanner, A. C.; Paster, B. J.; Lu, S. C.; Kanasi, E.; Kent, R., Jr.; Van Dyke, T.;
Sonis, S. T., Subgingival and Tongue Microbiota during Early Periodontitis. J Dent Res
2006, 85, (4), 318-23.
75.
Guthmiller, J. M.; Novak, K. F., Periodontal diseases. In Polymicrobial diseases,
Brogden, K. A.; Guthmiller, J. M., Eds. ASM Press: Washington, D.C., 2002; pp 137152.
76.
Kolenbrander, P. E., Coaggregation of human oral bacteria: potential role in the
accretion of dental plaque. J Appl Bacteriol 1993, 74, (Suppl), 79S-86S.
77.
Offenbacher, S., Periodontal diseases: pathogenesis. Ann Periodontol 1996, 1, (1),
821-78.
78.
Socransky, S. S.; Smith, C.; Haffajee, A. D., Subgingival microbial profiles in
refractory periodontal disease. J Clin Periodontol 2002, 29, (3), 260-8.
79.
Slots, J.; Ting, M., Systemic antibiotics in the treatment of periodontal disease.
Periodontol 2000 2002, 28, 106-76.
80.
Haffajee, A. D.; Socransky, S. S.; Gunsolley, J. C., Systemic anti-infective
periodontal therapy. A systematic review. Ann Periodontol 2003, 8, (1), 115-81.
81.
Herrera, D.; Sanz, M.; Jepsen, S.; Needleman, I.; Roldan, S., A systematic review
on the effect of systemic antimicrobials as an adjunct to scaling and root planing in
periodontitis patients. J Clin Periodontol 2002, 29 Suppl 3, 136-59; discussion 160-2.
82.
Qiu, X. Q.; Zhang, J.; Wang, H.; Wu, G. Y., A novel engineered peptide, a
narrow-spectrum antibiotic, is effective against vancomycin-resistant Enterococcus
faecalis. Antimicrob Agents Chemother 2005, 49, (3), 1184-9.
83.
Quirynen, M.; Teughels, W.; van Steenberghe, D., Microbial shifts after
subgingival debridement and formation of bacterial resistance when combined with local
or systemic antimicrobials. Oral Dis 2003, 9 Suppl 1, 30-7.
84.
Finlay, B. B.; Hancock, R. E., Can innate immunity be enhanced to treat
microbial infections? Nat Rev Microbiol 2004, 2, (6), 497-504.
85.
Hancock, R. E.; Patrzykat, A., Clinical development of cationic antimicrobial
peptides: from natural to novel antibiotics. Curr Drug Targets Infect Disord 2002, 2, (1),
79-83.
86.
Peschen, D.; Li, H. P.; Fischer, R.; Kreuzaler, F.; Liao, Y. C., Fusion proteins
comprising a Fusarium-specific antibody linked to antifungal peptides protect plants
against a fungal pathogen. Nat Biotechnol 2004, 22, (6), 732-8.
84
87.
Eckert, R.; He, J.; Yarbrough, D. K.; Qi, F.; Anderson, M. H.; Shi, W., Targeted
killing of Streptococcus mutans by a pheromone-guided "smart" antimicrobial peptide.
Antimicrob Agents Chemother 2006, 50, (11), 3651-7.
88.
Joly, S.; Maze, C.; McCray, P. B., Jr.; Guthmiller, J. M., Human beta-defensins 2
and 3 demonstrate strain-selective activity against oral microorganisms. J Clin Microbiol
2004, 42, (3), 1024-9.
89.
Steinberg, D. A.; Lehrer, R. I., Designer assays for antimicrobial peptides.
Disputing the "one-size- fits-all" theory. Methods Mol Biol 1997, 78, 169-86.
90.
Paster, B. J.; Boches, S. K.; Galvin, J. L.; Ericson, R. E.; Lau, C. N.; Levanos, V.
A.; Sahasrabudhe, A.; Dewhirst, F. E., Bacterial diversity in human subgingival plaque. J
Bacteriol 2001, 183, (12), 3770-83.
91.
Hutter, G.; Schlagenhauf, U.; Valenza, G.; Horn, M.; Burgemeister, S.; Claus, H.;
Vogel, U., Molecular analysis of bacteria in periodontitis: evaluation of clone libraries,
novel phylotypes and putative pathogens. Microbiology 2003, 149, (Pt 1), 67-75.
92.
Ledder, R. G.; Gilbert, P.; Huws, S. A.; Aarons, L.; Ashley, M. P.; Hull, P. S.;
McBain, A. J., Molecular analysis of the subgingival microbiota in health and disease.
Appl Environ Microbiol 2007, 73, (2), 516-23.
93.
Anderson, R. C.; Hancock, R. E.; Yu, P. L., Antimicrobial activity and bacterialmembrane interaction of ovine-derived cathelicidins. Antimicrob Agents Chemother
2004, 48, (2), 673-6.
94.
Giacometti, A.; Cirioni, O.; Del Prete, M. S.; Skerlavaj, B.; Circo, R.; Zanetti, M.;
Scalise, G., In vitro effect on Cryptosporidium parvum of short-term exposure to
cathelicidin peptides. J Antimicrob Chemother 2003, 51, (4), 843-7.
95.
Sambri, V.; Marangoni, A.; Giacani, L.; Gennaro, R.; Murgia, R.; Cevenini, R.;
Cinco, M., Comparative in vitro activity of five cathelicidin-derived synthetic peptides
against Leptospira, Borrelia and Treponema pallidum. J Antimicrob Chemother 2002, 50,
(6), 895-902.
96.
Lee, D. G.; Kim, P. I.; Park, Y.; Park, S. C.; Woo, E. R.; Hahm, K. S., Antifungal
mechanism of SMAP-29 (1-18) isolated from sheep myeloid mRNA against
Trichosporon beigelii. Biochem Biophys Res Commun 2002, 295, (3), 591-6.
97.
Brogden, K. A., Antimicrobial peptides: pore formers or metabolic inhibitors in
bacteria? Nat Rev Microbiol 2005, 3, (3), 238-50.
98.
Huang, H. W., Action of antimicrobial peptides: two-state model. Biochemistry
2000, 39, (29), 8347-52.
85
99.
Yang, L.; Harroun, T. A.; Weiss, T. M.; Ding, L.; Huang, H. W., Barrel-stave
model or toroidal model? A case study on melittin pores. Biophys J 2001, 81, (3), 147585.
100. Ehrenstein, G.; Lecar, H., Electrically gated ionic channels in lipid bilayers. Q
Rev Biophys 1977, 10, (1), 1-34.
101. Matsuzaki, K.; Murase, O.; Fujii, N.; Miyajima, K., An antimicrobial peptide,
magainin 2, induced rapid flip-flop of phospholipids coupled with pore formation and
peptide translocation. Biochemistry 1996, 35, (35), 11361-8.
102. Hallock, K. J.; Lee, D. K.; Ramamoorthy, A., MSI-78, an analogue of the
magainin antimicrobial peptides, disrupts lipid bilayer structure via positive curvature
strain. Biophys J 2003, 84, (5), 3052-60.
103. Pouny, Y.; Rapaport, D.; Mor, A.; Nicolas, P.; Shai, Y., Interaction of
antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid
membranes. Biochemistry 1992, 31, (49), 12416-23.
104. Yamaguchi, S.; Huster, D.; Waring, A.; Lehrer, R. I.; Kearney, W.; Tack, B. F.;
Hong, M., Orientation and Dynamics of an Antimicrobial Peptide in the Lipid Bilayer by
Solid-State NMR Spectroscopy. Biophys J 2001, 81, (4), 2203-14.
105. Bechinger, B., The structure, dynamics and orientation of antimicrobial peptides
in membranes by multidimensional solid-state NMR spectroscopy. Biochim Biophys Acta
1999, 1462, (1-2), 157-83.
106. Shai, Y., Mechanism of the binding, insertion and destabilization of phospholipid
bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic
peptides. Biochim Biophys Acta 1999, 1462, (1-2), 55-70.
107. Ladokhin, A. S.; White, S. H., 'Detergent-like' permeabilization of anionic lipid
vesicles by melittin. Biochim Biophys Acta 2001, 1514, (2), 253-60.
108. Oren, Z.; Shai, Y., Mode of action of linear amphipathic alpha-helical
antimicrobial peptides. Biopolymers 1998, 47, (6), 451-63.
109. Haynie, S. L.; Crum, G. A.; Doele, B. A., Antimicrobial activities of amphiphilic
peptides covalently bonded to a water-insoluble resin. Antimicrob Agents Chemother
1995, 39, (2), 301-7.
110. Guerra, N. P.; Araujo, A. B.; Barrera, A. M.; Agrasar, A. T.; Macias, C. L.;
Carballo, J.; Pastrana, L., Antimicrobial activity of nisin adsorbed to surfaces commonly
used in the food industry. J Food Prot 2005, 68, (5), 1012-9.
86
111. Cagri, A.; Ustunol, Z.; Ryser, E. T., Antimicrobial edible films and coatings. J
Food Prot 2004, 67, (4), 833-48.
112. Cutter, C. N.; Willett, J. L.; Siragusa, G. R., Improved antimicrobial activity of
nisin-incorporated polymer films by formulation change and addition of food grade
chelator. Lett Appl Microbiol 2001, 33, (4), 325-8.
113. Ganz, T., Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol
2003, 3, (9), 710-20.
114. Fujii, G.; Selsted, M. E.; Eisenberg, D., Defensins promote fusion and lysis of
negatively charged membranes. Protein Sci 1993, 2, (8), 1301-12.
115. Kagan, B. L.; Selsted, M. E.; Ganz, T.; Lehrer, R. I., Antimicrobial defensin
peptides form voltage-dependent ion-permeable channels in planar lipid bilayer
membranes. Proc Natl Acad Sci U S A 1990, 87, (1), 210-4.
116. Hristova, K.; Selsted, M. E.; White, S. H., Critical role of lipid composition in
membrane permeabilization by rabbit neutrophil defensins. Journal of Biological
Chemistry 1997, 272, (39), 24224-24233.
117. Wimley, W. C.; Selsted, M. E.; White, S. H., Interactions between human
defensins and lipid bilayers: evidence for formation of multimeric pores. Protein Science
1994, 3, 1362-1373.
118. Etienne, O.; Picart, C.; Taddei, C.; Haikel, Y.; Dimarcq, J. L.; Schaaf, P.; Voegel,
J. C.; Ogier, J. A.; Egles, C., Multilayer polyelectrolyte films functionalized by insertion
of defensin: a new approach to protection of implants from bacterial colonization.
Antimicrob Agents Chemother 2004, 48, (10), 3662-9.
119. Walker, C. B.; Karpinia, K.; Baehni, P., Chemotherapeutics: antibiotics and other
antimicrobials. Periodontol 2000 2004, 36, 146-65.
120. Brogden, K. A.; De Lucca, A. J.; Bland, J.; Elliott, S., Isolation of an ovine
pulmonary surfactant-associated anionic peptide bactericidal for Pasteurella haemolytica.
Proceedings of the National Academy of Science U.S.A. 1996, 93, 412-416.
121. McCray, P. B., Jr.; Bentley, L., Human airway epithelia express a defensin.
American Journal of Respiratory Cell and Molecular Biology 1997, 16, 343-349.
122. Schnapp, D.; Harris, A., Antibacterial peptides in bronchoalveolar lavage fluid.
Am J Respir Cell Mol Biol 1998, 19, (3), 352-6.
123. Kaser, M. R.; Skouteris, G. G., Inhibition of bacterial growth by synthetic SP-B178 peptides. Peptides 1997, 18, (9), 1441-1444.
87
124. Arima, H.; Ibrahim, H. R.; Kinoshita, T.; Kato, A., Bactericidal action of
lysozymes attached with various sizes of hydrophobic peptides to the C-terminal using
genetic modification. FEBS Letters 1997, 415, (1), 114-118.
125. Hiemstra, P. S.; Maassen, R. J.; Stolk, J.; Heinzel-Wieland, R.; Steffens, G. J.;
Dijkman, J. H., Antibacterial activity of antileukoprotease. Infection and Immunity 1996,
64, (11), 4520-4524.
126. Tomita, M.; Takase, M.; Wakabayashi, H.; Bellamy, W., Antimicrobial peptides
of lactoferrin. Advances in Experimental Medicine and Biology 1994, 357, 209-218.
127. Bals, R.; Wang, X. R.; Zasloff, M.; Wilson, J. M., The peptide antibiotic LL37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial
activity at the airway surface. Proceedings of the National Academy of Science U.S.A.
1998, 95, (16), 9541-9546.
128. Franken, C.; Meijer, C. J.; Dijkman, J. H., Tissue distribution of antileukoprotease
and lysozyme in humans. J Histochem Cytochem 1989, 37, (4), 493-8.
129. Porter, E. M.; van Dam, E.; Valore, E. V.; Ganz, T., Broad-spectrum
antimicrobial activity of human intestinal defensin 5. Infect Immun 1997, 65, (6), 2396401.
130. Huttner, K. M., Antimicrobial peptide expression is developmentally regulated in
the ovine gastrointestinal tract. American Society for Nutritional Sciences 1998, 297S299S.
131. Eisenhauer, P. B.; Harwig, S. S. L.; Lehrer, R. I., Cryptdins: antimicrobial
defensins of the murine small intestine. Infection and Immunity 1992, 60, 3556-3565.
132. Quayle, A. J.; Porter, E. M.; Nussbaum, A. A.; Wang, Y. M.; Brabec, C.; Yip, K.
P.; Mok, S. C., Gene expression, immunolocalization, and secretion of human defensin-5
in human female reproductive tract. American Journal of Pathology 1998, 152, (5), 12471258.
133. Huttner, K. M.; Kozak, C. A.; Bevins, C. L., The mouse genome encodes a single
homolog of the antimicrobial peptide human beta-defensin 1. FEBS Letters 1997, 413,
(1), 45-49.
134. Grandjean, V.; Vincent, S.; Martin, L.; Rassoulzadegan, M.; Cuzin, F.,
Antimicrobial protection of the mouse testis: Synthesis of defensins of the cryptdin
family. Biology of Reproduction 1997, 57, (5), 1115-1122.
135. Wu, E. R.; Daniel, R.; Bateman, A., RK-2: a novel rabbit kidney defensin and its
implications for renal host defense. Peptides 1998, 19, (5), 793-9.
88
136. Krisanaprakornkit, S.; Kimball, J. R.; Weinberg, A.; Darveau, R. P.; Bainbridge,
B. W.; Dale, B. A., Inducible expression of human beta-defensin 2 by Fusobacterium
nucleatum in oral epithelial cells: multiple signaling pathways and role of commensal
bacteria in innate immunity and the epithelial barrier. Infect Immun 2000, 68, (5), 290715.
137. Dale, B. A.; Kimball, J. R.; Krisanaprakornkit, S.; Roberts, F.; Robinovitch, M.;
O'Neal, R.; Valore, E. V.; Ganz, T.; Anderson, G. M.; Weinberg, A., Localized
antimicrobial peptide expression in human gingiva. J Periodontal Res 2001, 36, (5), 28594.
138. Tack, B. F.; Sawai, M. V.; Kearney, W. R.; Robertson, A. D.; Sherman, M. A.;
Wang, W.; Hong, T.; Boo, L. M.; Wu, H.; Waring, A. J.; Lehrer, R. I., SMAP-29 has two
LPS-binding sites and a central hinge. European Journal of Biochemistry 2002, in press.