Examination of the Antibacterial and Immunostimulatory Activity of a Wasp Venom Peptide

170 Pages·2013·2.83 MB·English

by  Yuvon Mobley

Examination of the Antibacterial and
Immunostimulatory Activity of a Wasp Venom Peptide
by
Yuvon R. Mobley
Department of Molecular Genetics and Microbiology
Duke University

Date: ___ _______
Approved:

___________________________
Soman Abraham, Supervisor

___________________________
Kenneth Kreuzer

___________________________
Margarethe Kuehn

___________________________
Herman Staats

___________________________
Raphael Valdivia

Dissertation submitted in partial fulfillment of
the requirements for the degree of
Doctor of Philosophy in the Department of
Molecular Genetics and Microbiology in the Graduate School of
Duke University

2013 i
v
ABSTRACT
Examination of the Antibacterial and
Immunostimulatory Activity of a Wasp Venom Peptide
by
Yuvon R. Mobley
Department of Molecular Genetics and Microbiology
Duke University

Date: __ _ _______
Approved:

___________________________
Soman Abraham, Supervisor

___________________________
Kenneth Kreuzer

___________________________
Margarethe Kuehn

___________________________
Herman Staats

___________________________
Raphael Valdivia

An abstract of a dissertation submitted in partial
fulfillment of the requirements for the degree of
Doctor of Philosophy in the Department of
Molecular Genetics and Microbiology in the Graduate School of
Duke University

2013
Copyright by
Yuvon R. Mobley
2013
Abstract
Antimicrobial peptides (AMPs) are part of the innate immune system that
is widely distributed in nature, acting as a defense mechanism against invading
microorganisms. AMPs have potent antimicrobial activity against a range of
microorganisms including fungi, bacteria, and viruses. In view of growing
multidrug resistance, AMPs are increasingly being viewed as potential
therapeutic agents with a novel mechanism of action. Mastoparan is a natural,
highly positively charged AMP derived from the venom of wasps. It was
originally of interest based on its inherent mast cell degranulation activity.
Previously, mastoparan has been shown to exhibit antimicrobial activity in vitro;
however, these studies have been limited in scope. Here we hypothesize that
mastoparan possess the capacity to be a potent broad-spectrum antibacterial
agent including activity against multidrug resistant bacteria.
We examined the scope of antibacterial activity exhibited by mastoparan
using a variety of antimicrobial susceptibility tests and have utilized a bacterial
skin infection (Staphylococcus aureus) model to determine the potential of
mastoparan to serve as a therapeutic agent. We tested mastoparan against four

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Gram-positive clinical isolates (e.g., Staphylococcus aureus, and Enterococcus
faecium), nine Gram-negative clinical isolates (e.g., Escherichia coli, Pseudomonas
aeruginosa, and Burkholderia cepacia), and four multidrug resistant clinical isolates
(e.g., MRSA, ESBL Escherichia coli, and ESBL Klebsiella pneumonia). These studies
reveal that mastoparan exhibits broad-spectrum activity against both Gram-
negative (MIC: 1.9 – 125 μg/ml) and Gram-positive (MIC: 15.6 – 125 μg/ml)
bacteria and against multidrug resistant bacteria (MIC: 7.8 – 125 μg/ml). We also
demonstrated that mastoparan disrupts the bacterial membrane, exhibits fast
acting antibacterial activity, and is highly effective against both multiplying and
non-multiplying bacteria. Furthermore, we have shown that mastoparan
demonstrates efficacy as a topical antimicrobial agent reducing lesion size by up
to 79% and the amount of bacteria recovered from skin lesions by up to a 98%
reduction. Based on these results we conclude that mastoparan is a highly
effective antibacterial agent and is therefore a potential alternative to currently
antibiotics. Mastoparan offers a promising new therapeutic option for treating
bacterial infections.
.

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Table of Contents
Abstract............................................................................................................................. iv
List of Tables ..................................................................................................................... x
List of Figures .................................................................................................................. xi
Acknowledgements ...................................................................................................... xiii
Chapter 1: Introduction .................................................................................................. 1
1.1 Bacterial Infections ............................................................................................... 1
1.1.1 Bacterial host-pathogen interaction in humans .......................................... 4
1.1.2 Bacterial skin infections .................................................................................. 8
1.2 Antimicrobial Agents .......................................................................................... 9
1.2.1 Antimicrobial agents’ mechanism of action .............................................. 13
1.2.2 Bacterial resistance to antimicrobial agents .............................................. 17
1.2.3 Factors contributing to antimicrobial resistance ...................................... 21
1.2.4 Limitations of current antimicrobial agents .............................................. 26
1.2.5 Current perspectives on antimicrobial agent development ................... 28
1.2.5.1 Natural antimicrobial compounds ...................................................... 29
1.2.5.2 Immune stimulatory compounds ........................................................ 29
1.3 Antimicrobial Peptides ...................................................................................... 30
1.3.1 Immunomodulatory roles of antimicrobial peptides .............................. 34
1.3.2 Antimicrobial peptides in wasp venom..................................................... 35

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1.3.3 Mastoparans ................................................................................................... 37
1.4 Impetus for This Work ...................................................................................... 39
1.4.1 Evaluation of the antibacterial properties of mastoparan ....................... 40
1.4.2 Evaluation of the immunostimulatory properties of mastoparan ......... 41
1.4.3 Evaluation of the therapeutic potential of mastoparan ........................... 43
Chapter 2: Dual Action Immunomodulatory and Broad-Spectrum Antibacterial
Activity of Wasp Venom Peptide ................................................................................ 46
2.1 Introduction ........................................................................................................ 46
2.2 Materials and Methods ..................................................................................... 50
2.2.1 Bacteria strains and growth conditions ..................................................... 50
2.2.2 Antimicrobial agents .................................................................................... 50
2.2.3 Mice ................................................................................................................. 51
2.2.4 Broth microdilution bacteria susceptibility test ........................................ 51
2.2.5 Time-kill kinetic assay .................................................................................. 52
2.2.6 Fluorescence microscopy assay to measure cell membrane permeability
................................................................................................................................... 53
2.2.7 Election microscopy ...................................................................................... 54
2.2.8 Lactate dehydrogenase (LDH) cytotoxicity assay .................................... 54
2.2.9 β-Hexosaminidase assay for in vitro mast cell degranulation ................ 55
2.2.10 Mastoparan treatment of mice intraperitoneally.................................... 56
2.2.10.1 Microscopic analysis of peritoneal lavage ........................................ 56

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2.2.10.2 Microscopic analysis of mesenteric mast cells ................................. 57
2.2.10.3 Myeloperoxidase assay for neutrophils ............................................ 58
2.2.11 Superficial skin colonization and infection models ............................... 59
2.2.12 Dermal toxicity study ................................................................................. 61
2.3 Results .................................................................................................................. 62
2.3.1 Mastoparan exhibits broad-spectrum antibacterial activity against
Gram-negative and Gram-positive bacteria ....................................................... 62
2.3.2 Mastoparan has a broader range of activity against bacteria in
comparison to selected marketed antibiotics ..................................................... 64
2.3.3 Mastoparan exhibits antibacterial activity against resistant clinical
isolates ..................................................................................................................... 67
2.3.4 Mastoparan disrupts bacterial membrane integrity ................................ 68
2.3.5 Mastoparan does not elicit cytotoxicity at the concentrations necessary
for its antibacterial activity ................................................................................... 74
2.3.6 Mastoparan exhibits a kinetically rapid mechanism of bacteria growth
inhibition ................................................................................................................. 77
2.3.7 Mastoparan is a potent mast cell activator in vitro and in vivo ............... 81
2.3.8 Mastoparan treatment elicits the recruitment of neutrophils ................. 85
2.3.9 Mastoparan is effective as a topical antibacterial agent utilizing its dual
action direct antibacterial and immunomodulatory activities ........................ 88
2.3.10 Mastoparan does not exhibit dermal toxicity ......................................... 93
2.3.11 Mastoparan promotes mast cell-dependent wound healing in mice .. 93
2.4 Discussion ......................................................................................................... 100

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Chapter 3: Perspectives and Conclusion .................................................................. 105
3.1 Broad-spectrum antibacterial and immunomodulatory activity exhibited
by mastoparan ........................................................................................................ 106
3.2 Mastoparan is a highly effective therapeutic agent against bacterial skin
infections ................................................................................................................. 111
3.3 Mastoparan promotes wound healing .......................................................... 114
3.4 Conclusions ....................................................................................................... 116
References ..................................................................................................................... 118
Biography ...................................................................................................................... 155

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List of Tables
Table 1: Mastoparan peptides identified from wasps. ............................................. 38
Table 2: Mastoparan exhibits broad-spectrum antibacterial activity against Gram-
positive and Gram-negative bacteria. . ....................................................................... 63
Table 3: Mastoparan is more active against bacteria (both Gram-negative and
Gram-positive) than selected marketed antibiotics.. ................................................ 65
Table 4: Mastoparan exhibits antibacterial activity against multidrug resistant
bacteria.. ........................................................................................................................... 68

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