Evading Glycopeptide Antibiotic Resistance

101 Pages·2017·7.92 MB·English

EVADING GLYCOPEPTIDE ANTIBIOTIC RESISTANCE
EVADING GLYCOPEPTIDE ANTIBIOTIC RESISTANCE

By

JASON F BACK, B. Sc

A Thesis

Submitted to the School of graduate Studies

In Partial Fulfillment ofthe Requirements

For the Degree

Master of Science

McMaster University

© Copyright by Jason F Back, April2006
MASTER OF SCIENCE (2006) McMaster University
(Biochemistry) Hamilton, Ontario
TITLE: Evading Glycopeptide Antibiotic Resistance

AUTHOR: Jason F. Back, B. Sc. (McMaster University)

SUPERVISOR: Professor G.D. Wright

NUMBER OF PAGES: x, 90

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Abstract
Glycopeptide Antibiotics (GPAs) such as vancomycin are often used clinically as
antibiotics of last resort against infections due to Gram-positive bacteria that are resistant
to more commonly used antibiotics such as methicillin. The clinical emergence of
vancomycin resistant enterococci (VRE) and vancomycin resistantS. aureus (VRSA)
necessitates methods to evade this resistance.
GP As consist ofa heptapeptide backbone that is cross-linked to create a pocket
that binds the D-Alanyl-D-Alanine terminus of peptidoglycan intermediates, inhibiting
strengthening ofthe cell wall and resulting in susceptibility to osmotic stress. Resistance
to GPAs occurs when D-Ala-D-Lactate replaces D-Ala-D-Ala and the GPA pocket can
no longer bind effectively. In order to create novel binding pockets, we must understand
the specificity ofthe P450 monooxygenase enzymes that have been shown to catalyze the
cross-links. The 4 P450-encoding genes ofthe GPA A47934 biosynthetic cluster of
Streptomyces toyocaensis as well as genes encoding electron transport proteins necessary
for P450 function from Streptomyces coelicolor were cloned in Escherichia coli for
heterologous expression and characterization. One P450, StaJ was purified and shown to
bind CO as expected using spectrophotometric tests.
The genes responsible for GP A resistance are regulated by a two component
regulatory system consisting ofa sensor kinase (VanS) and a response regulator (V an.R).
In order to probe the events leading to VanS autophosphorylation and ultimately
resistance activation we utilize a series of GP A derivatives harbouring the photo labile
group benzophenone as well as the fluorescent and affinity moieties BODIPY and biotin.
Benzophenone permits light controlled covalent binding of the GP A to proteins that bind
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them while BODIPY allows fluorescence detection and biotin allows enrichment and
detection by Western analysis. We report that this system was insufficient to clearly
identify vancomycin binding proteins due to background signals despite multiple rounds
oftroubleshooting. It must be our conclusion that under the conditions tested, there are
no proteins that bind the GP A derivative used in this study.
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Acknowledgements
I first would like to thank my mother, without whom I would be lost. Thank you
for keeping me focused and moving forward despite many obstacles in recent years.
Thank you for your strength and support through all aspects ofmy life.
I would also like to sincerely thank my supervisor Dr. Gerry Wright for the
opportunity to work in a wonderful lab. Thank you for your guidance and understanding,
I have learned much from you and it is greatly appreciated. In addition I think my
committee, Dr. J. Ortega and Dr. J. Nodwell for useful advice despite numerous
roadblocks.
Finally I thank all members of the Wright lab, especially Dr. K. Koteva for
extensive help with all issues chemistry and Dr. XD Wang for so much help with the
never ending molecular biology problems. From Team Streptomyces to the people ofthe
Learning Centre I thank you all.
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Table of Contents
Abstract 111

Acknowledgements v

Table of Contents
VI

List of Figures vm

List of Tables x

Chapter 1 - Introduction 1

1.0-Introduction to glycopeptide antibiotics 2

1.1 - Current knowledge of GPA biosynthesis 5

1.1.1 -Unusual amino acid biosynthesis in GPA production 9

1.1.2-Glycopeptide backbone assembly 13

1.1.3-GPA backbone cross-linking 16

1.1.4 - Backbone modification in A47934 18

1.2 - GPA Mode of action 20

1.3 -Resistance to glycopeptide antibiotics 24

1.4 - Overcoming GPA resistance 27

1.4.1 -Differential backbone modification in GPAs 27

1.4.2-Altered D-Ala-D-Ala binding pocket in GPAs 31

1.4.3 - GPA resistance enzymes as drug targets 33

1.5 -Research Objectives 34

Chapter 2 - Exploration of P450-mediated cross-linking in 35

Glycopeptide antibiotic biosynthesis

2.0-Introduction to P450-mediated GPA backbone 36

cross-linking in A47934

2.1 - CytochromeP450 monooxygenase activity and mechanism 38

2.2-Goals ofthe A47934 cross-linking study 42

2.3 - Materials and methods for cross-lining study 43

2.3.1-Cloning ofP450s, ferredoxins and 43

ferredoxin reductases

2.3.2-P450, ferredoxin and ferredoxin reductase 44

protein expression

2.3 .3 - Preparation of"co-expression constructs 44

2.3 .4 - Expression, purification and solubilization of StaJ 45

2.3 .5 - Reduced CO binding difference spectrum of StaJ 46

2.3.6 -7-ethoxycoumarin biotransformation by P450s 46

2.4-Results ofP450 study 47

2.5 -Conclusions and future work for P450 study 53

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Chapter 3 - Investigating methods of inhibiting the GP A resistance mechanism 54

3.0-Introduction to inhibiting the GPA resistance mechanism 55

3.1-Goals ofthe GPA-binding study 57

3.2-Materials and methods for GPA-binding study 61

3.2.1-Bacterial strains, growth and membrane preparation 61

3.2.2-Using bodipy-7-aminocephalosporanic acid to label PBPs 61

3.2.3-Using Bh-vanc-Lys-Btn-Bod to detect vancomycin 62

binding proteins

3 .2.4 - Enriching post-photolysis samples for biotinylated products 62

3.2.5-Depletion of background signal with streptavidin-agarose 63

and using stringent washes before elution from the resin

3.3 -results of GPA-binding study 64

3.4-Conclusions and future work for GPA-binding study 79

4.0 - Conclusions and fmal remarks 82

References 85

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List of Figures
1.1 - Structures ofvancomycin and teicoplanin 3

1.2-Summary ofthe structures ofGPA and GPA-like compounds 7

1.3-Predicted A47934 retrobiosynthesis 8

1.4 - HPG biosynthesis 10

1.5-DHPG biosynthesis 11

1.6-OH-Tyr biosynthesis 12

1.7-Outline ofNRPS domains 14

1.8-Inactivation mutants used to determine GPA cross-linking order 17

1.9-Summary ofbackbone structures ofthe two GPA classes 19

1.10 - Final stages of peptidoglycan maturation 21

1.11-Vancomycin binding to D-Ala-D-Ala 22

1.12 - V anH, V anA and V anX activities 25

1.13 - V anRS two-component regulatory system 26

1.14-Chlorobiphenyl chloroeremomycin and Oritavancin 28

1.15-Damaged vancomycin and non-GPA chlorobiphenyl compound 29

1.16-Telavancin 30

1.17 - Synthetic macrocycles active against VRE 32

2.1 -Cross-linking order in teicoplanin class GPAs 37

2.2-P450 catalytic cycle 38

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2.3-Proposed P450 radical mechanism for cross-linking 40

2.4 - Summary of OxyB in vitro activity 41

2.5 - StaT purification showing insoluble StaT 49

2.6 - StaT solubilization 49

2.7-StaT absorbance spectrum 50

2.8-Reduced CO binding difference spectrum of StaT 51

2.9-TLC analysis of7-ethoxycoumarin biotransformation by P450s 52

2.10-SDS-PAGE separation oflysates after overnight induction ofP450s 52

3.1-Mechanism by which benzophenone inserts into CH bonds 57

3.2-GPA derivatives used in this study 58

3.3-Structure of7aca-Bodipy 63

3.4 -7aca-Bodipy binding experiment 64

3.5-Bh-vanco-bodipy binding experiment 65

3.6 -7aca-biotin binding experiment 67

3.7-Bh-vanco-biotin binding experiment 69

3.8-Bh-vanco-biotin competition experiment 71

3.9-Bh-A47934-biotin binding experiment 73

3.10-Bh-vanco-Biotin binding with B. subtilis membranes 74

3.11 -Untreated B. subtilis membrane fraction dilutions 75

3.12-Pre-depleted, untreated B. subtilis and S. coelicolor membranes 76

3.13 - Depleted, untreated, stringently washed B. subtilis and S. coelicolor 77

membranes

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