Pseudomonas aeruginosa PATHOGENICITY AND ANTIBIOTIC RESISTANCE

175 Pages·2004·2.68 MB·English

by  Lin Zeng

Pseudomonas aeruginosa PATHOGENICITY AND ANTIBIOTIC RESISTANCE

By

LIN ZENG

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2004
Copyright 2004

by

Lin Zeng
This dissertation is dedicated to my parents and my wife Adrian.
ACKNOWLEDGMENTS
With great pleasure and sincere appreciation, I take this opportunity to present my
gratitude to the following people, without whose selfless assistance this dissertation could
never have been finished.
I thank my mentor, Dr. Shouguang Jin, for his excellent guidance and
encouragement that inspired my research and personal life here in a foreign country. I
appreciate the opportunity to make my life part of the development of science.
I thank all the current members of Dr. Jin’s laboratory (Weihui, Rose, Jinghua,
Xiaoling, Mounia, Dr. Jung, Dr. Kim, and Dr. Min) and several former members (Hwan,
Yang, Yanping, and Scott) who provided me with all the help I ever needed and gave me
a place to work, to think, and to grow. Special thanks also go to Dr. Unhwan Ha and Dr.
Yanping Wang, who started the project on antibiotic resistance and were willing to accept
me as part of the team.
I would like to thank my committee (Dr. Shouguang Jin, Dr. Paul A. Gulig, Dr.
Reuben Ramphal, and Dr. Thomas A. Bobik) whose insightful advice in the last 5 years
has made a great difference in my research progress and in my view of being a serious
scientist. Thanks also go to members in neighboring Dr. Gulig and Dr. Ramphal’s
laboratories, for letting me share their facility and space; more importantly, brilliant ideas
about research.
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Last but not least, I would like to thank my parents, my family, and my wife Adrian
and her family, for all the emotional and material support I have received throughout my
graduate study.
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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS.................................................................................................iv
LIST OF TABLES...............................................................................................................x
LIST OF FIGURES...........................................................................................................xi
ABSTRACT.....................................................................................................................xiv
1 INTRODUCTION........................................................................................................1
Pseudomonas aeruginosa.............................................................................................1
Basic Bacteriology.................................................................................................1
Infections...............................................................................................................2
Virulence Factors...................................................................................................2
Extracellular toxins........................................................................................2
Flagellum, pilus and alginate.........................................................................2
Type III secreted exotoxins............................................................................4
Biofilm...........................................................................................................4
Cystic Fibrosis..............................................................................................................5
Genetic Background and the Physiological Defect...............................................5
Bacterial Infections in CF Patients........................................................................5
Treatments.............................................................................................................6
Serine/Threonine Protein Kinases................................................................................7
Family Portrait of Hanks-Type Kinases................................................................8
A New Frontier: Research on Prokaryotic Ser/Thr Protein Kinases.....................8
Serine/Threonine Protein Kinases in P. aeruginosa..............................................9
Bacterial Antibiotic Resistance...................................................................................10
Membrane Permeability......................................................................................10
Efflux Pumps.......................................................................................................11
Enzymatic Modification......................................................................................11
Mutational Resistance.........................................................................................11
Research Overview.....................................................................................................12

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2 CHARACTERIZATION OF PPKA, AN IN VIVO INDUCIBLE
SERINE/THREONINE PROTEIN KINASE OF Pseudomonas aeruginosa:
BIOCHEMISTRY, TOPOLOGY, AND FUNCTION...............................................13
Introduction.................................................................................................................13
Material and Methods.................................................................................................15
Bacterial Strains and Media.................................................................................15
Plasmid Constructs..............................................................................................16
Overexpression and Purification of Recombinant PpkA and ExsA in E. coli....18
Kinase Assay.......................................................................................................19
Acid Hydrolysis of PpkA and Two-Dimensional Thin-Layer Chromatography
(TLC)...............................................................................................................19
Cell Fractionation................................................................................................20
Western Blot Analysis.........................................................................................21
Protease Digestion Protection Assay...................................................................22
Cross-Linking of PpkA-Expressing Cells...........................................................22
RNA Isolation and Micro-Array Assay...............................................................23
β-Galactosidase Assay.........................................................................................23
ELISA..................................................................................................................24
HeLa Cell Lifting Assay......................................................................................24
Twitching Motility Test.......................................................................................25
Results.........................................................................................................................25
PpkA Is Autophosphorylated at Serine and Threonine Residues........................25
PpkA Is a Transmembrane Protein......................................................................27
Intact Type II Secretion Apparatus Is Required for PpkA Stability....................30
PpkA Forms Homodimer When Expressed in P. aeruginosa.............................34
The Role of VWA Domain in the Stability and Kinase Activity of PpkA..........36
Constitutively Expressed PpkA Inhibits the Lifting of HeLa Cells by P.
aeruginosa........................................................................................................37
PpkA Inhibits P. aeruginosa Type III Secretion System....................................41
Expression Level of exsA in P. aeruginosa is Reduced When ppkA is
Constitutively Expressed.................................................................................42
Recombinant PpkA Phosphorylates ExsA in vitro..............................................43
Constitutively Expressed PpkA Inhibits P. aeruginosa Twitching Motility.......45
Important control experiments............................................................................49
Other Observations Associated With Constitutive Expression of ppkA.............51
Discussion...................................................................................................................51
Biochemistry and Topology of PpkA..................................................................53
Function of PpkA................................................................................................55
Kinase PpkA and type III secretion..............................................................55
Kinase PpkA and Twitching Motility..........................................................59
Conclusions.........................................................................................................60

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3 A TWO-COMPONENT REGULATORY SYSTEM INVOLVED IN
Pseudomonas aeruginosa ANTIBIOTIC RESISTANCE..........................................61
Introduction.................................................................................................................61
Materials and Methods...............................................................................................63
Strains and Plasmids............................................................................................63
Overexpression and Purification of Recombinant PprA and PprB.....................66
Whole-cell Alkaline Phosphotase Assay for Assessment of Outer-Membrane
Permeability.....................................................................................................67
Biofilm Formation Assay....................................................................................68
Twitching Motility Test.......................................................................................68
Minimum Inhibitory Concentration (MIC) Test.................................................68
Miscellaneous Assays..........................................................................................69
Results.........................................................................................................................69
Characterization of a Spontaneous Mutant of P. aeruginosa with Decreased
Sensitivities to Antibiotics...............................................................................69
A Novel Two-component Regulatory System Controls Antibiotic Sensitivity in
PAK1-3............................................................................................................70
PprA and PprB Form a Functional Two-Component Regulatory System..........72
MexXY-OprM Efflux Pump Is Not the Cause of the Increased Resistance in
PAK1-3, Nor the Sensitivity Associated with PprB Overexpression..............73
PprA and PprB Regulate Outer-Membrane Permeability...................................75
Overexpression of the pprB Gene in Clinical Isolates Renders Increased
Sensitivities to Aminoglycosides.....................................................................77
PprA and PprB Are Controlled by Quorum Sensing and RpoS..........................79
Growth-phase-dependent expression of both pprA and pprB genes in P.
aeruginosa...............................................................................................80
RpoS positively regulates pprB expression..................................................81
Both pprA and pprB genes are under the quorum sensing regulation..........82
Possible presence of negative regulator for pprB.........................................85
Over-Expression of pprB Leads to Increased Biofilm Formation in P.
aeruginosa........................................................................................................87
Over-Expression of pprB Leads to Decreased Twitching Motility in P.
aeruginosa........................................................................................................88
Discussion...................................................................................................................89

4 A GENE ENCODING AMINOGLYCOSIDE-MODIFYING ENZYME, APH(3’)-
IIB, IS UNDER THE POSITIVE CONTROL OF A SURROGATE REGULATOR
HPAA.........................................................................................................................95
Introduction.................................................................................................................95
Materials and Methods...............................................................................................97
Materials, Strains, and Media..............................................................................97
Construction of Mutations and Plasmids.............................................................99
Neomycin Resistance Tests...............................................................................100
Miscellaneous Assays........................................................................................101
Results.......................................................................................................................101
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Isolation of aph and Its Upstream Putative Regulatory Gene hpaA..................101
Regulation of aph Gene by the Auto-Regulator HpaA.....................................102
4-HPA Induces Neomycin Resistance in P. aeruginosa...................................106
HpaA Controls Metabolism of 4-HPA, while Aph Is Not Required.................108
The 4-HPA Analogue 3-HPA Is Incapable of Inducing hpaA-aph Operon......111
The Presence of hpaA-aph Locus and Induction of Neomycin Resistance in
Various P. aeruginosa Isolates......................................................................111
Additional Data.................................................................................................112
Induction of hpaA-aph operon by 3,4-DHPA, an effect independent of
HpaA activation....................................................................................113
PA4094 is not required for 3,4-DHPA-mediated aph activation...............114
Examination of the possible catabolic repression in hpaA-aph regulation115
Discussion.................................................................................................................118

5 CONCLUSIONS AND DIRECTIONS FOR FUTURE STUDY............................124
The PpkA, an in vivo Inducible Repressor for TTSS and Twitching Motility.........124
The PprA-PprB Two-Component Regulatory System.............................................129
The HpaA-Aph Operon............................................................................................132
Closing Remarks.......................................................................................................134

LIST OF REFERENCES.................................................................................................136
BIOGRAPHICAL SKETCH...........................................................................................156

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LIST OF TABLES
Table page

2-1. Strains and plasmids..................................................................................................17
2-2. Microarray result showing the genes/ORFs with changed expression level.............45
3-1. Strains and plasmids..................................................................................................64
3-2. The MICs for PAK and PAK1-3 derivatives.............................................................69
3-3. Effect of elevated levels of pprB expression on MICs for clinical isolates..............79
4-1. Strains, plasmids, and primers...................................................................................98
4-2. The MICs for PAK and mutants..............................................................................108

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