Investigation of the Role of the RNA Binding Protein CsrA in the Virulence of Enteropathogenic Escherichia coli Public

Abstract

The CsrA holoprotein is an RNA-binding protein that affects the stability and/or translation of transcripts. The gene was originally isolated in E. coli as a repressor of glycogen production. CsrA orthologs have since been discovered in numerous bacteria. For examples, in Erwinia carotovora subsp. carotovora, Pseudomonas aeruginosa, and Salmonella Typhimurium, besides regulating conserved ancestral processes, CsrA controls diverse virulence-associated traits. Despite its global regulatory role in innocuous and pathogenic bacteria, CsrA remains uncharacterized in any pathovar of E. coli.

We investigated the role of CsrA in the virulence of enteropathogenic Escherichia coli (EPEC). Inactivation of csrA profoundly diminished the infectivity of EPEC as evident from reduced pedestal formation on tissue culture cells. Molecular analysis revealed that the observed defect resulted from reduced transcript levels of the LEE4 operon, which primarily encodes for the regulatory and structural components of a type 3 secretion system (T3SS). Purified CsrA protein specifically bound to the LEE4 transcript, suggesting that CsrA presumably stabilizes the transcript and promotes pedestal formation. Intriguingly, modest overexpression of csrA, like inactivation, also repressed the LEE4 operon. However, unlike inactivation, overexpression of csrA also silenced the other LEE-encoded transcription units. Overexpression of csrA exerted its effect by repressing the global activator of LEE, GrlA, as evident from reduced transcript levels. Furthermore, CsrA appeared to exert a direct effect on GrlA as the purified protein specifically bound to the grlRA transcript. Thus, CsrA appears to modulate the LEE in a dose-dependent manner. Besides virulence, CsrA also controlled flagellar-based motility and glycogen production in EPEC.

Additionally, csrA mutation abolished the ability of EPEC to synthesize diffusible exotoxins that paralyze and kill the worm Caenorhabditis elegans. Overexpression of tnaA, which encodes for the toxin-synthesizing enzyme tryptophanase, in the csrA mutant, but not vice-versa, rescued the ability of the mutant to kill worms. Moreover, tryptophanase activity was abolished in the csrA mutant. Collectively, these results suggest that CsrA is upstream of tnaA in a regulatory circuit that is essential for exotoxin synthesis and the killing of worms by EPEC.

In summary, our results suggest that csrA is a pleiotropic broad-host range virulence determinant of EPEC.

Table of Contents

TABLE OF CONTENTS

Chapter 1 .................................................................................................... 1

Background and Significance ……..………….......................................... 2

Escherichia coli - From Saprotropism to Parasitism ...................... 2

EPEC and EHEC: Discovery, pathogenesis, epidemiology,

and drug development …........................................................... 3

Genetic organization of the LEE in EPEC and EHEC ........................ 8

Supramolecular structure of the LEE-encoded T3SS ..................... 12

Core transcriptional architecture of the LEE ............................... 16

Mechanism of action of Ler ..................................................... 19

Posttranscriptional and Posttranslational regulation

of the LEE ............................................................................. 20

Carbon Storage Regulatory (Csr) system in Eubacteria .................. 22

Structural Analysis of CsrA ....................................................... 29

Physiological Roles of CsrA amongst eubacteria ........................... 31

Rationale for the putative role of CsrA in the virulence of

EPEC and EHEC ....................................................................... 35

Bibliography……………….................................................................... 38

Chapter 2: The RNA Binding Protein CsrA is a

Pleiotropic Regulator of the Locus of Enterocyte

Effacement Pathogenicity Island of Enteropathogenic

Escherichia coli ............................................................................................ 74

Abstract ...................................................................................... 75

Introduction ................................................................................ 76

Materials and Methods................................................................... 81

Bacterial strains, Plasmids, Primers & Media ................................ 81

Cell Culture and Immunofluorescence Microscopy .......................... 87

Preparation of Cell Lysates, TCA Precipitation and

Western Blotting ...................................................................... 87

RNA Isolation .......................................................................... 88

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) ........ 89

In vitro transcription and RNA Electrophoretic Mobility

Shift Assay ............................................................................. 90

Transepithelial Resistance Assay ................................................. 91

Motility Assay .......................................................................... 92

Glycogen biosynthesis assay ...................................................... 92

Results ......................................................................................... 94

csrA regulates adherence and pedestal formation on

mammalian cells... ................................................................... 94

Disruption of Transepithelial resistance across

polarized Caco-2-BBE cells depends on csrA. ................................. 95

csrA is necessary for synthesis and secretion of

translocators, but only affects the secretion of the effector Tir ........ 96

csrA regulates the transcript levels of the architectural

components of the TTSS. ........................................................... 97

Purified CsrA binds to the leader segment of the

sepLespADB but not escD transcript.... ..........................................99

Overexpression of csrA globally represses the expression

from the LEE .......................................................................... 100

CsrA binds to the 5' leader segment of the grlRA transcript ............. 103

csrA regulates motility and glycogen biosynthesis in EPEC .............. 103

Discussion ................................................................................... 105

Acknowledgements ...................................................................... 110

Bibliography ................................................................................. 131

Chapter 3: CsrA and TnaB coregulate tryptophanase

activity to promote exotoxin-induced killing of

Caenorhabditis elegans by enteropathogenic Escherichia

coli ............................................................................................................. 147

Abstract ...................................................................................... 148

Introduction ................................................................................ 149

Results and Discussion ................................................................. 153

Acknowledgements ...................................................................... 159

Bibliography ................................................................................. 167

Chapter 4: Honing the Message: Posttranscriptional

and Posttranslational Control in Attaching and

Effacing Pathogens ........................................................................................ 182

Abstract ....................................................................................... 183

What are EPEC and EHEC? ........................................................ 184 Coordinated regulation of virulence ............................................ 184

Intrinsic transcriptional control of the LEE .................................... 185

Why control the LEE at the posttranscriptional and

posttranslational levels? ............................................................ 187

RNA-binding proteins regulate the LEE ......................................... 189

sRNA-mediated regulation of the LEE ........................................... 193

Posttranslational control of the LEE .............................................. 194

Other extra-transcriptional mechanisms affecting the LEE ................ 195 Elucidating the posttranscriptional and posttranslational "virulence regulome" of A/E pathogens ......................................... 196

Concluding remarks ................................................................... 199

Outstanding Questions ................................................................ 199

Acknowledgements ........................................................................ 201

Bibliography ................................................................................... 206

Chapter 5:

Overall Conclusions and Future Directions ......................................... 218

Bibliography .................................................................................. 221

Appendix: Additional Publications

1. The rhomboid protease AarA cleaves TatA and is

required for function of the twin-arginine translocase

in Providencia stuartii ...................................................................................... 227

Abstract ........................................................................................ 228

Introduction .................................................................................. 229

Results .......................................................................................... 231

Identification of a high copy suppressor that restores

extracellular signal production to an aarA mutant of

P. stuartii................................................................ ................. 231

AarA mutants are defective in Tat function and rescued

by tatA in multicopy ................................................................... 232

Isolation and expression of the P. stuartii tat operon ....................... 233

Expression of the tat operon is not dependent on aarA ..................... 234

The P. stuartii TatA protein is processed by AarA............................. 234 Purified AarA cleaves TatA in vitro................................................ 236

A truncated form of TatA, missing seven N-terminal

amino acids is able to restore Tat function in an

AarA-independent manner..................................................... ....... 237

Role of the P. stuartii Tat system in extracellular signal

production................................................................................. 237

Discussion ...................................................................................... 239

Materials and Methods ..................................................................... 241

Bacterial growth conditions .......................................................... 241

Preparation of conditioned media (CM) ........................................... 241

Isolation of tatA from P. mirabilis .................................................. 241

Isolation of the P. stuartii tatA region ............................................. 242

Northern blot analysis .................................................................. 243

Construction of pET.TatA-His ......................................................... 243

Western blot analysis ................................................................... 244

Purification of TatA-His6 and N-terminal sequencing .......................... 244

Expression and Purification of AarA ................................................ 245

In-vitro rhomboid cleavage assays ................................................. 246

Site directed mutagenesis of tatAPs. .............................................. 247

Construction of tatC null allele ....................................................... 247

Acknowledgements .......................................................................... 248

Bibliography ..................................................................................... 255

2. Pathogenic Bacteria Induce Colonic PepT1 Expression:

An Implication in Host Defense Response ............................................................ 261

Abstract .......................................................................................... 262

Background and Aims ................................................................... 262

Methods..................................................................................... 262

Results...................................................................................... 262

Conclusions................................................................................ 263

Introduction ..................................................................................... 264

Materials and Methods ...................................................................... 266

Cell culture ................................................................................. 266

Bacteria growth and infection ......................................................... 266

Plasmid construction and transfection .............................................. 266

Lipid raft isolation from HT29-Cl.19A cells ........................................ 266

Dual-luciferase reporter assay ........................................................ 267

RNA extraction and RT-PCR ............................................................ 267

Real-time RT-PCR ......................................................................... 267

Cloning of full-length cDNA encoding PepT1 expressed in EPEC-infected HT29-Cl.19A cells .................................................. 268

Immunofluerescence staining ......................................................... 269

Electrophoretic mobility shift assay (EMSA) ...................................... 269

Chromatin immunoprecipitation assay (ChIP) .................................... 269

Nuclear run-on assay .................................................................... 270

Protein extraction, Western blot and dot blot ..................................... 270

Uptake experiments ...................................................................... 271

Measurement of EPEC attachment to LRs ........................................... 271

ELISA.......................................................................................... 271

Ex vivo experiments ...................................................................... 272

Statistical analysis ......................................................................... 272

Results .............................................................................................. 273

EPEC induces PepT1 promoter activity and PepT1

transcription................................................................................. 273

EPEC induces PepT1 protein expression and transport

activity........................................................................................ 273

EPEC induces PepT1 expression in lipid rafts ....................................... 274

The transcription factor Cdx2 is important for EPEC

-induced PepT1 expression .............................................................. 274

EPEC-induced PepT1 expression is dependent on

intimate adherence of EPEC to host cells through LRs ........................... 275

PepT1 plays a role in EPEC adherence and

EPEC-induced inflammation ............................................................. 276

PepT1 has a role in Citrobacter rodentium adherence

and intestinal inflammation in mouse colon ........................................ 278

Discussion ......................................................................................... 279

Acknowledgements ............................................................................ 283

Bibliography ....................................................................................... 292

TABLES AND FIGURES

Chapter 1 - Figure 1.1 EPEC forming pedestals on the surface of infected 3T3 cells ................... 7 Figure 1.2

Genetic and regulatory architecture of the LEE .................................... 11

Figure 1.3 Supramolecular structure of the type III secretion system (T3SS) .......... 15 Figure 1.4

The Csr regulatory architecture in gammaproteobacteria ...................... 27

Table 1.1 The Csr regulatory architecture in gammaproteobacteria ...................... 28 Figure 1.5

NMR solution structure of CsrA/RsmA/RsmE in complex with

two hcnA molecules ....................................................................... 30

Chapter 2 - Table 2.1

Bacterial strains and plasmids used in this study ................................. 84

Table 2.2

Oligonucleotides used in this study .................................................... 86

Figure 2.1 (A-E) Inactivation of csrA does not affect growth but

profoundly diminishes adherence, pedestal formation

and disruption of transepithelial resistance (TER) by EPEC ....................111

Figure 2.2 csrA is necessary for the disruption of TER

across Caco-2 BBE ........................................................................ 114

Figure 2.3 (A-B) Secretion of EspA, EspB, EspD and Tir is diminished in

the csrA mutant ............................................................................ 115

Figure 2.4 (A-B) sepL, espA, espD, espB, escD, and escF, but not tir,

transcript levels are reduced in the csrA mutant ................................. 116

Figure 2.5 (A-G)

CsrA binds to the leader segment of the LEE4 operon .......................... 118

Figure 2.6 (A-E)

Expression of csrA from a multicopy plasmid globally

represses the expression from the LEE via GrlA .................................. 121

Figure 2.7 (A-E)

Purified CsrA binds to the leader segment of the grlRA

transcript .................................................................................... 124

Figure 2.8 (A-C) CsrA activates motility and represses glycogen

biosynthesis in EPEC ...................................................................... 127

Figure 2.9

Model for the CsrA mediated regulation of the LEE .............................. 129

Chapter 3 - Table 3.1

Bacterial strains and plasmids used in this study ................................. 160

Table 3.2

Oligonucleotides used in this study .................................................... 161

Figure 3.1 (A-E) CsrA regulates tryptophanase activity to promote the toxin-

mediated killing of C. elegans by EPEC .............................................. 162

Figure 3.2 (A-D) TnaB, but not AroP or Mtr, imports tryptophan into EPEC to

stimulate toxin-dependent killing of C. elegans .................................... 164

Figure 3.3

Model for the role of CsrA and TnaB in the regulation of

tnaA and toxin production ................................................................ 166

Chapter 4 -

Figure 4.1 (A-B)

Stages of pedestal formation by A/E pathogens ................................... 202

Figure 4.2

Transcriptional and extratranscriptional control of the locus of

enterocyte effacement (LEE) ............................................................ 204

Appendix - Additional Publication #1 Table A1.1

Tat phenotypes of various mutants .................................................... 249

Figure A1.1 Overexpression of tatA from P. mirabilis restores signal

production to P. stuartii aarA mutant ................................................. 250

Figure A1.2 (A-B)

TatA processing in wild-type and an aarA mutant ................................. 251

Figure A1.3 (A-C)

In vitro cleavage of TatA by purified AarA protease ............................... 252

Figure A1.4 (A-B)

Role of the Tat transport system in extracellular signal

production ..................................................................................... 254

Additional Publication #2 Figure A2.1 (A-F)

EPEC transcriptionally induces PepT1 expression

and transport activity in HT29-Cl.19A cells .......................................... 284

Figure A2.2 (A-C)

EPEC induces PepT1 expression in lipid rafts of HT29-Cl.19A

cells ............................................................................................. 285

Figure A2.3 (A-F)

Cdx2 is important for EPEC-induced PepT1 expression ........................... 286

Figure A2.4 (A-E)

EPEC-induced PepT1 expression requires Tir, intimin and

intact host lipid rafts ........................................................................ 288

Figure A2.5 (A-C)

PepT1 associated with lipid rafts modulates EPEC

adherence to HT29-Cl.19A cells ......................................................... 289

Figure A2.6 (A-C)

Over-expression of PepT1 in HT29-Cl.19A cells has a role

in EPEC-induced inflammation ........................................................... 290

Figure A2.7 (A-E)

Citrobacter rodentium induces PepT1 expression in mouse

colon . Role of PepT1 in C. rodentium adherence and

C. rodentium-induced keratinocyte-derived chemokine

production in mouse colon ................................................................ 291

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Microbiology & Molecular Genetics
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Microbiology
Mot-clé	tnaA EHEC post-translational post-transcriptional CsrA EPEC tnaB
Committee Chair / Thesis Advisor	Kalman, Daniel, Emory University
Committee Members	Rather, Phil N, Emory University Weiss, Bernard, Emory University Moran Jr., Charles, Emory University Benian, Guy, Emory University Romeo, Tony, Emory University

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