Investigation of the Role of the RNA Binding Protein CsrA in the Virulence of Enteropathogenic Escherichia coli Público
Bhatt, Shantanu (2011)
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 ……..………….......................................... 2Escherichia 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 EnteropathogenicEscherichia coli ............................................................................................ 74
Abstract ...................................................................................... 75
Introduction ................................................................................ 76
Materials and Methods................................................................... 81
Bacterial strains, Plasmids, Primers & Media ................................ 81Cell 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) ........ 89In 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 ............................................ 184Intrinsic 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 ......................................... 196Concluding 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 translocasein 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 rescuedby tatA in multicopy ................................................................... 232
Isolation and expression of the P. stuartii tat operon ....................... 233Expression 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................................................ 236A 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 signalproduction................................................................................. 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 .................................................. 268Immunofluerescence 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 ..................................... 270Uptake 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 1.2Genetic and regulatory architecture of the LEE .................................... 11
Figure 1.3 Supramolecular structure of the type III secretion system (T3SS) .......... 15 Figure 1.4The Csr regulatory architecture in gammaproteobacteria ...................... 27
Table 1.1 The Csr regulatory architecture in gammaproteobacteria ...................... 28 Figure 1.5NMR 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 2.2Oligonucleotides used in this study .................................................... 86
Figure 2.1 (A-E) Inactivation of csrA does not affect growth butprofoundly diminishes adherence, pedestal formation
and disruption of transepithelial resistance (TER) by EPEC ....................111
Figure 2.2 csrA is necessary for the disruption of TERacross Caco-2 BBE ........................................................................ 114
Figure 2.3 (A-B) Secretion of EspA, EspB, EspD and Tir is diminished inthe 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 glycogenbiosynthesis in EPEC ...................................................................... 127
Figure 2.9Model for the CsrA mediated regulation of the LEE .............................. 129
Chapter 3 - Table 3.1
Bacterial strains and plasmids used in this study ................................. 160
Table 3.2Oligonucleotides 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 tostimulate toxin-dependent killing of C. elegans .................................... 164
Figure 3.3Model 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 4.2Transcriptional 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 signalproduction 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
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