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Transcriptome Analysis of Escherichia Coli O157:H7 During in Vivo Mimicking Conditions Michael Dwight Carruthers Iowa State University

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Transcriptome Analysis of Escherichia Coli O157:H7 During in Vivo Mimicking Conditions Michael Dwight Carruthers Iowa State University Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2009 Transcriptome analysis of Escherichia coli O157:H7 during in vivo mimicking conditions Michael Dwight Carruthers Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Veterinary Preventive Medicine, Epidemiology, and Public Health Commons Recommended Citation Carruthers, Michael Dwight, "Transcriptome analysis of Escherichia coli O157:H7 during in vivo mimicking conditions" (2009). Graduate Theses and Dissertations. 10872. https://lib.dr.iastate.edu/etd/10872 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Transcriptional analysis of Escherichia coli O157:H7 during in vivo mimicking conditions by Michael D. Carruthers A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Microbiology Program of Study Committee: F. Chris Minion, Major Professor Jeff Beetham Nancy Cornick Larry Halverson Dan Nordman Iowa State University Ames, Iowa 2009 Copyright © Michael Carruthers, 2009. All rights reserved. ii TABLE OF CONTENTS LIST OF FIGURES vi LIST OF TABLES vii ABSTRACT viii CHAPTER 1. GENERAL INTRODUCTION 1 Introduction 1 Dissertation Organization 2 Literature Review 3 Escherichia coli 3 Pathogenic Escherichia coli 3 Escherichia coli O157:H7 5 Reservoirs 6 Major virulence factors 7 Diagnostic and food safety considerations 11 Transcriptional analysis of Escherichia coli O157:H7 using microarrays 12 Heat shock 13 Anaerobiasis 15 Microarrays 15 History 15 Types of DNA microarrays 16 Target preparation methods and hybridization 17 Microarray bioinformatics 19 Data acquisition 20 Data processing and statistics 21 Validation of results 23 Summary with Goals and Hypothesis 25 References 26 iii CHAPTER 2. TRANSCRIPTOME ANALYSIS OF ESCHERICHIA COLI O157:H7 DURING HEAT SHOCK 40 Abstract 40 Introduction 41 Materials and Methods 42 Strains and culture conditions 42 Microarray construction 42 RNA isolation 43 Labeled cDNA generation and hybridization 43 Microarray experimental design 44 Image acquisition and data analysis 44 Validation of microarray data 45 Results 46 Array development and construction 46 Heat shock 46 Discussion 51 Overview 51 Classic HS response 52 E. coli O157:H7 specific genes 52 Acknowledgements 53 References 53 CHAPTER 3. TRANSCRIPTIONAL RESPONSE OF ESCHERICHIA COLI O157:H7 EDL933 DURING ANAEROBIC GROWTH AND EXPOSURE TO RUMEN FLUID 56 Abstract 56 Introduction 57 Materials and Methods 59 Strains and culture conditions 59 Rumen fluid 60 iv Recombinant DNA techniques 60 β-galactosidase assay 60 RNA isolation 61 Labeled cDNA generation and hybridization 61 Microarray experimental design 61 Image acquisition and data analysis 61 Validation of microarray data 62 Microarray data accession 62 Results 63 Anaerobic vs. aerobic microarrays 63 Rumen fluid microarrays 66 β-galactosidase assay 70 Discussion 71 Acknowledgements 75 References 75 CHPATER 4. EXPLORING THE RESPONSE OF ESCHERICHIA COLI O157:H7 EDL933 WITHIN ACANTHAMOEBA CASTELLANII BY GENOME-WIDE TRANSCRIPTIONAL PROFILING 80 Abstract 80 Introduction 81 Materials and Methods 82 Strains and culture conditions 82 RNA isolation 83 Image acquisition and data analysis 84 Validation of microarray data 84 Microarray data accession 85 Results 85 Intracellular survival 85 Microarrays 86 v Discussion 93 Acknowledgements 89 References 99 CHAPTER 5. GENERAL CONCLUSIONS 105 General Discussion 105 Recommendations for Future Study 108 Up-regulated E. coli O157:H7 genes not found in E. coli K-12 108 Characterization of genes induced as a result of E. coli O157:H7 within Acanthamoeba 109 Iron acquisition/metabolism 109 Quorum sensing 109 SOS response and antibiotic resistance 110 Role of virulence genes and their maintenance 110 APPENDIX A. TABLE 2.S1. Differentially expressed genes of E. coli O157:H7 during heat shock 112 APPENDIX B. Table 3.S1. Differentially expressed genes of E. coli O157:H7 during anaerobic growth 121 APPENDIX C. Table 3.S2. Differentially expressed genes of E. coli O157:H7 during rumen fluid treatment 139 APPENDIX D. Table 4.S1. Differentially expressed genes of E. coli O157:H7 within Acanthamoeba ACKNOWLEDGEMENTS 144 vi LIST OF FIGURES Figure 2.1. Volcano plot pf transcriptional differences in E. coli O157:H7 47 Figure 3.1. Temporal growth of E. coli O157:H7 EDL933 in LB broth under anaerobic and normal atmospheric oxygen conditions as measured by OD600 64 Figure 3.2. Volcano plots of transcriptional differences of E. coli O157:H7 during anaerobic growth (A) and rumen fluid treatment (B) 67 Figure 3.3. Differentially expressed transcripts of Clusters of Orthologous Groups of proteins 68 Figure 3.4. Validation of microarray results by qRT-PCR. All genes were differentially expressed at p < 0.01 69 Figure 3.5. β-galactosidase assay results of a csgBA fusion under anaerobic and aerobic conditions 71 Table 4.1. qRT-PCR primers used to validate microarray results 86 Figure 4.2. Volcano plot of transcriptional differences of E. coli O157:H7 within A. castellanii 88 Figure 4.3. Clusters of Orthologous Groups of proteins of the differentially transcribed genes 88 Figure 4.4. Validation of microarray results by qRT-PCR. All results were significant at p < 0.008 93 vii LIST OF TABLES Table 2.1. qRT-PCR Primers 45 Table 2.2. Top twenty-five up-regulated genes based on p value of microarray data 48 Table 2.3. Top twenty-five down-regulated genes based on p value of microarray data 49 Table 2.4. Differentially transcribed genes not found in E. coli K-12 50 Table 3.1. qRT-PCR primers used to validate microarray results 63 Table 3.2. Select up-regulated transcripts as a result of anaerobiasis at FDR < 10% 65 Table 3.3. Select down-regulated transcripts as a result of anaerobiasis at FDR < 10% 66 Table 3.4. Significantly differentially expressed transcripts common to both microarray experiments at FDR < 10% 70 Table 4.1. qRT-PCR primers used to validate microarray results 85 Table 4.2. Differentially expressed genes involved in virulence as annotated in the ERIC database 89 Table 4.3. Differentially expressed genes involved in antibiotic resistance, the SOS response and iron acquisition/metabolism viii Abstract Most Escherichia coli reside harmlessly in the animal intestine and share a mutualistic interaction with its human host. This relationship endures until the scales are shifted toward the pathogen such as a break in the intestinal mucosal barrier, host immunosuppression, the introduction of the species into a non-native environment such as the urinary tract, acquisition of a new phenotype, appearance of a strain with the ability to cause disease in a healthy host, etc. The focus of this dissertation is the interaction of the pathogen E. coli O157:H7 with the ruminant host. Four separate microarray studies were conducted to ascertain the transcriptome of E. coli O157:H7 under environmental conditions found within the ruminant host. These conditions were heat shock, growth under anaerobic conditions, exposure to rumen fluid, and interaction with a protozoan. The response to elevated temperature (heat shock) is highly conserved. The transcriptome of Escherichia coli K-12 has been studied over a variety of conditions while such studies involving E. coli O157:H7 are just now being conducted. To better understand the impact of heat shock on E. coli O157:H7, global transcript levels of strain EDL933 cells shifted from 37°C to 50°C for 15 min were compared to cells left at 37°C by microarray. Using a mixed model analysis, 193 genes were found to be differentially transcribed at p < 0.0042 with a q-value of < 0.1. The 111 down-regulated genes include the curli pili associated genes csgABCDEFG, maltose transport associated proteins malEFK, and NADH dehydrogenase subunit encoding nuoCEHIJN. The 82 genes up- regulated include the heat shock induced genes rpoH, dnaK, dnaJ, groEL, groES, and grpE along with two LEE encoded genes; hypothetical gene Z5121 and sepZ. Twenty- three additional genes located in O-islands were found to be differentially expressed. qRT-PCR was performed to validate microarray results. Also, samples subjected to a 30˚C to 42˚C shift were examined by qRT-PCR to confirm differential transcription of selected genes. These results indicate that this pathogen may regulate its virulence factors in response to temperature changes. ix Escherichia coli O157:H7 encounters two environmental conditions within the ruminant gastrointestinal tract, limiting oxygen and rumen fluid. Both of these conditions may have implications in the general physiology of E. coli and its survival during passage through the rumen. The impact of these environmental conditions on the transcriptome of E. coli O157:H7 is unclear. To study this further, the transcriptome of E. coli O157:H7 strain EDL933 grown under anaerobic conditions was compared to cells exposed to rumen fluid and to cells grown aerobically. Four hundred nineteen genes were differentially expressed during anaerobic growth at p < 0.014 with a false discovery rate < 10%. Among the 280 up-regulated genes were the curli pili associated genes csgBA and csgDEFG, adherence associated genes ompA, tdcA, and cadA, heat shock genes dnaK, dnaJ, groEL, and groES and the σ38 encoding gene rpoS. Ten genes, 8 up- and 2 down- regulated, located in E. coli O157:H7 virulence-related O-islands were differentially transcribed with a fold change >1.3 suggesting that these genes may play a role in host adaptation. Translational β-galactosidase fusions with csgBA confirmed its regulation during anaerobic conditions. As a result of rumen fluid exposure, 83 genes were differentially transcribed at p < 0.002 with a false discovery rate < 10%, 38 up- and 45 down-regulated.
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