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The C-Di-Gmp Regulatory Network in Clostridioides Difficile and Its Role in Modulating Surface Adherence and Persistence in the Mammalian Gut

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The C-Di-Gmp Regulatory Network in Clostridioides Difficile and Its Role in Modulating Surface Adherence and Persistence in the Mammalian Gut THE C-DI-GMP REGULATORY NETWORK IN CLOSTRIDIOIDES DIFFICILE AND ITS ROLE IN MODULATING SURFACE ADHERENCE AND PERSISTENCE IN THE MAMMALIAN GUT Robert Woodrow McKee A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor in Philosophy in the Department of Microbiology and Immunology. Chapel Hill 2018 Approved by: Peggy A Cotter Jonathan J. Hansen Virginia L. Miller Rita Tamayo Mathew C Wolfgang © 2018 Robert Woodrow McKee ALL RIGHTS RESERVED ii ABSTRACT Robert Woodrow McKee: The c-di-GMP regulatory network in Clostridioides difficile and its role in modulating surface adherence and persistence in the mammalian gut (Under the direction of Rita Tamayo) Clostridioides difficile (Clostridium difficile) is a spore-forming bacterial pathogen responsible for hundreds of thousands of infections each year in the United States. C. difficile outbreaks are common in hospitals because C. difficile spores can persist for months on surfaces and are resistant to many disinfectants. Despite the significant disease burden that C. difficile represents, we know surprisingly little about the factors necessary for C. difficile to colonize and persist in the mammalian intestine. Previous work demonstrated that the signaling molecule cyclic diguanylate (c-di-GMP) regulates a variety of processes in C. difficile including production of the toxins that are required for disease symptoms. Using monolayers of human intestinal epithelial cells, we demonstrate that c-di-GMP promotes attachment of C. difficile to intestinal epithelial cells. We also demonstrate that regulation of type IV pili (TFP) by c-di-GMP promotes prolonged adherence of C. difficile to epithelial cells in vitro. C. difficile mutants lacking TFP were cleared more quickly than the parental strain during single strain mouse infections and were outcompeted by the parental strain during in vivo competition experiments in mice. Thus, our data provides evidence that TFP promote persistence of C. difficile in the intestine. To determine what other genes c-di-GMP regulates in C. difficile, we performed RNA- sequencing comparing the transcriptome of C. difficile with elevated c-di-GMP to that of C. difficile with basal levels of c-di-GMP. We demonstrate that c-di-GMP regulates the expression iii of 166 genes greatly expanding the known members of the c-di-GMP regulon. We demonstrate that c-di-GMP regulation of several transcripts in C. difficile is dependent on c-di-GMP sensing riboswitches present in the 5’ untranslated regions of these transcripts. Our results also show that c-di-GMP regulates a number of cell envelope proteins in addition to TFP and flagella. These data suggest a broader role for c-di-GMP in remodeling the C. difficile cell surface. iv ACKNOWLEDGEMENTS I would like to thank my advisor, Rita Tamayo, for taking me under her wing and showing me how to be a scientist. I would not be where I am today without her outstanding mentorship and guidance. I would also like to thank the many members of the Tamayo lab who have provided helpful feedback and advice throughout my years in the lab. I would especially like to thank Naira Aleksanyan and Liz Garrett for their contributions to Chapter 2, and Carissa Harvest for her contributions to Chapter 3. I am also grateful for the outstanding support that the UNC-CH Microbiology and Immunology program gives to its graduate students and the work the faculty and staff do to make the department an outstanding place to do science. I want to thank my friends and family who have supported me during my graduate career. Lastly, I want to thank my partner Mike, whose loving support has been critical to my success both in and beyond the lab. v TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix LIST OF ABBREVIATIONS ........................................................................................................ xi CHAPTER 1: INTRODUCTION ................................................................................................... 1 DISCOVERY OF CLOSTRIDIOIDES DIFFICILE ................................................................... 1 THE C. DIFFICILE LIFECYCLE AND TRANSMISSION ...................................................... 3 RISK FACTORS FOR CDI ........................................................................................................ 5 PATHOGENESIS OF CDI ......................................................................................................... 7 REGULATION OF TOXIN PRODUCTION ............................................................................. 8 COLONIZATION FACTORS OF C. DIFFICILE ................................................................... 10 C-DI-GMP SIGNALING .......................................................................................................... 12 C-DI-GMP SIGNALING IN C. DIFFICILE ............................................................................ 15 FIGURES .................................................................................................................................. 18 REFERENCES .......................................................................................................................... 20 CHAPTER 2: TYPE IV PILI PROMOTE CLOSTRIDIUM DIFFICILE ADHERENCE AND PERSISTENCE IN A MOUSE MODEL OF INFECTION ............................................... 33 SUMMARY .............................................................................................................................. 33 INTRODUCTION ..................................................................................................................... 34 MATERIALS AND METHODS .............................................................................................. 37 RESULTS .................................................................................................................................. 41 vi DISCUSSION ........................................................................................................................... 48 FIGURES .................................................................................................................................. 51 REFERENCES .......................................................................................................................... 64 CHAPTER 3: TRANSCRIPTIONAL REGULATION IN C. DIFFICILE BY THE SECOND MESSENGER C-DI-GMP ........................................................................... 73 SUMMARY .............................................................................................................................. 73 INTRODUCTION ..................................................................................................................... 73 RESULTS .................................................................................................................................. 77 DISCUSSION ........................................................................................................................... 82 MATERIALS AND METHODS .............................................................................................. 86 FIGURES .................................................................................................................................. 92 REFERENCES .......................................................................................................................... 99 CHAPTER 4: DISCUSSION ...................................................................................................... 104 SUMMARY OF RESULTS AND SIGNFICANCE ............................................................... 104 IMPORTANCE ....................................................................................................................... 111 CONCLUSIONS AND FUTURE DIRECTIONS .................................................................. 114 REFERENCES ........................................................................................................................ 115 APPENDIX: SUPPLEMENTAL FIGURES FOR CHAPTER 3 ............................................... 121 vii LIST OF TABLES Table 2.1. Primers used in this study. ........................................................................................... 62 Table 2.2. Strains and plasmids used in this study. ...................................................................... 63 Table 3.1. Putative cell envelope proteins whose expression is regulated by c-di-GMP. ............ 97 Table 3.2. Changes in transcript abundance for c-di-GMP riboswitches and the downstream genes. ........................................................................................................... 97 Table 3.3 Fold changes in transcripts for genes controlled by class II c-di-GMP riboswitches ... 98 Table 3.4. Fold changes in transcripts for genes controlled by GEMM riboswitches. ................. 98 Table A1.1 Genes regulated by c-di-GMP in C. difficile ........................................................... 121 Table A1.2 Plasmids and Strains used in this study ................................................................... 126 Table A1.3 Olignucleotides used in this study ..........................................................................
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