A Dissertation entitled Characterization of the Vibrio cholerae Phage Shock Protein Response by Cara Marie DeAngelis Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Science ___________________________________________ Jyl Matson, Ph.D., Major Advisor ___________________________________________ Robert Blumenthal Ph.D, Committee Member ___________________________________________ Mark Wooten, Ph.D, Committee Member ___________________________________________ Jason Huntley, Ph.D, Committee Member ___________________________________________ David Giovannucci, Ph.D, Committee Member ___________________________________________ Cyndee Grudgen, Ph.D., Dean College of Graduate Studies The University of Toledo May 2019 © 2019 Cara Marie DeAngelis This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Characterization of the Vibrio cholerae Phage Shock Protein Response by Cara Marie DeAngelis Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Science The University of Toledo May 2019 Cholera is a severe intestinal infection characterized by voluminous, watery diarrhea that can be fatal within hours. It is caused by the marine bacterium Vibrio cholerae of serogroups O1 and O139. While rare in the United States and other industrialized nations, cholera is endemic in more than 50 countries. In both its aquatic and intestinal life cycles, V. cholerae encounters various stressful conditions, such as fluctuating pH, bacteriophage predation, and exposure to antimicrobial peptides that may disrupt the cell envelope. The phage shock protein (Psp) system is a stress response pathway that senses and responds to inner membrane damage. The genetic components of the Psp system are present in several clinically relevant Gram-negative bacteria, including V. cholerae. However, most of the current knowledge about the Psp response stems from in vitro studies in Escherichia coli and Yersinia enterocolitica. In fact, the Psp response in V. cholerae has remained completely uncharacterized. In this dissertation, it is demonstrated that V. cholerae does have a functional Psp response system. The overexpression of GspD (EpsD), the type II secretion system secretin, induces the Psp response, whereas other V. cholerae secretins do not. In addition, several environmental conditions were identified as inducers of this stress response. Experiments on the genetic iii regulation and induction of the Psp system in V. cholerae suggest that the key regulatory elements are conserved with those of other Gram-negative bacteria. While a psp null strain is fully capable of colonizing the infant mouse intestine, it exhibits a colonization defect in a zebrafish model, indicating that this response may be important for disease transmission in the environment. Overall, these studies provide an initial understanding of a stress response pathway that has not been previously investigated in V. cholerae. iv To my parents v Acknowledgements There are a vast number of individuals that supported me throughout my predoctoral studies. First, I would like to acknowledge my mentor, Dr. Jyl Matson. While I still have a lot to learn, I will leave her laboratory with the ability to proudly call myself a scientist. I would like to thank my committee members for counseling me on my research project for the last three years. I would also like to thank Dr. Rande Worth for his mentorship and accepting the position of serving as my faculty representative at my defense. In addition, I would like to thank our collaborators, Dr. Jeff Withey and Dr. Dhrubajyoti Nag at Wayne State University. I am thankful for the continued patience, guidance and support I have received from my previous lab members, Jess Saul-McBeth, Sarah Plecha and Laura Stanbery- Nejedlik. In addition, I am grateful to past and present MMI students and the bonds of solidarity we formed over the years and monthly munches. I am also deeply grateful for the Glass City Readers and my fairy godmothers for cultivating my personal growth outside of science. Lastly, I would like to thank my family. They supported me through all the highs and lows. In addition, Nitro has been a loving companion every single day. vi Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ............................................................................................................ vi Table of Contents .............................................................................................................. vii List of Tables ................................................................................................................... ix List of Figures ......................................................................................................................x List of Abbreviations ........................................................................................................ xii Chapter 1 General Introduction ....................................................................................1 1.1 Cholera History and Global Impact ..........................................1 1.2 V. cholerae and its environment ...............................................5 1.3 V. cholerae Pathogenesis ..........................................................8 1.4 Extracytoplasmic stress response systems ..............................11 1.5 Summary and Aims….............................................................23 Chapter 2 Vibrio responses to extracytoplasmic stress ..............................................24 2.1 Summary…. ............................................................................24 2.2 Introduction…. ........................................................................25 2.3 The σE response …. .................................................................27 2.4 The Cpx response..…. .............................................................34 2.5 Stress relief via OmpU…. .......................................................38 2.6 Concluding remarks…. ...........................................................44 vii 2.7 Acknowledgments…...............................................................44 Chapter 3 Characterization of the Vibrio cholerae phage shock protein response ....45 3.1 Abstract…. ..............................................................................46 3.2 Importance…. .........................................................................46 3.3 Introduction…. ........................................................................47 3.4 Results..…. ..............................................................................50 3.5 Discussion…. ..........................................................................65 3.6 Materials and Methods…. .......................................................72 3.7 Acknowledgments…...............................................................82 3.8 Supplemental Figures…..........................................................83 Chapter 4 Additional Studies on the V. cholerae Psp response ..................................86 4.1 Introduction…. ........................................................................86 4.2 Growth phenotypes of psp mutants..........................................86 4.3 PspC requires PspB for stability .............................................88 4.4 Investigating the relationship between the Psp and σE response…………………………………………………………..91 4.5 Discussion……………………………………………………92 Chapter 5 Discussion ..................................................................................................97 5.1 Overview…. ............................................................................97 5.2 Discussion and future directions. ............................................99 5.3 Concluding Remarks…. ........................................................108 References ........................................................................................................................109 viii List of Tables 1.1 RNA sequencing reveals psp transcripts are upregulated in O395ΔrpoE in comparison to O395 ...............................................................................................14 2.1 Stress-associated phenotypes of the RpoE response in Vibrio species ..................31 2.2 Role of RpoE in host-induced response to stress ...................................................32 3.1 Strains and Plasmids ..............................................................................................73 4.1 Comparison of RNA-seq and qRT-PCR results of psp levels in O395 versus O395ΔrpoE. ...........................................................................................................91 ix List of Figures E 2 – 1 Model of σ , Cpx, and OmpU involvement in extracytoplasmic stress relief .......43 3 – 1 Genetic organization of Psp systems and PspA sequence similarity .....................51 3 – 2 The T2SS secretin, GspD, induces the Psp response in V. cholerae .....................55 3 – 3 GspD forms heat resistant multimers that mislocalize to the inner membrane, where the other V. cholerae secretins do not .........................................................57 3 – 4 GspD overexpression