Characterization of the Cpx Response in Vibrio cholerae by Paula Nicole Acosta Amador A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Biotechnology Department of Biological Sciences University of Alberta © Paula Nicole Acosta Amador, 2015 Abstract The gram negative bacterial cell envelope is composed of the outer membrane, the periplasm and the inner membrane. These compartments are exposed directly to changes in the environment that are sensed and adapted to through different signaling transduction pathways. This often occurs through two-component signal transduction systems (TCS), which are broadly distributed among different bacterial species. The Cpx pathway is a TCS that employs the sensor histidine kinase CpxA and the response regulator CpxR, and regulates crucial adaptations to envelope stress response that affects many functions, including antibiotic resistance, across bacterial species. This system has also been implicated in the regulation of a number of envelope localized virulence determinants across bacterial species. The first goal of this thesis was to characterize the Cpx regulon members in the human pathogen Vibrio cholerae when the Cpx pathway is activated. For this purpose I characterized the transcriptional profile of the pandemic V. cholerae El Tor strain C6706 upon overexpression of cpxR, and the inducing cues that lead to the activation of the Cpx pathway. My data shows that the Cpx regulon of V. cholerae is enriched for genes encoding membrane localized and transport proteins, including a large number of genes known or predicted to be iron-regulated. The V. cholerae Cpx regulon included three strongly Cpx-regulated, putative ferric reductases that are likely directly regulated by CpxR. I present evidence that the function of these ferric reductases is likely tied to the up-regulation of iron- related genes by the Cpx response. Activation of the Cpx pathway also led to the expression of TolC, the major outer membrane pore, and components of two resistance-nodulation-division (RND) efflux systems in V. cholerae. I found that iron chelation, toxic compounds, or deletion of specific RND efflux components lead to Cpx pathway activation. Further, mutations that eliminated the Cpx response or members of its regulon resulted in growth phenotypes in the ii presence of these inducers that, together with Cpx pathway activation, are partially suppressed by iron. Cumulatively, these results suggest that a major function of the Cpx response in V. cholerae is to mediate adaptation to envelope perturbations caused by toxic compounds and the depletion of iron. A second goal of this thesis was to characterize the effect of Cpx pathway activation on V. cholerae virulence factors. I found that activation of the Cpx pathway leads to a decrease in expression of the major virulence factors in this organism, cholera toxin (CT), and the toxin- coregulated pilus (TCP). The Cpx response controls virulence factor expression by repressing expression of the ToxT and TcpP regulators. I showed that the effect of the Cpx response on ctxB and tcpA expression is mostly abrogated in a cyclic adenosine monophosphate (cAMP) receptor protein (CRP) mutant, although expression of the crp gene is unaltered. These observations indicate that CRP function is affected by Cpx response activation. Altogether, the data presented here suggest a model whereby the Cpx response reduces production of CT and TCP by controlling the expression and function of regulators that function on the ToxR regulon in V. cholerae. iii Preface Some of the research conducted for this thesis forms part of a research collaboration, led by Professor T. L. Raivio at the University of Alberta, with Professor S. Pukatzki being the lead collaborator at the Department of Medical Microbiology & Immunology, University of Alberta. The ex-vivo animal model experiments referred to in Chapter 4 were performed by members of Professor S. Pukatzki’s research group. The high throughput killing screen referred to in the Appendix was design and performed by myself and D. Unterweger, a PhD student in Professor S. Pukatzki research group. Most of the content of Chapter 2 of this thesis has been published as N. Acosta, S. Pukatzki, and T.L. Raivio, “The Vibrio cholerae Cpx envelope stress response senses and mediates adaptation to low iron,” Journal of Bacteriology, vol. 197, issue 2, doi:10.1128/JB.01957-14. I was responsible for the data collection and analysis as well as the manuscript composition. S. Pukatzki contributed to manuscript edits. T.L. Raivio was the supervisory author and was involved with concept formation and manuscript composition and edits. iv Acknowledgments I would like to thank my supervisor Dr. Tracy Raivio for, first, giving me the opportunity of joining her lab, and also for all her support and feedback during my PhD studies. It was a pleasure to work with you not only for all your knowledge, experience and coaching, but also for your incredible kindness. I learnt many things from you; you gave me the tools to become a scientist and the trust to develop my projects. Thank you so much for all your advice and being part of this important step in my life. Thanks to my committee members, Dr. Stefan Pukatzki, for the great support in all aspects on the projects that we worked together, you were an important part in my academic development; it was a real pleasure to work in collaboration with you, I learnt valuable things from you and your lab members; and Dr. Christine Szymanski, for all your time, valuable and helpful comments, advices, and feedback that contributed a lot in this thesis. Thanks also to my thesis examiners, Dr. Mario Feldman and Dr. Victor J. DiRita, for accepting being part of my committee and for your time and effort. Thank you to all my lab members, past and present. Stef, Roxana, Shannon, Julia, Stefan, Suey, Junshu, Randi, Margarita, and Alex- I really appreciate all your help and advices that contributed to this thesis. I learnt many things from each of you such as your kindness, collaboration and most importantly have fun doing science. It was a pleasure to work with this wonderful team. Also thanks to all Pukatzki’s lab members, for your collaboration in many steps of this thesis, especially to Daniel who was a great person to work with. Mom you have always been my engine, my motivation to go a step further; I really appreciate and thanks all your effort and support through my entire life. Thanks to you I am the person who v I am. Natalia, Melissa and Juan Sebastian you are not only my siblings but also my friends, I know I can always count on you. Last but not least, David I thank you for being always with me, for supporting me and teaching me how to be a better person during any time no matter how difficult it is. We came here to reach this important academic step in our life but along this process we began a big journey full of joy and love. I can’t wait to see what our next adventure is, but something that I know for sure is that no matter what, it will always be special and full of love. vi Table of Contents 1 Chapter 1: General Introduction .................................................................................... 1 1.1 Gram-Negative Bacterial Cell Envelope ................................................................ 2 1.1.1 Outer membrane (OM) ................................................................................... 2 1.1.2 Periplasm ........................................................................................................ 3 1.1.3 Inner membrane (IM) ..................................................................................... 5 1.1.4 Gram-negative surfaces structures .................................................................. 6 1.2 Two-Component Signal Transduction Systems (TCS) ......................................... 8 1.2.1 Sensor histidine kinases .................................................................................. 8 1.2.2 Response regulators ........................................................................................ 9 1.2.3 Phosphotransfer specificity........................................................................... 10 1.3 Cell Envelope Stress Responses ............................................................................ 10 1.3.1 The σE pathway ............................................................................................. 11 1.3.2 Bae stress response ....................................................................................... 13 1.3.3 Rcs stress response ....................................................................................... 14 1.3.4 Phage-shock protein (Psp) stress response ................................................... 17 1.3.5 Cpx envelope stress response ....................................................................... 20 1.4 The Cpx Envelope Stress Response ...................................................................... 20 1.4.1 Cpx signal transduction ................................................................................ 20 1.4.2 Cpx inducing cues ........................................................................................ 23 vii 1.4.3 Physiological role of the Cpx response ........................................................ 25 1.4.4 The Cpx pathway in other bacterial species ................................................. 31 1.5 Vibrio cholerae .......................................................................................................