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Listeriolysin O Activates Listeria Monocytogenes Internalization Into Human Hepatocytes

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Listeriolysin O Activates Listeria Monocytogenes Internalization Into Human Hepatocytes Listeriolysin O activates Listeria monocytogenes internalization into human hepatocytes through a novel pore-dependent mechanism Dissertation Presented in partial fulfillment of the requirements for the degree doctor of philosophy in the Graduate School of The Ohio State University By Stephen Vadia, M.S. Graduate Program in Microbiology The Ohio State University 2014 Dissertation Committee: Dr. Stephanie Seveau, Advisor Dr. Robert Munson Dr. Larry Schlesinger Dr. Susheela Tridandapani Copyright by Stephen Vadia 2014 Abstract The causative agent of listeriosis, Listeria monocytogenes, is a frequent contaminant of fruits, vegetables, cheeses, and processed foods. In certain high risk groups, L. monocytogenes can cause serious and potentially fatal infections. The bacterium can traverse the intestinal epithelial barrier and spread throughout the body via the bloodstream and lymphatic system. From there, it can infect the liver, cross the blood- brain barrier, and in pregnant women, the placental barrier. L. monocytogenes is able to cross these biological barriers due to its ability to proliferate within certain phagocytic and nonphagocytic cells. Bacterial invasins, most notably internalin (InlA) and InlB, bind to receptors on the surface of nonphagocytic cells to induce endocytosis of the bacterium through a zipper-like mechanism. L. monocytogenes can then escape from the endocytic vacuole and enter the cytosol through the activity of the pore-forming toxin listeriolysin O (LLO). In the cytosol, the bacterium can replicate and disseminate to adjacent cells by cell-to-cell spreading. LLO belongs to the cholesterol-dependent cytolysin (CDC) family of bacterial pore-forming toxins. These toxins are secreted as monomers, oligomerize on cholesterol- rich membranes, and form large transmembrane pores. In addition to mediating vacuolar escape, LLO stimulates a number of signaling pathways in host cells. We hypothesized ii that the signaling activity of extracellular LLO affects the intracellular lifecycle of L. monocytogenes. Using quantitative immunofluorescence microscopy to assess the importance of LLO for bacterial association to and internalization into epithelial cells, we found that LLO is required for efficient L. monocytogenes internalization into hepatocytes, and is sufficient to induce internalization of noninvasive bacteria and polystyrene beads. Using novel LLO variants that bind to host cells but are unable to form the pore complex, we demonstrated that pore formation was required for LLO- mediated internalization. The CDC streptolysin O is known to stimulate a Ca2+-dependent membrane repair response that involves endocytosis of the toxin from the perforated plasma membrane. We found that LLO stimulates a similar Ca2+-dependent membrane repair response, but that this response is not sufficient to account for L. monocytogenes internalization. LLO- mediated internalization and membrane repair share the requirement for the influx of extracellular Ca2+, but differ in their requirement for F-actin and K+ efflux. Surprisingly, using ionophores to stimulate Ca2+ and K+ fluxes, as happens when the plasma membrane is perforated by LLO, was sufficient to induce internalization of large particles. LLO mediates bacterial internalization in all human hepatocytes we tested and in additional epithelial cell lines. However, LLO does not induce bacterial internalization in all epithelial cells that it can perforate. In light of this data, LLO emerges as a significant invasion factor expressed by L. monocytogenes, which is coexpressed with InlA and InlB during infection. With the same approaches used to assess the role of LLO, we also determined the contribution of InlA and InlB to bacterial association, internalization, and intracellular viability in iii nonphagocytic cells that express the InlA and InlB receptors. We found significant variability in the involvement of each of these invasins in bacterial association, internalization, and intracellular viability, demonstrating that the activity of invasins is not governed solely by the presence or absence of their receptors. iv Dedication This dissertation is dedicated to my mom for her neverending support and her encouragement to pursue a career in science. I would never have started or finished this journey without her there to help me along the way. And to my Grandma and Grandpa Iacobucci, for believing in me at times when I did not believe in myself. v Acknowledgements First and foremost, I would like to thank my advisor, Dr. Stephanie Seveau, for her guidance, mentorship, and patience with me during my graduate career. I would also like to acknowledge my committee members, Dr. Robert Munson, Dr. Susheela Tridandapani, and Dr. Larry Schlesinger, for their comments and insights during committee meetings, and for recommendations which have supported me greatly in securing funding while at Ohio State and during my job search. Former lab members have helped immensely with this project. Anne-Cecile Haghighat began this work before I joined the lab, trained me as an incoming graduate student, and with the help of Andrew Bruggeman, constructed LLO variants used in this project. Dr. Eusondia Arnett performed invaluable experimental work for these studies during particularly busy times. This work also benefitted greatly from the help of many collaborators here at Ohio State University. I would like to thank Dr. Prosper Boyaka for his immunological expertise and for performing mouse immunizations. From the Boyaka lab, Astrid Bonnegarde-Bernard, and Mike Fial, provided critical hands on assistance with mouse infections. We were also greatly assisted in these experiments by Dr. Abhay Satoskar, as well as Dr. Steve Oghumu and Sanjay Varikuti from the Satoskar lab. Dr. John Robinson vi and Dr. William Ackerman provided protocols, advice, cell lines, and placental villi. Brian Kemmenoe at the OSU Campus Microscopy and Imaging Facility provided precious help with scanning electron microscopy. Dr. Elizabeth Wilson-Kubalek (The Scripps Research Institute, La Jolla, CA) performed transmission electron microscopy and provided us with wonderful images. I also wish to thank Dr. Daniel Portnoy (University of California, Berkeley, CA), Dr. Pascale Cossart (Pasteur Institute, Paris, France), Dr. Rodney Tweten (University of Oklahoma Health Sciences Center, Oklahoma City, OK), and Dr. Nancy Freitag (University of Illinois at Chicago, USA) for generously providing us with bacterial strains and plasmids. I also must thank Dr. Chad Rappleye and members of his lab, especially Dr. Eric Holbrook and Dr. Jessica Edwards, for all of their advice over the years, as well as Dr. Mary Anne Rubio and Dr. Juan Alfonzo for support, recommendations, and the occasional pep talk. vii Vita 2003.........................................................................................................B.A. Anthropology Case Western Reserve University 2007 to 2008.......................................................................................University Fellowship The Ohio State University 2008 to 2012...........................................................................Graduate Teaching Associate Department of Microbiology The Ohio State University 2010.........................................................................................................M.S. Microbiology The Ohio State University 2012 to 2013.....................................................................................Presidential Fellowship The Ohio State University 2013 to present........................................................................Graduate Teaching Associate Department of Microbiology The Ohio State University viii Publications Vadia S, Arnett E, Haghighat AC, Wilson-Kubalek EM, Tweten RK, and Seveau S. 2011. The pore-forming toxin listeriolysin O mediates a novel entry pathway of L. monocytogenes into human hepatocytes. PLoS Pathogens, 7:e1002356. Chapter 2. Vadia S, Boyaka PN, Arnett E, Satoskar AR, Tweten RK, and Seveau S. 2013. Immunization with a listeriolysin O toxoid protects mice against Listeria monocytogenes. (Manuscript submitted for publication). Chapter 3. Vadia, S and Seveau, S. 2013. Fluxes of Ca2+ and K+ are required for the LLO-dependent internalization pathway of Listeria monocytogenes. (Manuscript submitted for publication). Chapter 4. Arnett E, Vadia S, Nackerman CC, Oghumu S, Satoskar A, McLeish KR, Uriarte SM, and Seveau S. 2013. The pore-forming toxin listeriolysin O is degraded by neutrophil proteases and fails to protect L. monocytogenes against intracellular killing. (The Journal of Immunology. In press). ix Vadia S, Arnett E, and Seveau S. Assessing the roles of LLO, InlA, and InlB during the L. monocytogenes intracellular lifecycle in hepatocytes and placental cells (Manuscript in preparation). Chapter 5. Fields of Study Major Field: Microbiology x Table of Contents Abstract...............................................................................................................................ii Dedication........................................................................................................................... v Acknowledgements........................................................................................................... vi Vita................................................................................................................................... viii Table of Contents.............................................................................................................
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