1 Identification of mammalian cell signaling in response to plasma membrane perforation: Endocytosis of Listeria monocytogenes and The Repair Machinery Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jonathan Gat-Tze Lam Graduate Program in Microbiology The Ohio State University 2018 Dissertation Committee Stephanie Seveau, Advisor Dan Wozniak John Gunn Li Wu 2 Copyrighted by Jonathan Gar-Tze Lam 2018 3 Abstract The animal plasma membrane is a semi-fluid structural platform that maintains cellular homeostasis by regulating the passage of ions and small molecules in and out of the cell and modulating cell signaling activities. Disruption of its barrier function via mechanical damage or perforation by a pore-forming toxin is quickly followed by a sudden influx of extracellular Ca2+, which triggers efficient plasma membrane repair processes, the mechanisms of which are, to date, not fully elucidated. Efforts to understand cellular responses to plasma membrane damage have resulted in several non- mutually exclusive models of repair, each realized by the use of various cell types damaged using approaches that attempt to replicate normal physiological damage (mechanical, osmotic, and sheer stress) or damage that occurs under infectious conditions (bacterial pore-forming toxins). In the context of infection, evolutionarily distinct pathogens including the parasite Trypanosoma cruzi, the Gram-positive bacterium Listeria monocytogenes, and the non- enveloped Adenovirus have been shown to damage the plasma membrane of non- professional phagocytic cells in order to co-opt the subsequent cellular responses to facilitate their entry into target cells. It was concluded that T. cruzi and Adenovirus mechanically damage or perforate the host cell plasma membrane in order to co-opt a i Ca2+ influx-dependent repair mechanism involving the exocytosis of lysosomes, release of acid sphingomyelinase, invagination of the host plasma membrane and endocytosis of the invading pathogen along with the damaged membrane. It was revealed that addition of the cholesterol-dependent cytolysin (CDC) pore-forming toxin Streptolysin O, which forms ~30 nm diameter proteinaceous pores in cholesterol-containing membranes, could further facilitate the efficient entry of these pathogens. Studies using the related CDC pore-forming toxin listeriolysin O (LLO) showed that L. monocytogenes entry into hepatocyte epithelial cells also requires Ca2+ influx subsequent to LLO-mediated perforation of the target cell suggesting that like T. cruzi and Adenovirus, L. monocytogenes could co-opt the repair machinery to gain entry. Using a combination of biochemical assays and live-cell fluorescence resonance energy transfer imaging, we found that LLO-mediated plasma membrane perforation and influx of extracellular Ca2+ activates a signal cascade involving the recruitment and activation of a conventional protein kinase C at the plasma membrane, activation of the central actin regulator, the Rho GTPase Rac1, and induction of Arp2/3-dependent F-actin polymerization leading to bacterial internalization. Inhibition of the cPKC/Rac1/Arp2/3 pathway prevents L. monocytogenes entry, but does not prevent membrane resealing, revealing that, in contract to the mechanism of entry of T. cruzi and Adenovirus, LLO-dependent L. monocytogenes endocytosis is distinct from the resealing machinery. An additional focus of this work has been on unifying the mutually non-exclusive models of plasma membrane repair, which include the Patch hypothesis, lysosomal ii exocytosis and subsequent endocytosis, exosome release, and ectocytosis/microvesicle shedding. Much of the pioneering work that led to these models required the use of morphologically unique cells (Xenopus laevis oocytes, sea urchin eggs, squid giant axons) damaged via microneedle puncture, laser ablation, or transection techniques. Further studies using various somatic mammalian cells including myocytes, neurons, epithelial and endothelial cells damaged via mechanical or sheer stress, or exposed to pore-forming toxins have also led to the identification of numerous proteins involved in the resealing process, many of which are cell type specific. The goal of this work was to provide a platform in which to study plasma membrane resealing in any cell type in a high-throughput manner in order to a) quantify the efficiency of resealing, b) identify new proteins involved in the resealing process, c) determine any overlap across different cell types, and d) unify the various models or resealing into a global system of repair mechanisms. Here we describe a high-throughput microplate-based assay to quantify the membrane resealing efficiency of cells exposed to the pore-forming toxin listeriolysin O using a spectrofluorometric plate reader. Additionally, image cytometry was incorporated to automatically enumerate target cells expressing nuclear localized histone 2B-GFP (HeLa H2B-GFP) before and after damage to account for differences in cell counts due to damage-induced cell detachment. Lastly, as a proof of concept, a siRNA library covering 287 gene targets involved in membrane trafficking, autophagy, lysosome biogenesis and function, ectocytosis, and cytoskeletal dynamics was used to identify proteins involved in the resealing of LLO-perforated HeLa cells. 26/287 knockdown conditions caused a defect in repair, which correspond to clathrin-mediated endocytosis (adaptor related iii proteins, dynamin), vesicular fusion and fission (Rab, SNARE, and exocyst proteins), plasma membrane stabilization (Annexins), and vesicle packaging (COP and ESCRT proteins). Surprisingly 19/287 knockdown conditions actually improved repair indicating that plasma membrane repair can potentially be negatively regulated. These preliminary findings confirmed the applicability of our high-throughput assay in identifying proteins involved in plasma membrane repair, but confirmation screens, pathway analyses, use of different cell types and damaging conditions are still required to meet the goals of this work. iv Dedication Dedicated to my parents Dr. Juan Lam and Dr. Flora Yeung for their guidance and support that have made me the man and scientist I am today. To my sister Jennifer Lam- Gerjarusak who has been a model of dedication and hard work. And lastly, to my beautiful wife Katy Elizabeth Lam, for whom this work lays the future for our family. v Acknowledgments I would like to acknowledge the following people who have been supportive in and out of the lab: Christopher Phelps, Dr. Jordan Angle, Garrett Smith, Dr. Joanna Marshall, Lauren Johnson, Siavash Azari, Jasneet Singh, Madison McQuate, Megan Linz and Bella Cho. I’d like to thank all of the professors in the Department of Microbiology and Department of Microbial Infection and Immunity who have been influential in my academic and technical training. To the researchers who have provided the scientific breakthroughs that helped my projects come to fruition: Dr. Adam Hoppe and Dr. Joel Swanson (University of Michigan Medical School) who developed the fluorescence resonance energy transfer (FRET) stoichiometry method; Dr. Xiaoli Zhang, Eric McLaughlin, and Dr. Chi Song (The Ohio State University) for the statistical analyses, Dr. Alexandra Newton (University of California San Diego) for her guidance on the study of protein kinase C activation and providing us with protein kinase C vectors; Dr. Stephen Vadia (The Ohio State University), a former graduate student of the laboratory, for his contribution to the identification of the novel listeriolysin O-dependent internalization mechanism of L. monocytogenes, and Dr. Sarika Pathak-Sharma (The Ohio State University), a former postdoctoral trainee of the laboratory, for the initial development of the high-throughput membrane resealing assay. I would also like to thank Dr. Robert Tabita and Dr. Jesse Kwiek (The Ohio State University) for allowing me to vi use their French Press and SpectraMax i3X + MiniMax300, respectively. To my committee members Dr. John Gunn, Dr. Dan Wozniak, and Dr. Li Wu (The Ohio State University), your guidance and input throughout my graduate career have ensured the success of my projects. Most importantly, to Dr. Stephanie Seveau developed the present research program, which was funded by the National Institutes of Health (RO1AI107250), and dedicated her time to train me as a research scientist. vii Vita 2009-2011 Laboratory Technician, Department of Biology, University of California San Diego 2011 B.S. Biochemistry/Chemistry, University of California San Diego Revelle College 2011-2012 Contracted Research Associate Synthetic Genomics Inc. La Jolla, Ca 2013-2015 Graduate Teaching Associate Department of Microbiology, The Ohio State University 2016-2017 Vice President of the Students for the Advancement of Microbiology, The Ohio State University Publications Lam, J., et al., High-Throughput Measurement of Plasma Membrane Resealing Efficiency. Journal of Visualized Experiments, in Press. Lam, J., et al., Host cell perforation by listeriolysin O (LLO) activates a Ca(2+)- dependent cPKC/Rac1/Arp2/3 signaling pathway that promotes L. monocytogenes internalization independently of membrane resealing. Molecular Biology of the Cell, 2017. 29(3): p.270-284. Pathak-Sharma, S., et al., High-Throughput Microplate-Based Assay to Monitor Plasma Membrane Wounding and Repair. Frontiers in Cellular and Infection Microbiology, 2017. 7: p. 305. Fields of Study Major