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Salmonella Enterica Serovar Typhimurium Infection in Mouse Pregnancy

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Salmonella Enterica Serovar Typhimurium Infection in Mouse Pregnancy SALMONELLA ENTERICA SEROVAR TYPHIMURIUM INFECTION IN MOUSE PREGNANCY by Shuhiba Mohammad A thesis submitted to the Department of Biomedical and Molecular Sciences In conformity with the requirements for the degree of Master of Science Queen’s University Kingston, Ontario, Canada December, 2016 Copyright ©Shuhiba Mohammad, 2016 Abstract Salmonella are Gram-negative, intracellular food-borne pathogens that cause pregnancy complications. In pregnant mice, Salmonella enterica serovar Typhimurium (S.Tm) infection results in placental bacterial replication, inflammation, necrosis, and fetal loss by unknown mechanisms. Necroptosis, or programmed necrosis mediated by RIPK3 (receptor-interacting protein kinase 3), an inflammatory cell death pathway, is implicated in the pathogenesis of S.Tm in non-pregnant mice. This goal of this thesis was to investigate the role of necroptosis in the pathogenesis of S.Tm infection during mouse pregnancy. I hypothesized that elimination of the key necroptotic cell death protein RIPK3 would decrease placental inflammation and trophoblast cell death, and increase conceptus survival compared to controls. Mice expressing a functional Slc11a1 (encodes the natural resistance-associated macrophage protein 1, NRAMP1) gene with or without RIPK3 function (Ripk3-/-Slc11a1+/+ compared to Slc11a1+/+) were infected with 103 S.Tm by tail vein injection on gestational day (GD) 12. Mice were euthanized on GD 14 (48h post-infection) or GD 15 (72h post-infection) and implantation sites (IS) and maternal serum were harvested for analyses. In nearly all challenged mice (except one outlier), S.Tm were detected in most IS within a litter but there was limited immune cell infiltration, placental damage or cell death in Slc11a1 competent mice regardless of Ripk3 gene deletion. Maternal serum cytokine analyses confirmed lack of maternal immune responses to S.Tm infection. IS amongst the litter of a single dam (Ripk3-/-Slc11a1+/+ at 72h post- infection) displayed heavy but not universal placental S.Tm infection of decidual tissues and spongiotrophoblast, associated with elevated maternal serum pro-inflammatory cytokines. S.Tm infection of the fetal yolk sac (YS) was observed in 54.5% of IS from this dam. YS infection was confirmed in archival samples in mice expressing Ripk3 with intact Slc11a1 and in mice lacking functional Slc11a1. In Slc11a1 incompetent mice, S.Tm were detected in placental labyrinthine trophoblast. Based on the available data, this thesis suggests that Ripk3 and necroptosis have no significant roles in either promotion ii or prevention of progressive Salmonella infection during mouse pregnancy. It also provides pilot data that NRAMP1 controls placental localization and lethality due to YS infection. iii Acknowledgements This thesis has come to fruition through the unrelenting support of so many. Firstly, from my advisor, Dr. Anne Croy, for which her mentorship is and always will be, unmatched. Her undying support and counsel have had a massive role in my success. The effect of her guidance and support will impact my work for a lifetime. I will continue to exemplify her attitudes towards the pursuit of science by responding to emails within minutes of receipt (no matter the time), attending as many scientific meetings as humanely and financially possible, and to educate and discuss with everyone the wonders of this world. I also hope to emulate her impeccable style and fashion-sense, by which I am constantly inspired. Dr. Croy will always be my role model and she is the best advisor I could have ever hoped for. I would also like to acknowledge Dr. Neil MacLusky, former Chair of Biomedical Sciences at the University of Guelph for believing in me, especially when it was difficult to believe in myself. He supported and guided my passion for science by connecting me with Queen's University and Dr. Croy, and for that I am eternally grateful. Thank you to my collaborators at the University of Rochester, Dr. Shawn Murphy and Ian Perry. I will miss our lengthy discussions and the beautiful images of the microscopic monster we've been chasing for forever in "Where's Waldo"-like fashion. I would also like to thank Dr. Lakshmi Krishnan and Kristina Wachholz at the University of Ottawa for sample preparation. Thank you, Dr. Nancy Martin, for all of your expertise and help as part of my advisory committee. Thank you to all the mice sacrificed for this endeavor. I would like to thank my Croy lab mates Ashley Martin, Allison Felker, Mackenzie Redhead, Vanessa Kay, Nathália Portilho, Rayana Luna, Ernesto Figueiro-Filho, Matt Rätsep and countless others for their guidance and friendship. Thank you to all my colleagues in the Department of Biomedical and Molecular Sciences at Queen's University for which there are far too many to name here. A special thank you to my undergraduate volunteers, Zain Saleem and Olivia Jeon for their many hours spent running intricate histology protocols to my exact specifications. Many of the images included in this thesis were a direct result of their work. Their tireless efforts will not be forgotten; my wrist and sanity thanks them. Thank you to my enormous network of roommates, friends and my incredible family for their constant support throughout this thesis. Madar and Padar, thank you for supporting me through this journey. I know I will always be your curious baby that stuck keys in sockets, and would never believe in monsters lurking behind curtains. Thank you for supporting me in the pursuit of my dreams. "Somewhere, something incredible is waiting to be known" - Carl Sagan iv Table of Contents Abstract ......................................................................................................................................................... ii Acknowledgements ...................................................................................................................................... iv List of Figures ............................................................................................................................................ viii List of Tables .................................................................................................................................................x List of Abbreviations ................................................................................................................................... xi Chapter 1 Introduction and Literature Review ..............................................................................................1 1.1 Salmonella ............................................................................................................................................1 1.1.1 Natural course of Salmonella infection .........................................................................................1 1.1.2 Salmonella infection in human pregnancy ....................................................................................3 1.2 Pregnancy-enhanced pathogenesis of Salmonella ...............................................................................3 1.2.1 Immunological shifts during pregnancy .......................................................................................3 1.2.2 Placental Salmonella tropism ........................................................................................................5 1.3 Mouse Pregnancy .................................................................................................................................9 1.3.1 Mouse placental anatomy .............................................................................................................9 1.3.2 Immune cells at the mouse maternal-fetal interface ...................................................................13 1.4 Salmonella enterica serovar Typhimurium infection during murine pregnancy ...............................17 1.5 Salmonella-induced cell death mechanisms ......................................................................................22 1.5.1 Necroptosis .................................................................................................................................23 1.6 Rationale, hypothesis and main objectives ........................................................................................26 Chapter 2 Materials and Methods ................................................................................................................27 2.1 Mice ...................................................................................................................................................27 2.2 Genotyping .........................................................................................................................................28 2.3 Mating strategy and pregnancy timing ..............................................................................................29 2.4 Bacterial preparation and in vivo infection ........................................................................................29 2.5 Sample collection ...............................................................................................................................29 2.6 Tissue processing for histology .........................................................................................................30 2.7 Salmonella enterica serovar Typhimurium immunohistochemistry ..................................................32 2.7.1 S.Tm Immunofluorescence .........................................................................................................34
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