Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 5-2011 Stress Response And Pathogenesis of Salmonella enterica serovar Typhimurium Jigna D. Shah Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Microbiology Commons Recommended Citation Shah, Jigna D., "Stress Response And Pathogenesis of Salmonella enterica serovar Typhimurium" (2011). All Graduate Theses and Dissertations. 900. https://digitalcommons.usu.edu/etd/900 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. STRESS RESPONSE AND PATHOGENESIS OF SALMONELLA ENTERICA SEROVAR TYPHIMURIUM by Jigna D. Shah A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Nutrition and Food Sciences Approved by: ____________________ ___________________ Bart C. Weimer Marie Walsh Major Advisor Co Advisor ___________________ ___________________ Dong Chen John Stevens Committee Member Committee Member ___________________ ___________________ Joanie Hevel Byron Burnham Committee Member Dean of Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2011 ii Copyright © Jigna D. Shah All Rights Reserved iii ABSTRACT Stress Response and Pathogenesis of Salmonella enterica serovar Typhimurium by Jigna D. Shah, Doctor of Philosophy Utah State University, 2011 Major Professor: Dr. Bart Weimer Department: Nutrition, Dietetics, and Food Sciences Salmonella is a food-borne pathogen that leads to substantial illness worldwide. The clinical syndromes associated with Salmonella infection are enteric (typhoid) fever and gastroenteritis, in healthy humans. Typhoid fever is caused by host-adapted S. Typhi and S. Paratyphi. Gastroenteritis is caused by serovars usually referred to as non typhoidal Salmonellae (NTS). In recent years, an increasing number of outbreaks due to NTS, despite increased efforts in food safety, were reported because of persistence of Salmonella in the food chain. Thus I hypothesized that Salmonella is able to withstand stresses in the environment and treatments used during food processing for its elimination and thereby able to develop resistance against subsequent stress encounters. The effect of cold, peroxide, and acid was tested on survival of S. Typhimurium and the survival was persistent under cold stress (5°C) for up to 240 h. Pre-adaptation to cold stress (5°C, 5 h) also increased survival of S. Typhimurium during subsequent exposure to acid stress (pH 4.0, 90 min) by repressing hydroxyl radical formation. Cold stress (5°C, 48 h) to S. Typhimurium significantly (p < 0.05) increased its adhesion and invasion in intestinal iv epithelial cells. This phenotype was attributed to a pair of protein-protein interactors acting as receptors on microbial (STM2699) and host cell surface (SPTAN1). Cold stress significantly (q < 0.05) induced STM2699 in S. Typhimurium and SPTAN1 was significantly (q < 0.05) induced in epithelial cells upon infection with cold-stressed S. Typhimurium. Cold stress to S. Typhimurium also significantly (q < 0.05) induced genes related to virulence such as type 3 secretion system apparatus and effectors genes, prophage genes, and plasmid genes and they remain induced upon infection of epithelial cells with additional induction of spv genes on the plasmid. Infection of epithelial cells with cold-stressed S. Typhimurium significantly (p < 0.05) increased activation of caspase 9 and 3/7. Cold-stressed S. Typhimurium switched metabolism from aerobic respiration to fermentation and it persisted during infection of epithelial cells. As a result, short chain fatty acids formate and acetate, which act as diffusible signal for invasion, were detected in significantly (q < 0.05) high amounts in extracellular media of cells infected with cold-stressed S. Typhimurium supporting the phenotype of high adhesion and invasion of cold-stressed S. Typhimurium in epithelial cells. (241 pages) v ACKNOWLEDGMENTS With deep gratitude and sincerity, I would like to thank my advisor, Dr. Bart Weimer, for his guidance, expertise, support, and patience throughout this study. He provided constant encouragement and motivation with great understanding. The scientific discussions with him were very fruitful. I wish to extend my sincere appreciation to my co-advisor, Dr. Marie Walsh, for her kind support all the way through and my committee members, Dr. Dong Chen for his help with proteomics study, Dr. John Stevens for his help with statistics, and Dr. Joanie Hevel for her valuable suggestions. I sincerely thank Dr. Giovanni Rompato and Ninglin Yin from Genomics core at the Center of Integrated Biosystems for their assistance in running GeneChips. I am grateful to Prerak Desai for his extensive discussions around my work, and his interesting explorations in the datasets have been very helpful for this study. I wish to thank Dr. Balasubramanian Ganesan and Sweta Rajan for their friendly help and scientific debates during my stay at Logan. I am thankful to all my friends and lab mates at Logan for their help, especially Amrita and Ashwini for the care and support they provided. My deepest gratitude goes to my entire extended family for their unflagging affection and support throughout my life; this study would not have been possible without their continuous encouragement. Most importantly, I am indebted to my mother, Mrs. Ratan D. Shah, for her unconditional love and constant motivation. I sincerely dedicate this dissertation to my mother who has been instrumental in inspiring me at every step throughout my life on both personal and professional fronts. Jigna D. Shah vi CONTENTS Page ABSTRACT ....................................................................................................................... iii ACKNOWLEDGMENTS ...................................................................................................v LIST OF TABLES ............................................................................................................. xi LIST OF FIGURES ......................................................................................................... xiii LIST OF SYMBOLS AND ABBREVIATIONS ............................................................ xvi CHAPTER 1. INTRODUCTION ...........................................................................................................1 REFERENCES .......................................................................................................4 2. LITERATURE REVIEW ................................................................................................6 Phylogeny and serotypes ..............................................................................6 Genetics and genomics ................................................................................7 Salmonellosis in animals..............................................................................8 Salmonellosis in humans............................................................................10 Adhesion. ...................................................................................................11 Invasion. .....................................................................................................13 Prophages. ..................................................................................................16 Stress response of Salmonella ....................................................................16 Acid stress response ...................................................................................18 Peroxide stress response ............................................................................20 Cold stress response ...................................................................................22 HYPOTHESIS ......................................................................................................24 OBJECTIVES .......................................................................................................25 REFERENCES .....................................................................................................25 3. COLD STRESS TO S. TYPHIMURIUM INCREASES ITS SURVIVAL DURING SUBSEQUENT ACID STRESS ...................................................................................49 vii ABSTRACT .........................................................................................................49 INTRODUCTION ................................................................................................50 MATERIALS AND METHODS .........................................................................52 Bacterial strains and culture conditions .....................................................52 Stress treatments. .......................................................................................52 Proteome analysis ......................................................................................53 Gene expression analysis ...........................................................................54 Hybridization and normalization ...............................................................55 NAD+/NADH quantification .....................................................................56 Radical measurement .................................................................................56