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Iron- and Temperature-Dependent Regulation of Shigella Dysenteriae Virulence

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Iron- and Temperature-Dependent Regulation of Shigella Dysenteriae Virulence Iron- and Temperature-Dependent Regulation of Shigella Dysenteriae Virulence- Associated Factors A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Yahan Wei December 2016 © 2016 Yahan Wei. All Rights Reserved. 2 This dissertation titled Iron- and Temperature-Dependent Regulation of Shigella Dysenteriae Virulence- Associated Factors by YAHAN WEI has been approved for the Department of Biological Sciences and the College of Arts and Sciences by Erin R. Murphy Associate Professor of Bacteriology Robert Frank Dean, College of Arts and Sciences 3 ABSTRACT WEI, YAHAN, Ph.D., December 2016, Biological Sciences Iron- and Temperature-Dependent Regulation of Shigella Dysenteriae Virulence- Associated Factors Director of Dissertation: Erin R. Murphy Shigella is a genus of Gram-negative pathogenic bacteria that causes shigellosis, a severe form of bacillary dysentery in human with an infectious dose of less than 100 cells. The global burden of shigellosis is estimated to be no less than 125 million infections, with the majority of both infection and resulting deaths occurring in children under the age of five. These facts, in combination with the lack of a vaccine or universally effective treatment makes understanding the molecular mechanisms underlying the pathophysiology of Shigella of utmost importance. To survive and successfully colonize in the host, bacterial pathogens regulate the expression of multiple virulence-associated factors in response to the changes of environmental cues. This study focused on two of the important virulence-associated processes in the infections of S. dysenteriae, the most virulent species in the genus of Shigella: 1) acquisition of essential nutritional iron via the Shigella heme uptake (Shu) system from the iron-limited environment within the human host, and 2) secretion of effector proteins required for the invasion processes through the type III secretion system (T3SS). Investigations presented here identify the host-associated environmental factors that regulate the expression of the specific factors required to complete the processes listed above, and characterize the molecular mechanisms underlying each regulation. Specifically, studies focused on the 4 regulation of the Shu system demonstrate that its periplasmic binding component, ShuT, is subject to iron-dependent transcriptional regulation via the activity of the global transcriptional regulator Fur, and temperature-dependent post-transcriptionally mediated by an RNA thermometer located within the 5' untranslated region of the gene. Studies focused on the regulation of MxiG, a component of the T3SS, identify and characterize a functional RNA thermometer that mediates post-transcriptional temperature-dependent regulation. Findings in this study provide: 1) a unique example of Fur-mediated regulation via interacting with a sequence located downstream of the promoter region, and 1) the first evidence of RNA thermometer controlling expression of T3SS component. These findings could lead to revealing of further details about the molecular mechanisms of each regulatory system, and also contribute to the knowledge pool required in designing new strategies of defending against bacterial infections. 5 DEDICATION I would like to dedicate this work to my mother, for her love and support 6 ACKNOWLEDGMENTS I would like to acknowledge all the people and institutions for their support throughout my graduate career and numerous contributions to this work. Firstly, I want to express my sincere gratitude to my advisor Erin R. Murphy, who have been really patient, motivating, and continuously supporting me and guiding me through the project. I would also like to thank my committee members, Peter Coschigano, Jennifer Hines, Donald Holzschu, and Tomohiko Sugiyama, for their insightful comments and encouragement. My thanks also go to my lab mate Megan Firs, our lab technician Michelle Pate, and former lab members Andrew Kouse and William Broach. Finally, I would like to thank Ohio University and the American Heart Association for their financial support. 7 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Dedication ........................................................................................................................... 5 Acknowledgments............................................................................................................... 6 List of Tables .................................................................................................................... 11 List of Figures ................................................................................................................... 12 Chapter 1: Introduction ..................................................................................................... 14 1.1 Significance ............................................................................................................ 14 1.2 Shigella ................................................................................................................... 16 1.2.1 Shigella species and evolutionary relationships .............................................. 16 1.2.2 Pathogenesis ..................................................................................................... 17 1.2.3 Shigella virulence factors ................................................................................. 19 1.3 Regulation of Shigella iron uptake systems ............................................................ 21 1.3.1 Iron uptake systems in Shigella ....................................................................... 21 1.3.2 Regulation of iron uptakes systems ................................................................. 29 1.4 Regulation of bacterial virulence-associated genes via RNA thermometers .......... 33 1.4.1 Families of RNA thermometers ....................................................................... 35 1.4.2 Bacteria virulence-associated genes regulated by RNA thermometer ............. 45 Chapter 2: Iron-dependent regulation of shuT .................................................................. 49 2.1 Abstract ................................................................................................................... 49 2.2 Introduction ............................................................................................................. 49 2.2.1 Heme uptake system and bacterial pathogenesis ............................................. 49 2.2.2 Shigella heme uptake (Shu) system ................................................................. 51 2.2.3 Transportation of heme via the Shu system ..................................................... 52 2.2.4 Fur-mediated iron-dependent regulation of the Shu system. ........................... 56 2.3 Methods and materials ............................................................................................ 58 2.3.1 Strains and culture conditions .......................................................................... 58 2.3.2 Oligonucleotide primers ................................................................................... 60 2.3.3 Reporter plasmid construction ......................................................................... 61 8 2.3.4 RNA extraction and DNA removal .................................................................. 62 2.3.5 Reverse transcriptase PCR ............................................................................... 64 2.3.6 Rapid amplification of cDNA 5' end analysis .................................................. 65 2.3.7 Beta-galactosidase assay .................................................................................. 66 2.3.8 Western blot analysis ....................................................................................... 67 2.3.9 Quantitative Real-time PCR analysis ............................................................... 68 2.3.10 Electrophoretic mobility shift assay ............................................................... 69 2.3.11 In silico analyses ............................................................................................ 71 2.3.12 Statistical analysis .......................................................................................... 72 2.4 Results ..................................................................................................................... 72 2.4.1 Fur mediates iron-dependent regulation of shuT ............................................. 72 2.4.2 Identification of shuT transcriptional start site ................................................ 74 2.4.3 Identification of shuT promoter region ............................................................ 77 2.4.4 Sequences within shuT promoter and 5' untranslated region mediates iron- dependent regulation by Fur ..................................................................................... 79 2.4.5 A functional Fur binding site is located immediately down-stream of the shuT transcription start site ................................................................................................ 81 2.5 Discussion ............................................................................................................... 89 Chapter 3: Temperature-dependent regulation of shuT ...................................................
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