Role of the Small Regulatory RNAs Lpr10 and Lpr17 in the Regulation of the Stress Response in Legionella pneumophila Joseph Saoud Department of Natural Resource Sciences Faculty of Agriculture and Environmental Sciences McGill University December 2020 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © Joseph Saoud 2020 Table of Contents Abstract .......................................................................................................................................... 7 Résumé ........................................................................................................................................... 9 Acknowledgements ..................................................................................................................... 12 Contribution to knowledge......................................................................................................... 18 Contribution of authors .............................................................................................................. 20 Contribution of Authors Chapter 3....................................................................................... 20 Contribution of Authors Chapter 4....................................................................................... 20 Contribution of Authors Chapter 5....................................................................................... 20 List of Abbreviations .................................................................................................................. 22 Chapter 1: Introduction ............................................................................................................. 25 Chapter 2: Literature Review .................................................................................................... 28 2.1 Introduction ................................................................................................................. 28 2.2 Legionella’s General Characteristics ........................................................................ 31 2.3 Life Cycle ..................................................................................................................... 33 2.4 Host Cell Infection ...................................................................................................... 34 2.4.1 Attachment and Entry ........................................................................................... 35 2.4.2 Establishment of the LCV ..................................................................................... 36 2.4.3 Replication ............................................................................................................ 36 2.4.4 Egress .................................................................................................................... 37 2.5 Immune Response: ...................................................................................................... 37 2.5.1 Cytokine Production ............................................................................................. 38 2.5.2 Detection ............................................................................................................... 38 2.5.3 Oxidative Burst ..................................................................................................... 41 2.6 Secretion Systems in Legionella ................................................................................. 42 2.6.1 Type I Secretion System (T1SS): Lss ................................................................... 42 2.6.2 Type II Secretion System (T2SS): Lsh ................................................................. 43 2.6.3 Type IVa Secretion System (TIVaSS): Lvh ......................................................... 44 2.6.4 Type IVb Secretion System (TIVbSS): Icm/Dot .................................................. 44 2.7 Major Regulators of Legionella ................................................................................. 48 2.7.1 Sigma Factors........................................................................................................ 49 2.7.2 Quorum Sensing.................................................................................................... 52 2.7.3 Stringent Response................................................................................................ 53 2.7.4 Two-Component Systems (TCS) .......................................................................... 54 2.8 Small Regulatory RNAs (sRNA)................................................................................ 58 2.8.1 Base-Pairing sRNAs ............................................................................................. 58 2.8.2 Protein-Binding sRNAs ........................................................................................ 59 2 2.8.3 RNA Chaperones .................................................................................................. 59 2.8.4 sRNAs in Other Bacteria ...................................................................................... 61 2.8.5 sRNAS in Legionella pneumophila ...................................................................... 67 2.9 Regulation by sRNAs .................................................................................................. 70 2.9.1 Negative Regulation by sRNAs ............................................................................ 71 2.9.2 Positive Regulation by sRNAs.............................................................................. 74 2.10 Thermal Stress Response ........................................................................................... 76 2.10.1 RpoH Regulon ...................................................................................................... 77 2.10.2 RpoE Regulon ....................................................................................................... 78 2.11 Protein Misfolding ...................................................................................................... 78 2.11.1 Protein Aggregation .............................................................................................. 79 2.11.2 Chaperones ............................................................................................................ 80 2.11.3 Toxicity of Protein Aggregates ............................................................................. 82 2.12 Tail-Specific Proteases ................................................................................................ 83 2.12.1 Tsp in Pseudomonas aeruginosa .......................................................................... 83 2.12.2 Tsp in Staphylococcus aureus ............................................................................... 84 2.12.3 Tsp in Chlamydia trachomatis .............................................................................. 84 2.12.4 Tsp in Salmonella ................................................................................................. 85 2.12.5 Tsps and Outer Membrane Proteins ...................................................................... 85 Connecting Text Chapter 3 ........................................................................................................ 87 The small regulatory RNA Lpr10 regulates the expression of RpoS in Legionella pneumophila ................................................................................................................................. 88 3.1 ABSTRACT ................................................................................................................. 89 3.2 INTRODUCTION: ..................................................................................................... 90 3.3 RESULTS .................................................................................................................... 95 3.3.1 Lpr10 is expressed in PE phase ............................................................................ 95 3.3.2 rpoS and several other genes are upregulated in the Lpr10 mutant ...................... 98 3.3.3 Deletion of Lpr10 improves survival in water .................................................... 101 3.3.4 Lpr10 pairs to the 5’ region of rpoS mRNA ....................................................... 103 3.3.5 A putative third and fourth rpoS TSS are located upstream of Lpr10 binding site .. ............................................................................................................................. 106 3.4 DISCUSSION ............................................................................................................ 110 3.5 EXPERIMENTAL PROCEDURES ....................................................................... 116 3.5.1 Bacterial strains and media ................................................................................. 116 3.5.2 Survival in water ................................................................................................. 117 3.5.3 Deletion of lpr10 and complementation of the mutant ....................................... 118 3.5.4 RNA extraction ................................................................................................... 122 3.5.5 Northern Blotting ................................................................................................ 122 3.5.6 5’ RACE.............................................................................................................