CRISPR-Cas Systems: New Players in Bacterial Gene Regulation, Innate Immune Evasion, Pathogenesis, and Beyond Öffentlichkeit

Abstract

CRISPR (clustered, regularly interspaced, short palindromic repeats) - Cas (CRISPR-associated) systems are a form of prokaryotic defense against invading foreign nucleic acids, particularly those derived from bacteriophages and plasmids. Such foreign nucleic acids are targeted and cleaved by CRISPR-Cas systems in an RNA-dependent, sequence-specific manner. Additionally, CRISPR-Cas systems are adaptive, providing protection against previously encountered foreign elements. Canonically, it has been thought that these restriction systems act solely in prokaryotic immunity against exogenous genetic elements. However, here, we reveal the very first demonstration of a unique role for CRISPR-Cas systems in the control of endogenous gene expression, a previously unappreciated form of prokaryotic gene regulation. We demonstrate that in the intracellular bacterial pathogen, Francisella novicida, the CRISPR-Cas endonuclease, Cas9, functions in association with two small RNAs to target and alter the stability of a particular endogenous transcript which encodes a bacterial lipoprotein (BLP). Since BLPs are recognized by the host innate immune receptor Toll-like Receptor 2 (TLR2), CRISPR-Cas-mediated repression of BLP expression dampens the activation of TLR2-dependent immune signaling. Furthermore, we demonstrate that control of BLP levels in F. novicida promotes resistance to antimicrobials and enhances the stability of the bacterial envelope, which additionally allows evasion of the host inflammasome complex. Dampening the activation of both TLR2 and the inflammasome by Cas9-mediate regulation ultimately promotes the successful survival of this pathogen in the mammalian host. Since ~45% of bacteria and ~83% of Archaea encode these machineries, this newly described regulatory function of CRISPR-Cas systems is likely to play a broad role in controlling the pathogenesis and physiology of diverse prokaryotes.

Table of Contents

Chapter 1 Introduction...................................................................................... 1 Thesis Overview............................................................................. 14 References...................................................................................... 15

Chapter 2 Repression of bacterial lipoprotein production by Francisella novicida facilitates evasion of innate immune recognition

Abstract.............................................................................. 24

Introduction........................................................................ 25

Results................................................................................ 26

Discussion.......................................................................... 38

Materials & Methods.......................................................... 41

References.......................................................................... 47

Figures................................................................................ 52

Supplemental Information.................................................. 58

Chapter 3 A CRISPR-Cas system mediates bacterial innate immune evasion and virulence

Abstract.............................................................................. 64

Introduction........................................................................ 65

Results................................................................................ 66

Discussion.......................................................................... 71

Materials & Methods.......................................................... 72

References.......................................................................... 77

Figures................................................................................ 80

Chapter 4 A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion

Abstract.............................................................................. 86

Introduction........................................................................ 87

Results................................................................................ 90

Discussion.......................................................................... 97

Materials & Methods........................................................ 102

References........................................................................ 106

Figures.............................................................................. 109

Supplemental Information................................................ 113

Chapter 5 Degeneration of a CRISPR-Cas system and its regulatory target during the evolution of a pathogen

Abstract............................................................................ 124

Introduction...................................................................... 125

Results & Discussion........................................................ 128

References........................................................................ 136

Figures.............................................................................. 139

Chapter 6 Discussion of CRISPR-Cas - mediated gene regulation

Summary & Predicted Mechanism of RNA Targeting ...... 141

Role of CRISPR-Cas gene regulation in pathogenesis....... 145

Role of CRISPR-Cas in the response to envelope stress

......................................................................................... 147

Role of other CRISPR-Cas systems in bacterial physiology

......................................................................................... 148

Conclusions...................................................................... 152

References........................................................................ 153

Figures.............................................................................. 157

Chapter 7 Cas9 as a platform for genome engineering and implications for Cas9-mediated RNA targeting

Cas9 in genome engineering............................................. 159

Engineering to control transcription.................................. 161

Engineering Francisella Cas9 to target viral RNA in eukaryotic cells 162

Thesis Conclusions........................................................... 165

References........................................................................ 166

Figures.............................................................................. 169

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Microbiology & Molecular Genetics
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Microbiology
Stichwort	Microbiology
Committee Chair / Thesis Advisor	Weiss, David S, Emory University
Committee Members	Shafer, William M, Emory University Rengarajan, Jyothi, Emory University Moran Jr., Charles, Emory University Tzeng, Yih-Ling, Emory University

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