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Mechanisms and Consequences of Staphylococcus Aureus Leukocidin Ab-Mediated Activation of the Host Nlrp3 Inflammasome

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Mechanisms and Consequences of Staphylococcus Aureus Leukocidin Ab-Mediated Activation of the Host Nlrp3 Inflammasome MECHANISMS AND CONSEQUENCES OF STAPHYLOCOCCUS AUREUS LEUKOCIDIN AB-MEDIATED ACTIVATION OF THE HOST NLRP3 INFLAMMASOME Jason H. Melehani A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctorate of Philosophy in the Department of Pharmacology in the School of Medicine Chapel Hill 2016 Approved by: Robert A. Nicholas Gary L. Johnson Claire M. Doerschuk Monte S. Willis Joseph A. Duncan © 2016 Jason H. Melehani ALL RIGHTS RESERVED ii ABSTRACT Jason H. Melehani: Mechanisms and consequences of Staphylococcus aureus Leukocidin AB- mediated activation of the host NLRP3 inflammasome (Under the direction of Joseph A. Duncan) The NLRP3 inflammasome is a critical innate immune sensor implicated in the pathogenesis of dozens of infectious and non-infectious diseases. Activation of the NLRP3 inflammasome causes IL-1β and IL-18 secretion and necrotic cell death. Staphylococcus aureus is a common cause of infections in humans. S. aureus produces a family of pore-forming toxins that are cytotoxic to human immune cells. One recently discovered pore-forming toxin, Leukocidin AB, is the focus of studies herein. Leukocidin AB is a human-specific, pore-forming toxin that binds CD11b to initiate pore formation. In order to characterize the mechanism of Leukocidin AB cytotoxicity and determine its significance, we evaluated the effects of Leukocidin AB on primary human monocytes and THP1 monocytic cells. Leukocidin AB was one of the most potent toxins in killing primary human monocytes. In THP1 cells, knockdown of NLRP3 or ASC by shRNA diminished Leukocidin AB- induced cytotoxicity and prevented secretion of IL-1β and IL-18. We also characterized the NLRP3 inflammasome in bacterial survival during phagocytosis. When S. aureus was phagocytosed by THP1 cells, LukAB triggered IL-1β secretion and cell death. shRNA-mediated depletion of NLRP3 or ASC suppressed IL-1β secretion but had no effect on Leukocidin AB- induced cell death. These data suggest that a separate mechanism is responsible for triggering iii cell death when Leukocidin AB binds CD11b on the phagosome membrane instead of the plasma membrane. We also initiated studies to characterize the role of kinases and phosphorylation in the response to Leukocidin AB. Using multiplex inhibitor bead chromatography and quantitative mass spectrometry, we identified eight kinases that rapidly decrease in activity during Leukocidin AB exposure. We demonstrated a role for Death-Associated Protein Kinase in the response to Leukocidin AB by showing that its inhibition suppressed Leukocidin AB-induced cytokine secretion and cytotoxicity. And finally, we used a novel transfection method for overexpressing mutant proteins in THP1 cells to show that the NLRP3 S198D/S201D mutant could spontaneously activate NLRP3 inflammasome signaling. In total, these studies make significant contributions to the understanding of S. aureus pathogenesis and the regulation of innate immune NLRP3 inflammasome signaling in response to a human specific, S. aureus pore-forming toxin. iv To my dear family, friends, and mentors for their unwavering support v ACKNOWLEDGEMENTS I would like to acknowledge and thank the directors of the UNC Medical Scientist Training Program past and present, the faculty of the Pharmacology Department with whom I interacted, the members of my thesis committee, the numerous faculty and staff at the University of North Carolina with whom I worked with, my collaborators at the University of North Carolina Chapel Hill and New York University, my colleagues in the Duncan Lab, my colleagues in the Medical Scientist Training Program and Pharmacology Graduate Program, the Infectious Disease Society of America for funding support, and my family and friends. vi TABLE OF CONTENTS LIST OF TABLES .............................................................................................................................. xiii LIST OF FIGURES ............................................................................................................................ xiv LIST OF ABREVIATIONS.................................................................................................................. xvi CHAPTER 1: Discovery, activation and regulation of the NLRP3 inflammasome ........................... 1 1.1 A general overview of NLRP3 inflammasome form and function ...................................... 1 1.2 Clinical relevance of NLRP3 inflammasome signaling ........................................................ 3 1.3 Interleukin-1β-driven monogenic hyperinflammatory fever disorders are caused by mutations in the NLRP3 inflammasome pathway ................................. 4 1.4 The NLRP3 inflammasome is a master innate immune sensor .......................................... 7 1.5 Cautions in interpreting NLRP3 inflammasome research................................................... 8 1.5.1 Studies frequently overlook important outcomes of NLRP3 inflammasome activity ..................................................................................................................... 8 1.5.2 Dozens of activators and regulators complicate a comprehensive model for NLRP3 inflammasome activation ......................................................... 11 1.5.3 Commonly used Caspase 1 knockout mice are also Caspase 11 deficient .............. 12 1.6 Priming the NLRP3 inflammasome for activation ............................................................. 13 1.6.1 Pattern-recognition receptor signaling upregulates transcription of NLRP3 inflammasome components ...................................................................... 13 1.6.2 Negative feedback loops respond to prolonged priming to reign in NLRP3 inflammasome signaling ............................................................................ 18 1.6.3 Kinase-mediated post-translational priming regulates NLRP3 inflammasome activity .......................................................................................... 22 1.6.4 Caspase 8 contributes to transcriptional and post-translational NLRP3 inflammasome priming ......................................................................................... 25 vii 1.6.5 Multiple ubiquitination signals regulate NLRP3 inflammasome priming and protein degradation .............................................................................................. 28 1.6.6 Post-transcriptional regulation of NLRP3 inflammasome mRNA stability .............. 32 1.6.7 NLRP3 inflammasome signaling without priming .................................................... 33 1.7 Critical features of NLRP3 inflammasome activation ....................................................... 34 1.7.1 Potassium efflux is a universal requirement for NLRP3 inflammasome formation .............................................................................................................. 35 1.7.2 Mitochondrial disruption triggers ROS production ................................................. 37 1.7.3 Lysosome dysfunction liberates the cysteine protease Cathepsin B ...................... 43 1.7.4 Endoplasmic reticulum dysfunction disrupts protein folding ................................. 47 1.7.5 Calcium acts as a second messenger ....................................................................... 49 1.7.6 Ubiquitination acts as a platform for regulator binding .......................................... 58 1.7.7 Phosphorylation controls inflammasome oligomerization ..................................... 62 1.7.7.1 Syk kinase couples C-type lectin domain family members to NLRP3 inflammasome signaling ...................................................................... 62 1.7.7.2 RIPK1/RIPK3- and NLRP3-dependent necrosis overlap .................................. 65 1.7.7.3 Kinases have a wide ranging role in regulating the NLRP3 inflammasome ... 68 1.8 Canonical inflammasome activators: nigericin, ATP and monosodium urate ................. 74 1.8.1 Nigericin is a potassium ionophore that activates the NLRP3 inflammasome ....... 75 1.8.2 Extracellular ATP triggers NLRP3 activation by binding P2RX7 ............................... 85 1.8.3 Monosodium urate disrupts lysosomes to trigger the NLRP3 inflammasome ....... 97 1.9 Consequences of NLRP3 inflammasome signaling in pulmonary diseases .................... 105 1.9.1 The NLRP3 inflammasome is a sentinel in pulmonary infections.......................... 108 1.9.1.1 NLRP3 inflammasome activation helps clear respiratory viruses ................ 108 1.9.1.2 The NLRP3 inflammasome is differentially required for clearing extracellular and intracellular bacterial infections ....................................... 110 1.9.1.3 The NLRP3 inflammasome responds to acute and chronic fungal infections............................................................................................ 113 viii 1.9.2 Chronic obstructive pulmonary disease is influenced by long term NLRP3 inflammasome stimulation ................................................................................. 115 1.9.3 NLRP3 may contribute to airway hyperreactivity in asthma, allergy and obesity .......................................................................................................... 117 1.9.4 The NLRP3 inflammasome is critically activated in acute lung injury ................... 122 1.9.5 The NLRP3 inflammasome drives fibrotic changes in pulmonary fibrosis ............ 126 1.9.6 NLRP3 inflammasome signaling
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