HOST-PATHOGEN INTERACTIONS IN PSEUDMONAS AERUGINOSA INFECTION By CHAIRUT CHARLES VAREECHON Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pathology CASE WESTERN RESERVE UNIVERSITY August 2017 Case Western Reserve University School of Graduate Studies We hereby approve the thesis/dissertation of Chairut Charles Vareechon Candidate for the degree of Doctor of Philosophy in Pathology Committee Chair: Man-Sun Sy Committee Members: Clive Hamlin Derek Abbott Arne Rietsch Eric Pearlman Date of Defense: May 15th, 2017 We also certify that written permission has been obtained for any proprietary material contained therein 2 Table of Contents List of Tables………………………………………………………………………….. 6 List of Figures......................................................................................................7 Acknowledgements………………………………………………………………….10 List of Abbreviations….….….….….….….….….….….….….….….……….….…11 Abstract….….….….….….….….….….….….….….….….….…....….….….……...14 Chapter 1: Introduction Pseudomonas aeruginosa and Disease………….………...........….….….18 Pseudomonas aeruginosa Type III Secretion System and Virulence...…19 Targeted cells of the P. aeruginosa T3SS...….….….….….….….….….…19 T3SS the molecular syringe…………………………….……….….…..…...20 The apparatus………………………………………...….………..…..21 The translocon…………………………………………………………22 The secreted effectors……………………………….……………….23 ExoS……………………………….…………………………….24 ExoT……………………….…………………………………….26 ExoU……………….…………………………………………….27 ExoY……….…………………………………………………….28 The chaperones…….………………………………………….…….29 Pseudomonas aeruginosa genome……….………………….………..…..29 Genomic islands………….…………………………………………....……..30 Neutrophils………………………………………………………………….…31 Neutrophil recruitment………………………………………………………..32 Neutrophil Antimicrobial Mechanisms…………………………...…...…….35 Neutrophil Granules………………………………………………….…….…36 Azurophil (Primary) Granules………………………………….……….38 3 Specific (Secondary) Granules…………………………………………38 Gelatinase (Tertiary) Granules…………………………………………39 NADPH oxidase System and ROS………………………………………….40 NADPH oxidase and susceptibility to P. aeruginosa infection……………45 PI3Kγ-mediated activation of ROS production……………………………..46 Neutrophils and Ras…………………………………………………………..47 Bacterial keratitis………………………………………………………………48 Clinical features of Pseudomonas aeruginosa keratitis……………..........49 P. aeruginosa keratitis and neutrophils…………………………….............51 Chapter 2: Pseudomonas aeruginosa ExoS inhibits NADPH oxidase assembly and ROS production in human neutrophils through ADP ribosylation of Ras………………………………………………………………………………………53 Abstract…………………………………………………………………………54 Introduction………………………………………………………………….....55 Material and Methods…………………………………………………………57 Results………………………………………………………………………….71 Discussion……………………………………………………………………...82 Supplemental Figures…………………………………………………….......86 Chapter 3: The ExoU genomic island of strain 19660 modulates type III secretion-dependent virulence…………………………………………….…………………………………90 Abstract…………………………………………………………………………91 Introduction…………………………………………………...………………..92 Material and Methods…………………………………………………………95 4 Results………………………………………………………………………...101 Discussion…………………………………………………………………….115 Supplemental Figure…………………………………………………….......118 Chapter 4: Data Summary, Future Directions and Preliminary Data……………………………………………………………………………………119 Data Summary……………………………………………………………….120 Future Studies on ADP-ribosylation by ExoS and ExoT (Chapter 2)…..122 Future experiments: Possible additional substrates of ExoS………123 Ezrin, radixin, and moesin………………………………………..123 Potential ADPRT substrate: Ral…………………………………128 Rab5 as a target for ADP-ribosylation…………………………..131 Rap1a as a target for ADP-ribosylation…………………………134 Rac as a substrate for ExoS………………………………..……134 Unknown Substrates………………………………………...……137 Targeted Substrates of ExoT………………………………………….138 CrkI/II as a substrate in neutrophils……………………………..138 Gelsolin as a potential substrate for ExoT…………….…….….140 Unknown Substrates…………………………………….…….….141 Characterization of 14-3-3 Proteins in Neutrophils…………………142 Identify neutrophil PRR that recognize P. aeruginosa……………..143 Future Studies based on Chapter 3………………………………………144 Putative NADH:flavin oxidoreductase and H2O-forming oxidases..145 Pathogenicity island screening………..………………………………147 ExoU and Impairment of Neutrophil Oxidative Burst………………..147 5 Preliminary Data..………………………………………………………….150 References……………………………………………………………………….….151 6 List of Tables Table 1.1 Type III secretion effector proteins 28 Table 2.1 Reagents and Strains 66 Table 3.1 Strains and plasmids 97 Table 3.2 Primers Used 99 7 List of Figures Figure 1.1: Type III secretion system of P. aeruginosa….….….…….….….….…20 Figure 1.2: Model of translocon organization and function….…….….….….……23 Figure 1.3: Molecular structures of ExoS, ExoT, ExoU, and ExoY.……………...24 Figure 1.4: Neutrophil extravasation….….….….….….….….….…….….….….….34 Figure 1.5: Microbicidal functions of neutrophil….….….….….….…….….….…...36 Figure 1.6: Granule fusion with the pathogen-containing phagosome....….….…37 Figure 1.7: Neutrophil granule contents and characteristics….….….….….….….40 Figure 1.8: Assembly of NADPH oxidase system….….….….….….…….….……41 Figure 1.9: Structures of cytosolic NADPH oxidase components….…….….…...43 Figure 1.10: p47phox structure and activation….….….….….….….….……..….…..44 Figure 1.11: Clinical characteristics of P. aeruginosa keratitis……………………51 Figure 2.1: NADPH oxidase mediates ROS production by neutrophils and facilitates clearance of P. aeruginosa during bacterial keratitis….………..….…..70 Figure 2.2: ExoS and ExoT ADPRT activities inhibit ROS production in human neutrophils...….….….….….….….….….….….….….….….….….….…..73 Figure 2.3: ExoS and ExoT ADP-ribosyl transferase activities interfere with PI3K signaling in neutrophils….….….….….…………..….….….…..….……..78 Figure 2.4: Tat-Ras(R41K) rescues ROS production in human neutrophils, resulting in increased killing of P. aeruginosa………………………………….…..81 Figure S2.1: NADPH oxidase mediates ROS production by neutrophils facilitate clearance of exoST(A-) strain during bacterial keratitis….….….…...….86 8 Figure S2.2: The type-III secretion system does not affect initial neutrophil phagocytosis or cell death….….…….…...….….….….….….…..87 Figure S2.3: ROS production in neutrophils infected by the ∆pscD mutant strain requires PI3-kinase, but ExoS ADP-ribosylation does not affect the GTP-Ras/Ras ratio in infected neutrophils…….……....….….88 Figure S2.4: Tat-Ras(R41K) and Tat-GFP do not induce ROS production in human neutrophils, nor does Tat-GFP restore ROS production in infected neutrophils………………………………………………………………………...……89 Figure 3.1: P. aeruginosa strain 19660 has greater virulence than PAO1….…103 Figure 3.2: Corneal virulence of strain 19660 depends on both ExoU and ExoT……………………………….……………………………….………………….105 Figure 3.3: P. aeruginosa 19660 is more virulent than PAO1 when expressing only ExoT….….….…..….….……………………...….….….….….…..107 Figure 3.4: The ExoU genomic island, though not the putative nitric oxide reductase, confers corneal virulence…….….….….…....….…...…...110 Figure 3.5: The role of NADH-flavin oxidoreductase in P. aeruginosa keratitis……………………………………………………………….112 Figure 3.6: EXB41 is not a secreted effector……………………………..….…...114 Figure S3.1: EXB35 and EXB39 complementation….….….…..….….….….…..119 Figure 4.1: The role of ExoS and ExoT in inhibiting ROS production…..……...121 Figure 4.2: ERM domain structure….….….….….….….….….….….…...….……124 9 Figure 4.3: Proposed model of ERM regulating granule fusion and protein trafficking………….….….……….….….….….….….….….….….….….…128 Figure 4.4: Proposed model of Ral regulating secondary granule mobilization……………………….…………………………………………131 Figure 4.5: Proposed model of Rab5 regulating granule mobilization………….133 Figure 4.6: Rac activation of NADPH oxidase complex….….….….….………...137 Figure 4.7: Proposed model of Crk signaling and ExoT mediated inhibition…..140 Figure 4.8: T3SS-dependent inhibition of granule fusion by strain PAO1……..150 10 Acknowledgements I want to say thank you to Dr. Trine Jorgensen and Dr. Alan Levine. Your guidance and tough love given during my undergraduate years were instrumental in my decision to pursue a Ph.D. in immunology. The mentorship provided by both of you two will never be forgotten or taken for granted. I want to say thank you to Dr. Eric Pearlman and Dr. Arne Rietsch for being amazing co-mentors during my Ph.D. I am the scientist I am today because of the training provided by both you two. I will always be thankful for the advice, support, and whiskey you two provided. Thank you to all my friends and members of the Pearlman and the Rietsch lab for providing support, laughter, and memories throughout the years. Thank you Tatiane Soares De Lima for the support and love these last two years. I would not be a clinical microbiology fellow at Children’s Hospital Los Angeles without your inspiration and persistence. Lastly, this Ph.D. is dedicated to the Vareechon family. Family is everything. Without my family, I would not have completed this long journey. Mom and Dad, thank you for all the sacrifices that were made to ensure that both of your boys received a higher education and better life opportunities. Giving up a life in Thailand and immigrating to the US with nothing on your backs has paid off. I am indebted to both of you and will always strive to make you two proud. Today, and as always, I am proud to be a Vareechon. Perge! 11 List of abbreviations ADPRT - ADP-ribosyl transferase AIR - auto-inhibitory