SENSING of HOST CELL CONTACT by the PSEUDOMONAS AERUGINOSA TYPE III SECRETION SYSTEM by ERIN ARMENTROUT Submitted in Partial
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SENSING OF HOST CELL CONTACT BY THE PSEUDOMONAS AERUGINOSA TYPE III SECRETION SYSTEM By ERIN ARMENTROUT Submitted in partial fulfillment of requirements for the degree of Doctor of Philosophy Department of Molecular Biology and Microbiology CASE WESTERN RESERVE UNIVERSITY August 2017 Case Western Reserve University School of Graduate studies We hereby approve the dissertation of Erin Armentrout Candidate for the degree of Doctor of Philosophy Committee Chair Piet de Boer Committee Member Arne Rietsch Committee Member Liem Nguyen Committee Member Pieter de Haseth Date of Defense June 30th, 2017 *We also certify that written approval has been obtained for any propriety material contain therein 1 Table of Contents List of Tables 4 List of Figures 5 Abstract 7 Chapter 1: Pseudomonas aeruginosa, a human pathogen, and its 9 virulence factor, the Type III Secretion System Effectors 12 T3SS Structure 14 Powering the T3SS and Control of Secretion Rate 16 Regulation of T3SS Gene Transcription 17 Regulation of Secretion 18 Conclusion 22 Chapter 2: Sensing of Host Cell Contact by the Type III Secretion 24 System Introduction 25 Translocator Interactions 26 Models of initiation of effector secretion 28 Materials and Methods 32 Bacterial strains, cells, growth conditions 32 Plasmid and strain construction 32 Translocation assay 33 Crosslinking 34 Hemolysis 34 Red blood cell membrane isolation 35 Results and Discussion 36 Confirming interactions 36 Assigning functions 39 Conclusion 44 Chapter 3: Cellular Contribution to Host Cell Contact Signaling 53 Introduction 54 Targeting cellular components 54 Lipid rafts and endocytosis 55 Membrane curvature 58 Materials and Methods 59 Bacterial strains, cells, growth conditions 59 Plasmid and strain construction 60 siRNA knockdown 60 Translocation assay 61 Membrane damage repair assay 62 Microwell assay 63 2 Results and Discussion 64 Determining cellular targets of ExoS 64 Blocking endocytic pathways 68 Effector secretion trigger mechanism 72 Conclusion 74 Chapter 4: Conclusion and Future Works 90 Bacterial protein interactions 91 Cellular targets 93 Cellular processes: endocytosis and beyond 96 Membrane curvature 100 References 105 3 List of Tables 3-1: Cellular targets of ExoS and ExoT 76 4 List of Figures 1-1: The Type III Secretion System 23 2-1: Pseudomonas/Yersinia mismatch 46 2-2: Crosslinking PcrV and PopD & Translocation Defect 47 2-3: PopD dimer formation and associated translocation defect 48 2-4: PopB dimer formation and associated translocation defect 49 2-5: PopD-PcrV interaction effect on pore formation and insertion 50 2-6: PcrV and PopD Translocation Defect is not due to slow translocation 51 2-7: Proposed model 52 3-1: Feedback inhibition during infection with phagocytosed vs. non- 77 phagocytosed bacteria 3-2: Membrane damage repair 78 3-3: ExoS feedback inhibition in the presence of ExoT 79 3-4: ExoS feedback inhibition in the absence of ExoT 80 3-5: The effect of ExoT-GAP on ExoS feedback inhibition 81 3-6: The MLD affects feedback inhibition and the GAP domain is responsible 82 for feedback inhibition in the ExoS(2RD-N) mutant 3-7: The role of calcium during infection 83 3-8: Translocation is not affected by the absence of ASM 84 3-9: Absence of Caveolin-1 has varying effects on translocation 85 3-10: Absence of Caveolin-1 has varying effects on translocation 86 3-11: Knockdown of Flotillin-1 has no effect on translocation 87 3-12: Knockdown of Flotillin-2 has slight effect on translocation 88 5 3-12: Microwell assay 89 4-1: Host cell contribution to infection 103 4-2: Interplay between cellular processes and bacterial infection 105 6 Sensing of Host Cell Contact by the Pseudomonas aeruginosa Type III Secretion System Abstract by ERIN ARMENTROUT Pseudomonas aeruginosa uses a type III secretion system (T3SS), a syringe- like apparatus, to inject bacterial effector proteins into the host cell cytoplasm. Upon cell contact the T3SS assembles the translocon, which is essential for effector injection. The translocon creates a conduit for effectors to pass from the bacterium to the host cell and consists of three proteins: PopB and PopD, pore-forming translocators, and PcrV, the needle tip. Mismatch studies with Yersinia translocators identified interactions between PopD and PcrV, as well as PopB and PopD. The PopD-PcrV interaction was confirmed by covalently crosslinking the interacting proteins during cell contact. We demonstrated that PopB and PopD also form homo- dimers. Tethering the PopD dimer or disrupting the PopD-PcrV interaction interferes with triggering of effector secretion. These data support the model that cell-contact is sensed by the translocation pore. The signal is then transmitted to the needle tip and from there propagated down the needle to initiate of effector secretion. The host cell component that mediates sensing of the host cell contact has not yet been identified. Preliminary data from the lab suggested that the phagosome causes constant triggering of translocation, which may be related to membrane curvature. We aimed to identify this aspect using a three-pronged approach. First, 7 we attempted to recapitulate in vitro the membrane curvature that the bacterium experiences during phagocytosis. A curved membrane composed of only phosphatidylserine and phosphatidylcholine did not cause triggering of effector secretion, suggesting that there must be additional factors involved in sensing of host cell contact. Second, we demonstrated that blocking membrane damage repair- related endocytosis decreased the level of effectors translocated. However, this decrease was minor and implies that the membrane damage repair response is not solely responsible for triggering of effector secretion. Third, because ExoS can regulate its own translocation into host cells through its catalytic function, we aimed to identify cellular components that contribute to translocation by detecting targets of ExoS. We found that localization of ExoS, as well as both the GTPase activating protein- and the ADP-ribosyltransferase domains contribute to proper regulation of translocation. 8 Chapter 1 Pseudomonas aeruginosa, a human pathogen, and its virulence factor, the Type III Secretion System 9 Pseudomonas aeruginosa is a Gram-negative, facultative anaerobic bacterium that is found in soil, in water, on surfaces, and as part of human skin biomes. It is an opportunistic human pathogen that is common in hospital-acquired infections. P. aeruginosa infects immunocompromised individuals, most commonly patients with cystic fibrosis, burn wounds, or ventilator-associated pneumonia. Cases of infection in healthy individuals are associated with damage at the site of infection; for example, contact lens-associated keratitis caused by P. aeruginosa occurs after the cornea becomes injured from contact lens wear (Wilcox, 2007). P. aeruginosa is a substantial problem in hospitals for two reasons 1) the bacterium likes to grow on surfaces, such as medical devices, and creates biofilms and 2) the bacterium has inherent antibiotic resistance. The high degree of antibiotic resistance in P. aeruginosa is due to two main factors. First, penetration of antibiotics is decreased by low permeability of its outer membrane, which is due to a lack of porins and inefficient transport by porins. Second, P. aeruginosa has a high adaptability to challenges by antibiotics, which is a result of efflux pumps, changes in gene expression, increases in production of beta-lactamase and other enzymes, and decreases in DNA damage repair that leads to an increase in mutation rate (Breidenstein et al., 2011). Both its intrinsic antibiotic resistance and propensity to create biofilms makes P. aeruginosa difficult to treat and remove to prevent spread in hospitals. These qualities have driven researchers to search for alternatives to antibiotics for preventing infection by P. aeruginosa. P. aeruginosa has several virulence factors. One of particular interest, the Type III Secretion System (T3SS) and its accompanying effectors, is correlated with 10 poor clinical outcomes in ventilator-associated pneumonia and blood stream infections (Hauser et al., 2002; El-Solh et al., 2012). In one study, T3SS gene expression was lower for P. aeruginosa strains isolated from patients with chronic respiratory infections compared to strains isolated from patients with acute infections, suggesting that the T3SS is associated with acute infections (Roy-Burman et al., 2001). More recently, Jorth et al. (2015) found that T3SS gene expression varies between regions of the lung in individual patients in response to different selective pressures. Additionally, research suggests that the T3SS is important for establishing biofilms at the epithelial barrier (Tran et al., 2014). Together these data suggest that studying the T3SS can be useful for treatment and prevention for patients with acute or chronic infections. The T3SS is related to the flagellar system and they likely share a common ancestor. This can be seen in the high degree of structural homology between the basal body of each structure (Saier, 2004). Both systems are designed to export bacterial proteins; however, the purpose of each system differs. Swimming motility in bacteria is controlled by the flagellum, which bacteria generally use in search of nutrients. On the other hand, T3SS is a molecular syringe that injects bacterial proteins called effectors into eukaryotic cells (Figure 1-1). Bacterial effectors manipulate eukaryotic cellular functions to increase survival. Genes encoding the