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Full text release has been delayed at the author's request until May 19, 2025

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Needle Tip-Pore Interactions in the Pseudomonas aeruginosa Type III Secretion System Translocon

Kundracik, Emma Caitlin
http://orcid.org/0000-0001-6648-1474
http://rave.ohiolink.edu/etdc/view?acc_num=case1676044568541758

Abstract Details

2023, Doctor of Philosophy, Case Western Reserve University, Molecular Biology and Microbiology.
The type III secretion system (T3SS, “injectisome”) is an important virulence factor for many Gram-negative pathogens including Pseudomonas aeruginosa. This complex nanomachine injects toxic effector proteins directly into the host cell. Despite decades of research, the portion of the system that interfaces with the host cell – the translocon – remains very poorly understood. Biochemical and structural studies of the translocon are limited by the fact that it is assembled via a complex process which cannot be replicated in vitro. In this work, we used genetic methods to study how the three translocon proteins – PopB, PopD, and PcrV – interact with each other. We took advantage of the incompatibility of homologous translocator proteins in P. aeruginosa and A. hydrophila to map protein-protein interactions that are critical for translocon function. We discovered a key interaction involving the tip domain of the needle tip protein PcrV and the pore protein PopB which is crucial to the formation of the pore in the host cell membrane (Chapter 3). Our data establish for the first time that PcrV is specifically required for the formation of the translocon pore, separately from insertion of the pore proteins in the membrane. I next turned my attention to methods for stabilizing PcrV oligomers in solution (Chapter 4). Physiologically, this protein functions as a pentamer at the tip of the T3SS needle. When purified in solution, however, it remains a monomer. I sought to stabilize the pentameric form of PcrV in order to create a reagent that could be used for biochemical studies of the translocon. I conclude the thesis by outlining some future projects to explore a potential prepore state for the translocation pore, conformational changes in PcrV, and two specific domains in PopB and PopD which could be functionally important (Chapter 5). The overarching goal of this research is to unlock new therapeutic targets for anti-virulence drugs against P. aeruginosa. The needle tip and translocon are required for delivery of T3SS effector proteins, and blockage of either of these components abolishes T3SS-mediated virulence. Anti-virulence targets are attractive as an infection control strategy because they impede infection rather than killing the bacteria directly, putting less selective pressure on the bacteria to evolve resistance mechanisms. This work has furthered our understanding of an elusive component of the T3SS which is an attractive drug target.
Arne Rietsch (Advisor)
Derek Taylor (Committee Member)
W Henry Boom (Committee Member)
Piet de Boer (Committee Chair)
185 p.
Microbiology; Molecular Biology
type III secretion system, T3SS, translocon, protein-protein interactions, pore formation

Recommended Citations

Citations

  • Kundracik, E. C. (2023). Needle Tip-Pore Interactions in the Pseudomonas aeruginosa Type III Secretion System Translocon [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1676044568541758

    APA Style (7th edition)

  • Kundracik, Emma. Needle Tip-Pore Interactions in the Pseudomonas aeruginosa Type III Secretion System Translocon . 2023. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1676044568541758.

    MLA Style (8th edition)

  • Kundracik, Emma. "Needle Tip-Pore Interactions in the Pseudomonas aeruginosa Type III Secretion System Translocon ." Doctoral dissertation, Case Western Reserve University, 2023. http://rave.ohiolink.edu/etdc/view?acc_num=case1676044568541758

    Chicago Manual of Style (17th edition)

case1676044568541758
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This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.