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Open Access Thesis
Molecular & Cellular Biology
Master of Science (M.S.)
Year Degree Awarded
Month Degree Awarded
The type III secretion system (T3SS) is employed by various Gram-negative pathogens to promote infection of host cells. Central to the function of the T3SS is the injectisome, a syringe-like membrane protein complex which allows direct translocation of cytotoxic effector proteins into the cytosol of a host cell. Contact between the needle tip and the plasma membrane of a host cell activates the Type III secretion system, promoting attachment of the bacteria to the host and secretion of T3SS substrates through the injectisome. In Pseudomonas aeruginosa, the proteins PopB and PopD are translocated through the injectisome to insert into the host membrane, forming a heterooligomeric, membrane-spanning translocon pore. Formation of the translocon provides a channel through which the attached bacteria can then translocate effectors into the host. Although the translocon pore is essential for T3SS-mediated infection, little is known about the structure and topology of PopB and PopD when associated with membranes in vivo.
In this work, I seek to develop a cysteine specific labeling assay to probe the transmembrane topology of PopD upon insertion into the membrane of a host cell. Several PopD variants were produced with single cysteines inserted within putative extracellular or intracellular loop domains. The transmembrane orientation of these cysteines was studied based on their accessibility to PEG maleimide, a cysteine specific, membrane impermeable labeling reagent.
By employing this method, I here characterize a loop domain of PopD that is accessible to host cytosol upon translocon formation. With optimization, the PEGylation assay may be used to further study the structure of PopD, as well as the other translocon protein PopB.
Mahan, Kyle A., "Development of a Site-Specific Labeling Assay to Study the Pseudomonas aeruginosa Type III Secretion Translocon in Native Membranes" (2021). Masters Theses. 1111.