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ORCID

https://orcid.org/0000-0002-0360-5668

Access Type

Open Access Thesis

Document Type

thesis

Degree Program

Molecular & Cellular Biology

Degree Type

Master of Science (M.S.)

Year Degree Awarded

2021

Month Degree Awarded

September

Abstract

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.

DOI

https://doi.org/10.7275/24588731.0

First Advisor

Alejandro Heuck

Second Advisor

Margaret Stratton

Third Advisor

Alice Cheung

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