Student Theses and Dissertations

Ryan Q. Notti

Date of Award

2016

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Stebbins Laboratory

Abstract

Once optimistically believed to be a relic of the pre-antibiotic era, bacterial pathogens remain a substantial threat to human health, and the growing epidemic of antibiotic resistance has raised concerns for the long term prospects of antimicrobial therapy. By understanding the mechanisms used by bacteria to manipulate their host and cause disease, it is hypothesized that we might more rationally approach anti-infective therapeutic design. Type III secretion systems (T3SS) are employed by some gram-negative human pathogens to manipulate the host environment. One T3SS subtype, known as the “injectisome,” delivers virulence factors directly into host cells. The other T3SS subtype secretes the the polymeric flagellar filament used for motility. While both systems share related elements of a cytoplasmic “sorting platform” that facilitates the hierarchical secretion of protein substrates, the structural mechanism of its assembly remains unclear. The work described in this thesis makes strides towards the mechanistic understanding of T3SS sorting platform assembly by applying structural, biochemical, and genetic techniques to the characterization of the SctQ/FliM/FliN protein family and their interactions with other sorting platform components. These proteins uniquely possess Surface Presentation Of Antigens (SPOA) folds, and I will present the molecular structures of distinct homotypic and heterotypic SPOA-SPOA interactions in the Salmonella typhimurium SPI-1 sorting platform protein SpaO (Chapter 2). In Chapter 3, I structurally characterize the interaction of the heterotypic SPOA complex with a regulator of the SPI-1 ATPase and demonstrate the necessity of the interaction for T3SS secretory function. Then, I will present the homologous structures from the S. typhimurium flagellar apparatus and compare and contrast them with their SPI-1 homologues, providing an explanation for the observed subtype specificity in sorting platform assembly (Chapter 4). In Chapter 5, biochemical evidence for an interaction of the SpaO amino-terminal domain(s) with the homotypic SPOA complex is presented. These results provide a model for the subtype-specific assembly of T3SS sorting platforms and will support further mechanistic analysis and anti-virulence drug design (Chapter 6).

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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