Date of Award:

5-2018

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Chemistry and Biochemistry

Advisor/Chair:

Nicholas E. Dickenson

Co-Advisor/Chair:

Robert S. Brown

Third Advisor:

Joan M. Hevel

Abstract

Shigella spp. are Gram-negative, non-motile bacterial pathogens that are the causative agent of bacillary dysentery in humans. During infection, Shigella utilize a complex type three secretion system (T3SS) to inject effector proteins and take over host cell functions. With the rise of multi-antibiotic resistant Shigella strains, the T3SS is a promising alternative therapeutic target. While the needle and syringe-like apparatus of the T3SS has been extensively studied in Shigella, several components and mechanisms of this system remain unclear. The research presented here addresses two major knowledge gaps in the current understanding of the T3SS ATPase, Spa47, and the initial host-pathogen interaction at the tip of the apparatus. In this work, high resolution crystal structures of Spa47 guided the creation of an oligomer model which suggested ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Mutagenesis experiments targeting predicted active site residues and the oligomerization domain revealed that active site residues alone are not responsible for Spa47 oligomerization while protein oligomerization is crucial for ATPase activity. Together with in vivo experiments, we show that ATP hydrolysis and proper Spa47 oligomer formation is critical for T3SS apparatus formation, effector secretion, and overall Shigella virulence. Additionally, we have combined the Langmuir Blodgett technique with fluorescent microscopy to visualize the interaction between key T3SS tip proteins with defined artificial phospholipid membranes. These membranes were generated using Langmuir Blodgett which provided control over lipid phase and composition. Lipid phase and protein localization were monitored using lipophilic dyes and selective fluorescent protein labeling. These experiments suggest a differential interaction between the tip protein IpaB with the membrane components cholesterol and sphingomyelin based on IpaB oligomerization. IpaC, another T3SS tip protein, was found to destabilize membranes when alone, but was stabilized in the presence of IpaB. These experiments suggest that IpaB confers IpaC stability within membranes and that tip protein localization is dependent on lipid phase and composition. Overall, these new insights into the T3SS ATPase and tip proteins provide a more complete understanding of Shigella virulence that will aid in future endeavors to identify alternative therapeutic targets for treatment.

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Available for download on Monday, May 01, 2023

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