Development of a Novel Method for Genome-wide Identification of Proteins Exported during Infection and Functional Studies of One in vivo Exported Protein in Mycobacterium tuberculosis Public Deposited

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  • March 19, 2019
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  • Perkowski, Ellen
    • Affiliation: School of Medicine, Department of Microbiology and Immunology
Abstract
  • Intracellular pathogens manipulate and outwit the host’s immune defenses and reprogram the hostile intracellular environment into a hospitable replicative niche. The intracellular pathogen Mycobacterium tuberculosis is responsible for the disease tuberculosis, which kills approximately 1.5 million people per year. M. tuberculosis produces many proteins that are exported: transported out of the bacterial cytoplasm to the bacterial cell surface and out into the host environment. Exported proteins are located at the host-pathogen interface, in an ideal location to manipulate the host response and allow for intracellular growth, and exported proteins contribute significantly to virulence. Unfortunately, approaches used to identify proteins exported by the bacteria are limited to bacteria growing in laboratory media (in vitro). Because in vitro conditions cannot mimic the complexity of the host environment, there are likely critical exported virulence factors that have been missed because they are only exported in the context of infection. The main objective of the research described in this dissertation was to develop a method to identify proteins that are exported by bacterial pathogens during infection of a host (in vivo). We developed a novel method that we refer to as EXIT, EXported In vivo Technology, and applied it to identify M. tuberculosis proteins exported during murine infection. EXIT identified 593 in vivo exported proteins, 100 that were experimentally shown to be exported for the first time, and 32 proteins with no in silico predicted export signals. EXIT identified 38 proteins exported significantly more in vivo than in vitro, suggesting that temporal or spatial control of their export is important to infection. 21 of these 38 proteins have unknown function, making them particularly interesting for future functional characterization. We focused on one of the EXIT identified in vivo exported proteins, OmasA, a protein of unknown function for further study. We demonstrated that OmasA was required for M. tuberculosis virulence in a mouse model of tuberculosis. We further demonstrated a function for OmasA in stabilizing multi-protein Mce transporters required for lipid import. Future studies will focus on assigning function to new EXIT identified exported proteins, in particular proteins whose export is significantly induced during infection.
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  • In Copyright
Advisor
  • Richardson, Anthony
  • Cotter, Peggy
  • Braunstein, Miriam
  • Goldman, William
  • Kawula, Thomas
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2015
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  • Chapel Hill, NC
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