Date

January 2010

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Molecular Microbiology and Immunology

Institution

Oregon Health & Science University

Abstract

Salmonella afflicts millions of people across the globe, is the leading cause of death among HIV infected individuals in sub-Saharan Africa, and is estimated to cost billions of dollars in lost productivity and wages every year. The relevance of this pathogen cannot be disputed and in light of emerging antibiotic resistant strains, it is of upmost importance that we understand the molecular mechanisms by which it mediates virulence. Salmonella is an intracellular pathogen and its virulence is in large part mediated by the secretion of effector proteins into host cells. Type III secretion systems (TSSS) are the best understood secretion mechanism, and they function as molecular syringes that directly inject effectors from bacteria to the eukaryotic cytosol. Salmonella enteriditis serovar Typhimurium has two TTSS which are encoded on Salmonella pathogenicity island 1 (SPI-1) and Salmonella pathogenicity island 2 (SPI-2). SPI-1 is required for the invasion of non-phagocytic cells and the intestinal phase of infection, while SPI-2 mediates intracellular survival and is required for systemic disease. Following oral infection, a small population of CD18+ cells rapidly carry Salmonella from the lumen of the intestine to the blood – this is the rapid septiciemia phenotype. My work demonstrates that the SPI-2 effector SrfH is responsible for rapid septicemia. Then I attempted to gain an understanding of the host mechanisms involved. I present evidence showing that rapid septicemia is mediated by SrfH interaction with host TRIP6, and that this phenotype may occur via a c-Src signaling cascade. To date 37 type III effectors have been reported in the literature, but when I initiated my thesis only a few had been described. However, such numbers underestimate the effector repertoire. Thus the second half of this thesis focuses upon the identification of novel secreted proteins. Two approaches were used to address this objective. First, I describe a transposon based strategy that was designed to generate effector fusions with an enzyme linked reporter. Although it was a sensitive way to evaluate secretion of individual effectors, only three novel effector proteins were identified. An alternative, mass-spectrometry based approach is presented next. We used LC-MS/MS to identify over 200 proteins secreted into SPI-2 inducing, defined media. By employing various genetic mutants, we selectively identified the majority of type III substrates and have developed the most efficient screen for Salmonella effector proteins to date. Moreover, this study identified several effector proteins that were secreted into host cells independent of TTS, suggesting that Salmonella employs alternative secretion systems during infection. It will take many years to define the function of these proteins.

Identifier

doi:10.6083/M498850G

School

School of Medicine

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