Date on Master's Thesis/Doctoral Dissertation

5-2020

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biology

Degree Program

Biology, PhD

Committee Chair

Worley, Micah

Committee Member

Perlin, Michael

Committee Member

Yoder-Himes, Deborah

Committee Member

Ewald, Paul

Committee Member

Graham, James E.

Author's Keywords

salmonella; dendritic cells; type 3 secretion system; microbiology

Abstract

Salmonella appears in the bloodstream of mice in as little as 15 minutes after oral inoculation and establishes persistent colonies in the spleen and liver. While its pathway to blood is undetermined, this phenomenon is dependent on the activity of Salmonella pathogenicity island 2 (SPI-2) coded type III secretion system (T3SS) and CD18+ phagocytes. We hypothesize that dendritic cells associated with the basal face of the gut epithelium, that are naturally migratory and known to sample for luminal antigens directly transport Salmonella to the bloodstream. This process comprises of at least two phases, dissociation and reverse transmigration. We define dissociation as the process were intraepithelial dendritic cells separate from the epithelium after picking up Salmonella from the gut. This is followed by reverse transmigration, a normal host process in which dendritic cells can reenter the bloodstream by traversing the vascular endothelium in the basal to apical direction. In vitro models of the two processes were developed to help identify T3SS effectors that could affect rapid extraintestinal dissemination of Salmonella in mice. Chronologically the in vitro reverse transmigration assay was developed first and allowed dendritic cells to migrate from the basal to apical face of endothelial cell monolayers cultured on filters with small pores. The T3SS effector SpvC was found to play an important role in the SPI-2 mediated in vitro reverse transmigration of dendritic cells and along with SrfH was demonstrated to greatly promote early extraintestinal dissemination of Salmonella in mice. However, SrfH failed to stimulate reverse transmigration in vitro. We were able to demonstrate that SrfH triggered dissociation of dendritic cells bound to the basal side of epithelial cell monolayers cultured on tissue culture inserts with small pores. The presence of a glycine residue at position 103 of SrfH was vital for dissociation and could potentially trigger this process through its association with the host adaptor protein TRIP6. These results combined with the observation that the srfH Gly103 and the spv operon are conserved amongst strains of non-typhoidal Salmonella capable of causing bacteremia in people suggests that this pathway to the bloodstream could be important for understanding human infections.

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