Date

December 2007

Document Type

Thesis

Degree Name

M.S.

Department

Dept. of Molecular Microbiology and Immunology

Institution

Oregon Health & Science University

Abstract

Salmonella enterica serotype Typhimurium is a Gram-negative facultative intracellular bacterium that causes a typhoid-like disease in mice. Salmonella invades the gut epithelium and establishes a systemic infection via invasion of phagocytes and replication within the Salmonella-containing vacuole (SCV). Salmonella's gene expression is a response to its environment and allows the bacterium to avoid macrophage killing and to establish persistence. Our understanding of the mechanisms and virulence factors necessary for Salmonella to invade and initiate infection are far better understood than those required for thwarting the adaptive immune response, preventing clearance and establishing a long-term infection. It was the aim of this study to identify novel genes required in the evasion of the adaptive immune response. To identify Salmonella genes responsible for evading the adaptive immune response, we performed a microarray-based negative selection screen. Using a mutagenesis library, we infected RAG- mice that are missing 8 cells and T cells, as well as RAG+ mice, and compared the presence of mutants from spleens recovered at days five, six and seven. Following transposon detection, labeling, hybridization, quantitation, normalization and analysis, we identified a group of candidates. Using an allelic exchange protocol we individually knocked out these genes and used the strains in a competitive index experiment testing for persistence. Using qRT-PCR, we quantified bacterial numbers throughout the course of infection for each mutant strain as compared to control. We identified two Salmonella factors that are likely to be involved in evading the adaptive immune response, granting the bacterium the ability to prevent its own clearance. Listed as coding for a putative outer membrane or exported protein (STM4242) and putative cytoplasmic protein (STM 111 0), these genes are good candidates for further analysis of function and mechanism.

Identifier

doi:10.6083/M46D5R0T

School

School of Medicine

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