Date of Degree
MS (Master of Science)
Salmonella enterica serovar Typhimurium is a common cause of bacterial food poisoning, and S. Typhimurium expresses type 1 fimbriae that enable the bacteria to bind to eukaryotic cells. Fimbrial proteins are encoded by the fim gene cluster (fimAICDHFZYW). The structural components of the fimbriae are FimA (major subunit), FimI, FimH (adhesin), and FimF (adaptor). In order to determine the genes required for fimbrial assembly in S. Typhimurium SL1344, mutations in fimA, fimI, fimH, and fimF were constructed and examined for their ability to produce fimbriae. While SL1344δfimI was able to assemble fimbriae, SL1344δfimA, δfimH, and δfimF were afimbriate, indicating that fimA, fimH, and fimF are each required for fimbrial formation in S. Typhimurium. These results suggest differences in the genetic requirements when comparing S. Typhimurium type 1 fimbrial and E. coli type 1 and Pap fimbrial systems. S. Typhimurium fim gene regulation was also examined. FimZ and FimY are positive regulators of fimbrial gene expression, and FimW is a negative regulator. FimZ is closely related to the family of response regulators of two-component systems. The response regulator activity of FimZ was examined by substituting the conserved aspartate-56 residue, the putative site of phosphorylation, with alanine, to generate an inactive phosphorylation site, or glutamate, to mimic a phosphorylated protein. Resulting strains were examined for fimbrial production and gene expression. It was observed that when the aspartate-56 is substituted with alanine fimbriae are not produced and when glutamate replaces aspartate-56 fimbriae are produced constitutively. Bacterial two-hybrid assays were also carried out to determine the effect of FimZ aspartate-56 substitutions on the previously described FimZ/FimW protein interaction. It was found that FimZD56A is unable to interact with FimW and FimZD56E is able to interact with FimW. Additionally, complementation studies were used to examine the roles of FimZ and FimY in relation to each other and results suggest that FimY acts upstream of FimZ.
Copyright 2012 Sarah Ann Zeiner