Abstract

Bovine mastitis is a common disease among dairy cattle characterized by the inflammation of the udders and loss of milk production. Mastitis-associated Escherichia coli (MAEC) are frequent causes of the disease, but the features that distinguish them from other E. coli strains remain enigmatic. MAEC infections can range from sub-clinical to severe, acute cases that can be fatal. Historically, the severity of mastitis has been attributed to host factors but more recently, a few bacterial genes have been shown to contribute to virulence in mastitis infections. In a large-scale genomic analysis of >100 MAEC isolates the gene for Chitinase A (ChiA) was positively associated with robust growth in the mammary glands during a mouse model of mastitis. This correlation suggests the hypothesis that ChiA contributes directly to MAEC fitness. The regulation of chiA has not been documented in contexts relevant to bovine mastitis. In the lab strain K-12, chiA is not expressed during aerobic growth in rich media. However, previous work with enterotoxigenic E. coli strain H10407 indicated that expression may be induced by hypoxic environments and the presence of bile salts. To measure expression of chiA, I created a chiA-GFP reporter plasmid and measured changes in fluorescence using flow cytometry. My results indicate promoter activity of chiA in MAEC is significantly increased in hypoxic conditions and the presence of bile salts, but not both. Adhesion to host tissues is an important characteristic of successful pathogens. Since ChiA facilitates adhesion between adherent-invasive E. coli and intestinal epithelial cells, I investigated its role in adhesion to bovine mammary epithelial cells in four MAEC strain backgrounds. Isogenic mutants lacking chiA were made in 2 mild (M45 and M93) and 2 severe (M111 and G1) clinical isolates. Loss of chiA resulted in significant reduction of adherence of M45, M93, and G1 to epithelial cells, but not M111. Wild type levels of adhesion were restored upon reintroduction of chiA into mutants through a plasmid vector. Additionally, the genomes of each MAEC isolate were analyzed for the presence of genes that could possibly influence the adhesion and virulence. Strain M111 contained genes for 2 distinct fimbriae that were not present in the other MAEC strains, possibly reducing its reliance on ChiA. The interaction of ChiA with mammary epithelial cells in MAEC could possibly offer an advantage for certain strains to be better suited to colonize and persist in mammary glands. Increased understanding of the regulation of chiA and its role in adherence can lead to novel targets for more effective treatment and prevention of bovine mastitis.

Degree

College and Department

Life Sciences; Microbiology and Molecular Biology

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