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Celia, Laura Katherine

Animal Science

2005

M.S.

Bovine mastitis is an inflammatory reaction of the mammary gland usually in response to a microbial infection. This disease has a significant economic impact on the U.S. dairy industry, as it is estimated to cost approximately $2 millon per year due to decreased milk production, discarded milk, and veterinary bills. With the advent of genetic engineering, it was proposed that the generation of a transgenic cow, capable of producing antibacterial proteins in its milk, could reduce the occurrence of bovine mastitis. In 2005, it was demonstrated that the production of lysostaphin in the milk of transgenic cows was capable of enhancing the resistance of these animals to intramammary infusion ofS. aureus. However, while these cows display enhanced resistance to S. aureus mastitis, they remain susceptible to other mastitis pathogens. In an effort to further this approach of mastitis prevention, the purpose of this study was to identify other antibacterial proteins, specifically effective against S. uberis, for potential use in the generation of transgenic cows. In recent studies, bacteriophage lysin enzymes have proven to be particularly potent killers of bacteria. This thesis investigates the ability of a S. uberis derived bacteriophage lysin to target and kill S. uberis. Mitomycin C was used to induce production of a temperate bacteriophage from a clinical isolate of S. uberis. Phage DNA was isolated and cloned for sequencing. An open reading frame homologous to other streptococcal bacteriophage lysins was identified. The fragment was amplified by PCR, cloned into the pBAD 18 prokaryotic expression vector, and expressed in E. coli. Purification of the recombinant protein, termed 7001ysin, by ion exchange column chromatography allowed for the characterization of enzyme activity. Approximately 12kbp of the phage genomic DNA was sequenced, and open reading frames annotated based on homology to phage proteins described in public databases. Sequence analysis of the lysin gene identified two different domains; an amino-terminal enzymatic domain (amidase), as well as a carboxy-terminal cell wall binding domain. Addition of the purified lysin to bacterial suspensions of S. uberis, Streptococcus agalactiae, and Streptococcus dysgalactiae caused a rapid decrease in the turbidity of these suspensions, indicating bacterial cell lysis. A direct measurement of bacterial viability was performed using a plate count assay. The effectiveness of the 7001ysin against these bacterial species in vitro was dependent on the pH, and presence of Ca 2+ in the media, as well as the growth stage of the bacteria. Characterization of the cell wall binding domain, by the production of a GFP fusion protein, determined that this domain was effective in targeting of the lysin to the S. uberis cell wall. Targeting of the lysin by this domain.was found to be dependent of the presence of Ca 2+ in the media. The ability of the 7001ysin enzyme to kill S. uberis in vitro demonstrates its potential to serve as an alternative antibacterial protein for the prevention/treatment of S. uberis mastitis. Further testing is required to determine the effectiveness of the lysin in an in vivo model.