|Foster frey, Juli|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/10/2006
Publication Date: 6/6/2006
Citation: Donovan, D.M., Foster Frey, J.A., Dong, S., Rousseau, G., Moineau, S., Pritchard, D.G. 2006. The cell lysis activity of the streptococcus agalactiae bacteriophage b30 endolysin relies on the chap endopeptidase domain. Applied Environmental Microbiology. 72:5108-5112. Interpretive Summary: Mastitis is an infection of dairy cattle mammary glands. It costs the US dairy industry $2 billion dollars annually. Streptococcus agalactiae is one of the most serious mastitis pathogens. In an effort to identify novel antimicrobial proteins for use against mastitis causing pathogens, bacteriophage endolysins are being examined. The bacteriophage B30 has an enzyme (endolysin) that can degrade the cell wall of S. agalactiae and thus kill the pathogen. The B30 endolysin can lyse the S. agalactiae in whey, so is a good candidate antimicrobial for mastitis infections. The B30 endolysin has two lytic domains, an endopeptidase and a glycosidase that both are highly conserved when compared to other endolysins and thus both were believed important for lysing the pathogen. However, when B30-derived protein truncations are made, or site-directed mutations in the two domains of the protein, we can show that only one of the domains, the endopeptidase, is functional and active in the lysis of the pathogen. This is unexpected and might something new about the function of the endolysin in phage biology.
Technical Abstract: The 443 amino acid Streptococcus agalactiae bacteriophage B30 endolysin gene contains a CHAP endopeptidase domain, an Acm lysozyme-like glycosidase, and a C-terminal SH3b cell wall binding domain. Although both hydrolase domains are enzymatically functional, it is unknown the degree to which each contributes to cell lysis of the host S. agalactiae. For the purpose of identifying novel anti-mastitis antimicrobial agents, a series of deletion mutants was constructed to localize the catalytically active regions of the lysin. From this analysis, the endopeptidase activity was located on a protein fragment containing the first 156 amino acids of the native lysin. It did not require the SH3b domain for activity. Truncated proteins harboring just the ACM hydrolase domain showed only weak activity in plate lysis assays and no activity in turbidity assays against streptococcal pathogens. These results were verified with site-directed point mutations that inactivate each domain. The ACM domain has virtually no lytic activity when lysing 'from without'. The N-terminal CHAP endopeptidase is responsible for almost 100% of the lytic activity. Even in the absence of the SH3b domain, the truncated CHAP domain maintains the same species specificity as the native B30 endolysin. Like the native lysin, the truncated 156 amino acid endopeptidase domain fragment lyses the mastitis pathogens S. dysgalactiae and S. uberis, the milk processing bacteria S. thermophilus, Leuconostoc cremoris as well as other streptococci such as S. salivarius and S. suis, and is inactivated during pasteurization.