Characterization of a bacteriophage lysin (Ply700) from Streptococcus uberis

• Published in: Veterinary microbiology 2008-07, Vol.130 (1), p.107-117
• Main Authors: Celia, Laura K., Nelson, Daniel, Kerr, David E.
• Format: Article
• Language: English
• Subjects: Antibacterial; antibacterial properties; Antibiotic; antimicrobial agents; bacterial adhesion; Bacteriology; bacteriophages; binding properties; Biological and medical sciences; bovine mastitis; cell adhesion; Cloning, Molecular; cows; cytolysis; disease prevention; DNA, Viral - genetics; enzymes; Escherichia coli; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Viral - physiology; in vitro studies; lysin; Mastitis; Microbiology; milk; Miscellaneous; Prophages - metabolism; Staphylococcus aureus; Streptococcus - virology; Streptococcus agalactiae; Streptococcus dysgalactiae; Streptococcus Phages - metabolism; Streptococcus pyogenes; Streptococcus uberis; Viral Proteins - metabolism; Virology
• ISSN: 0378-1135; 1873-2542
• DOI: 10.1016/j.vetmic.2007.12.004

The antibacterial properties of bacteriophage lytic enzymes may be of importance in future mastitis control programs. A prophage was isolated from a strain of Streptococcus uberis (ATCC 700407) following exposure to mitomycin C. Partial sequencing of the phage DNA revealed a putative lysin based on sequence similarity to other streptococcal phage lysins. The putative lysin (Ply700) was recombinantly expressed in Escherichia coli, and chromatographically purified. Addition of the purified Ply700 to bacterial suspensions of S. uberis, Streptococcus pyogenes, and Streptococcus dysgalactiae caused a rapid, calcium-dependent lysis while there was little activity against Streptococcus agalactiae, Staphylococcus aureus, or E. coli. Killing of S. uberis in milk by Ply700 (50 μg/ml) was confirmed by plate count assay. Activity was related to the initial concentration of bacteria in that 31% killing ( P < 0.05) was observed with an inoculating dose of approximately 4500 cfu/ml, while 81% killing ( P < 0.01) was observed when the inoculum was reduced to approximately 600 cfu/ml. In contrast, complete sterilization was observed in parallel cultures suspended in assay buffer indicating that factors in milk are able to neutralize the lysin. Functional characterization of the C-terminal domain, as a component of a GFP fusion protein, revealed its calcium-dependent ability to bind to S. uberis. The C-terminal domain may have utility in targeting S. uberis while it remains to be determined if the lysin by itself has sufficient potency in milk for effective use in the control of S. uberis mastitis.