Author
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Donovan, David |
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Foster Frey, Juli |
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Submitted to: FEMS Microbiology Letters
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 6/18/2008 Publication Date: 10/1/2008 Citation: Donovan, D.M., Foster-Frye,J., 2008. LambdaSa2 prophage endolysin requires Cpl-7 binding domains and Amidase-5 domain for antimicrobial lysis of streptococci. FEMS Microbiology Letter. 287(1):22-23. Interpretive Summary: Streptococcal pathogens contribute to a wide variety of human and livestock diseases. The routine use of antibiotics to battle these pathogens has produced a new class of superbug, multi-drug resistant streptococci. To treat these pathogens, there is a need for new antimicrobials. The newly identified LambdaSa2 prophage endolysin gene has been shown to have antimicrobial activity toward streptococcal pathogens. We have expanded the known pathogen target range of this antimicrobial to show that it also kills many bovine mastitis and human pathogens. Through deletion analysis we have identified that the N-terminal endopeptidase domain confers more than 90% of the lytic activity to the protein, and that the Cpl-7 cell wall binding domains are essential for this peptidoglycan hydrolase activity. Elucidation of which pathogens can be targeted and which domains are functional allows the use of just the active domains in the creation of fusion proteins active against the target pathogen(s). This aids in the creation of novel antimicrobials with multiple activities targeting the same pathogen, or fusion antimicrobials that target several pathogens simultaneously. Due to the high species specificity of the endolysin lytic domains, and the absence of resistant strain development by phage endolysins, these antimicrobials should help avoid resistance development in both non-streptococcal commensal bacteria and the target pathogen. Technical Abstract: Streptococcal pathogens contribute to a wide variety of human and livestock diseases. The routine use of antibiotics to battle these pathogens has produced a new class of superbug, multi-drug resistant streptococci. To treat these pathogens, there is a need for new antimicrobials. Bacteriophage endolysins demonstrating bactericidal peptidoglycan hydrolase activity comprise one group of new candidate antimicrobials that are reportedly refractory to resistance development. The LambdaSa2 prophage endolysin gene was recently isolated from a Group B Streptococcal genome, expressed on an E. coli plasmid, and shown by homology screening and biochemical analysis to harbor an amidase-5 (endopeptidase), an amidase-4 (glycosidase) domain and two Cpl-7 cell wall binding domains. It was reported that this endolysin can digest the cell walls of Streptococcus agalactiae, S. pneumoniae and Staphylococcus aureus. In this study, turbidity reduction and plate lysis assay testing indicates that this peptidoglycan hydrolase shows strong lytic activity toward S. pyogenes, S. dysgalactiae, S. uberis, S. equi, GES, and GGS. Deletion analysis on the His-tagged version of this gene further indicates that the N-terminal endopeptidase domain is minimally active in the absence of a Cpl-7 domain when lysing cells from without but can achieve a higher specific activity than the full length protein (on some strains) with both Cpl-7 domains. This truncated version shows weak activity against two Coagulase Negative Staphylococci, S. hyicus and S. xylosus. The truncated construct with just the glycosidase domain is virtually inactive when lysing cells from without and only shows weak activity on plate lysis assays. |
