United States of America, As Represented by the Secretary of Agriculture

Igor V Abaev

David M Donovan

Mathias Schmelcher

Increases in antibiotic resistant strains ofhave elicited efforts to develop novel antimicrobials. One potential treatment includes lytic enzymes produced by staphylococcal bacteriophage during the lytic cycle. The phage Twort endolysin (PlyTW) harbors three domains, a CHAP endopeptidase, an amidase-2 domain, and a SH3b-5 cell wall binding domain. The CHAP domain alone is necessary and sufficient for lysis of live; the amidase-2 domain alone is insufficient. Loss of the SH3b cell wall binding domain results in a 10 fold reduction of enzymatic activity in turbidity reduction and plate lysis assays compared to the full length protein. Deletion of the amidase-2 domain resulted in a protein (PlyTW &#x394;172-373) with lytic activity that exceeded the activity of the full length construct in both assays. Addition of Caenhanced activity in turbidity reduction assays. Chelation by the addition of EDTA or zinc inhibited the activity of all PlyTW constructs.

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is notorious for developing resistance to virtually all antibiotics to which it is exposed. Staphylococcal phage 2638A endolysin is a peptidoglycan hydrolase that is lytic forwhen exposed externally, making it a new antimicrobial candidate. It shares a common protein organization with over 40 other staphylococcal peptidoglycan hydrolases: a CHAP endopeptidase domain, a mid-protein amidase 2 domain and a C-terminal SH3b cell wall binding domain. It is the first phage endolysin reported with a cryptic translational start site between the CHAP and amidase domains. Deletion analysis indicates that the amidase domain confers most of the lytic activity and requires the full SH3b domain for maximal activity. It is common for one domain to demonstrate dominant activity over another; however, the phage 2638A endolysin is the first to show high amidase domain activity dominant over the N-terminal CHAP domain, an important finding for targeting novel peptidoglycan bonds.

Methicillin-resistant (MRSA) and multi-drug resistant strains ofare becoming increasingly prevalent in both human and veterinary clinics.-causing bovine mastitis yields high annual losses to the dairy industry. Treatment of mastitis by broad range antibiotics is often not successful and may contribute to development of antibiotic resistance. Bacteriophage endolysins are a promising new source of antimicrobials. The endolysin of prophage &#x3c6;SH2 ofstrain JCSC1435 (&#x3c6;SH2 lysin) shows lytic activity against live staphylococcal cells. Deletion constructs were tested in zymograms and turbidity reduction assays to evaluate the contribution of each functional module to lysis. The CHAP domain exhibited three-fold higher activity than the full length protein. Activity was further enhanced in the presence of bivalent calcium ions. The full length enzyme and the CHAP domain showed activity against multiple staphylococcal strains, including MRSA strains, mastitis isolates, and coagulase negative staphylococcal (CoNS) strains.

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Obolensk, Ukraine

Igor V Abaev