Specific lysis of staphylococcal pathogens by bacteriophage phi11 endolysin
AssigneeThe United States of America as represented by the Secretary of Agriculture
InventorDavid M. Donovan
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Abstract
The Staphylococcus aureus bacteriophage phi11 endolysin has two peptidoglycan hydrolase domains (endopeptidase and amidase) and a SH3b cell wall-binding domain. In turbidity reduction assays, the purified protein can lyse untreated staphylococcal mastitis-causing pathogens, S. aureus and coagulase negative staphylococci (S. chronogenes, S. epidermis, S. hyicus, S. simulans, S. warneri, and S. xylocus), making it a strong antimicrobial protein and an effective candidate for treating multidrug-resistant staphylococci. Lytic activity is maintained at the pH (6.7) and the ‘free’ calcium concentration (3 mM) of milk. Truncated endolysin-derived proteins, containing just the endopeptidase domain, also lyse staphylococci, in the absence of the SH3b-binding domain.
Claims9
An isolated or recombinant nucleic acid encoding an antimicrobial phi11 endolysin-derived peptidoglycan hydrolase enzyme molecule having specificity and exolytic activity for the peptidoglycan cell wall of untreated Staphylococcus aureus and coagulase negative staphylococci (CNS), said CNS comprising S. chronogenes, S. epidermis, S. hyicus, S. simulans, S. warneri, and S. xylocus, wherein said phi11 endolysin-derived peptidoglycan hydrolase is a truncated peptidoglycan hydrolase having endopeptidase and amidase activity and lacking a SH3b binding domain, and said nucleic acid encoding said truncated peptidoglycan hydrolase molecule has the sequence set forth in SEQ ID NO:
- 2.
An isolated or recombinant nucleic acid encoding an antimicrobial phi11 endolysin-derived peptidoglycan hydrolase enzyme molecule having specificity and exolytic activity for the peptidoglycan cell wall of untreated Staphylococcus aureus and coagulase negative staphylococci (CNS), said CNS comprising S. chronogenes, S. epidermis, S. hyicus, S. simulans, S. warneri, and S. xylocus, wherein said phi11 endolysin-derived peptidoglycan hydrolase is a truncated peptidoglycan hydrolase having endopeptidase activity and lacking a SH3b binding domain, and said nucleic acid encoding said truncated peptidoglycan hydrolase molecule has the sequence set forth in SEQ ID NO:
- 3.
A process for transforming a host cell, comprising stably integrating the nucleic acid of claim 1 or the construct of claim 9 into the host cell.
A method of making a recombinant antimicrobial peptidoglycan hydrolase protein having endopeptidase and amidase activity and lacking a SH3b binding domain, said method comprising steps:
- a. introducing into a host cell a nucleic acid or construct encoding a antimicrobial phi11 endolysin-derived peptidoglycan hydrolase enzyme molecule having specificity and exolytic activity for the peptidoglycan cell wall of untreated Staphylococcus aureus and coagulase negative staphylococci (CNS), said CNS comprising S. chronogenes, S. epidermis, S. hyicus, S. simulans, S. warneri, and S. xylocus, wherein said phi11 endolysin-derived peptidoglycan hydrolase is a truncated peptidoglycan hydrolase having endopeptidase and amidase activity and lacking a SH3b binding domain, and said nucleic acid encoding said truncated peptidoglycan hydrolase molecule has the sequence set forth in SEQ ID NO:2;
- b. culturing said cell under conditions suitable for expression of said protein;
- c. recovering the protein so expressed.
Dependencies are inferred from the claim text.