Triple Acting Antimicrobials That Are Refractory To Resistance Development

Abstract

Multi-drug resistant superbugs are a persistent problem in modern health care. This invention provides an antimicrobial endolysin-Lysostaphin triple fusion protein, comprising (1) an endolysin CHAP endopeptidase domain, (2) an endolysin amidase domain, and (3) a Lysostaphin glycyl-glycine endopeptidase domain. The domains are derived from two proteins that show antimicrobial synergy when used in combination. The protein has specificity and exolytic activity for the peptidoglycan cell wall of untreated, live Staphylococcus aureus from many growth phases i.e. stationary, logarithmic and biofilm growth. The recombinant triple fusion protein comprising the three functional antimicrobial domains is designed to be refractory to resistance development.

1 - 17 . (canceled) 
     
     
         18 . A recombinant antimicrobial  Staphylococcus -specific endolysin-Lysostaphin triple fusion protein, comprising (1) an endolysin CHAP endopeptidase domain, (2) an endolysin amidase domain, and (3) a Lysostaphin glycyl-glycine endopeptidase domain, said protein having specificity and exolytic activity for the peptidoglycan cell wall of untreated, live  Staphylococcus aureus.    
     
     
         19 . The recombinant triple fusion protein of  claim 18  wherein each domain of said protein cuts the peptidoglycan at a different, unique covalent bond of the peptidoglycan, and each is lytic in the presence of lysis by the others. 
     
     
         20 . The recombinant triple fusion protein of  claim 18  wherein the parental lysin of each domain of the triple fusion protein is synergistic in antimicrobial activity when the parental lysins of said domains are used in combination. 
     
     
         21 . The protein of  claim 18 , wherein the endolysin is a LysK endolysin-derived peptidoglycan hydrolase. 
     
     
         22 . The protein of  claim 18 , wherein the endolysin is a phi11 endolysin-derived peptidoglycan hydrolase. 
     
     
         23 . The protein of  claim 21  wherein said triple fusion protein comprises a polypeptide identified by SEQ ID NO:6. 
     
     
         24 . The protein of  claim 22  wherein said triple fusion protein comprises a polypeptide identified by SEQ ID NO:14. 
     
     
         25 . A recombinant antimicrobial  Staphylococcus -specific endolysin-Lysostaphin triple fusion protein, comprising (1) an endolysin-derived CHAP endopeptidase domain and (2) an endolysin-derived amidase domain, and (3) a Lysostaphin glycyl-glycine endopeptidase domain, said protein having specificity and exolytic activity for the peptidoglycan cell wall of untreated, live  S. aureus , wherein an endolysin-derived domain is truncated. 
     
     
         26 . The protein of  claim 25 , wherein the fusion protein has endopeptidase and amidase activity and does not require a SH3b binding domain. 
     
     
         27 . The protein of  claim 25 , wherein the fusion protein has endopeptidase activity and does not require a SH3b binding domain. 
     
     
         28 . The recombinant antimicrobial  Staphylococcus -specific endolysin-Lysostaphin triple fusion protein of  claim 25  wherein said protein comprises a polypeptide identified by SEQ ID NO: 8, SEQ ID NO:10, or SEQ ID NO:12. 
     
     
         29 . A composition useful for the treatment of a disease caused by multidrug-resistant staphylococci, wherein said composition comprises the protein of  claim 18  and a pharmaceutically acceptable carrier. 
     
     
         30 . A composition useful for the treatment of a disease caused by staphylococci, wherein said composition comprises the protein of  claim 25  and a pharmaceutically acceptable carrier. 
     
     
         31 . A method of treating infection and disease caused by multidrug-resistant staphylococci in an individual comprising:
 administering to said individual an effective dosage of a composition of  claim 18  or  claim 25 , wherein said composition comprises a recombinant antimicrobial  Staphylococcus -specific endolysin-Lysostaphin triple fusion protein, comprising (1) an endolysin CHAP endopeptidase domain, (2) an endolysin amidase domain, and (3) a Lysostaphin glycyl-glycine endopeptidase domain, said protein having specificity and exolytic activity for the peptidoglycan cell wall of untreated, live  Staphylococcus aureus , wherein said administration is effective for the treatment of said multidrug-resistant staphylococci.   
     
     
         32 . A method of treating mastitis in an animal comprising: administer to said animal an effective dosage of a composition of  claim 18  or  claim 25 , wherein said composition comprises a recombinant antimicrobial  Staphylococcus -specific endolysin-Lysostaphin triple fusion protein, comprising (1) an endolysin CHAP endopeptidase domain, (2) an endolysin amidase domain, and (3) a Lysostaphin glycyl-glycine endopeptidase domain, said protein having specificity and exolytic activity for the peptidoglycan cell wall of untreated, live  Staphylococcus aureus , wherein said administration is effective for reducing the severity of said mastitis.