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US 2016/0097044 A1Published application
Antimicrobial Enzyme Fusions Reduce Resistance and Kill Intracellular Staphylococcus aureus
OwnerDavid M. Donovaninventor
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OwnerDavid M. Donovaninventor
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Multi-drug resistant bacteria are a persistent problem in modern health care, food safety and animal health. There is a need for new antimicrobials to replace over-used conventional antibiotics. Here we describe engineered triple-acting staphylolytic peptidoglycan hydrolases wherein three unique antimicrobial activities from two parental proteins are combined into a single fusion protein, effectively reducing the incidence of resistant strain development. The fusion protein reduced colonization by S. aureus in a rat nasal colonization model, surpassing the efficacy of either parental protein. Modification of the triple-acting lytic construct with a protein transduction domain significantly enhanced both biofilm eradication and the ability to kill intracellular Staphylococcus aureus as demonstrated in cultured cells, and mouse models of staphylococcal mastitis and osteomyelitis. Bacterial cell wall degrading enzyme antimicrobials can be engineered to enhance their value as potent therapeutics.
A recombinant fusion nucleic acid encoding an antimicrobial Staphylococcus-specific triple fusion protein, wherein said encoded fusion protein comprises (1) a LysK endolysin CHAP endopeptidase domain, (2) a LysK endolysin amidase domain, and (3) a lysostaphin glycyl-glycine endopeptidase domain and a cell wall binding domain, wherein when in the K-L configuration, comprises:
The fusion nucleic acid encoding K-L-PTD1, K-L-PTD2, K-L-PTD3, K-L-PTD4, K-L-PTD6, K-L-PTD7, K-L-PTD8, K-L-PTD9, K-L-PTD10 and K-L-PTD11, said fusion nucleic acid having the sequence SEQ ID NO:53, 55, 57, 59, 61, 63, 65, 67, 69, and 71, respectively.
A recombinant fusion nucleic acid encoding an antimicrobial lysostaphin fusion protein, comprising lysostaphin and a protein transduction domain (PTD), said fusion protein having lytic activity for the peptidoglycan cell wall resulting in bactericidal activity toward untreated, live intracellular and extracellular Staphylococcus aureus, including multi-drug resistant strains (e.g. MRSA).
The fusion nucleic acid encoding L-PTD1, L-PTD2, L-PTD3, L-PTD4, L-PTD6, L-PTD7, L-PTD8, L-PTD9, L-PTD10, L-PTD11 and L-PTD12, said fusion nucleic acid having the sequence SEQ ID NO:9, 11, 13, 15, 17, 19, 21, 23, 25, 27 and 29, respectively.
A process for transforming a host cell, comprising stably integrating the nucleic acid or the construct into the host cell.
A recombinant antimicrobial Staphylococcus-specific triple fusion protein, wherein said fusion protein comprises (1) a LysK endolysin CHAP endopeptidase domain, (2) a LysK endolysin amidase domain, and (3) a lysostaphin glycyl-glycine endopeptidase domain and a cell wall binding domain, wherein when in the K-L configuration, comprises:
A lysostaphin-PTD fusion protein, wherein said fusion protein is lysostaphin and any one of PTD1-PTD4 and PTD6-PTD12, each lysostaphin-PTD fusion protein, L-PTD1, L-PTD2, L-PTD3, L-PTD4, L-PTD6, L-PTD7, L-PTD8, L-PTD, L-PTD10, L-PTD11 and L-PTD12 having the sequence SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30, respectively.
A LysK-PTD fusion protein, wherein said fusion protein is LysK and any one of PTD1-PTD4 and PTD6-PTD12, each LysK-PTD fusion protein, K-PTD1, K-PTD2, K-PTD3, K-PTD4, K-PTD6, K-PTD7, K-PTD8, K-PTD, K-PTD10, K-PTD11 and K-PTD12 having the sequence SEQ ID NO:32, 34, 36, 38, 40, 42, 44, 46, 48, 50 and 52, respectively.
A method of treating intracellular S. aureus infections in human brain microvascular endothelial cells (hBMEC) comprising treating hBMEC with an effective amount of a composition comprising lysostaphin-PTD12 fusion protein, whereby the number of live intracellular S. aureus is reduced in said cells as compared to untreated cells.
Dependencies are inferred from the claim text.