|Foster Frey, Juli|
Submitted to: Evergreen International Phage Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 6/17/2009
Publication Date: 9/13/2009
Citation: Becker, S.C., Pohl, C.S., Mohammadi, H., Schmelcher, M., Foster Frey, J.A., Lease, R.A., Don, S., Baker, J.R., Pritchard, D.G., Donovan, D.M. 2009. Engineering Antimicrobials Refractory to Resistance. Evergreen International Phage Meeting. Interpretive Summary: n/a
Technical Abstract: Multi-drug resistant superbugs are a persistent problem in modern health care, demonstrating the need for a new class of antimicrobials that can address this concern. Triple-acting peptidoglycan hydrolase fusions are a novel class of antimicrobials which have qualities well suited to avoiding resistance development. Lysostaphin is a peptidoglycan hydrolase (glycyl-glycine endopeptidase) secreted by S. simulans to kill S. aureus that has been shown to also be a potent antimicrobial for many antibiotic resistant strains of S. aureus, including multi drug resistant strains [MRSA and VRSA]. However, resistance to lysostaphin has been reported, reducing its potential as an antimicrobial. LysK is the phage K (staphylococcal bacteriophage) endolysin. It is a peptidoglycan hydrolase enzyme with two lytic activities (amidase and endopeptidase) that can also lyse many staphylococcal strains, including MRSA, and thus is a potent staphylococcal antimicrobial. To date, no resistant phenotypes have been observed for phage derived peptidoglycan hydrolases, making this class of enzymes ideal for antimicrobial development. We have demonstrated that in combination, lysostaphin and LysK target three unique bonds in the peptidoglycan of S. aureus. When used in combination, these proteins act synergistically to inhibit the growth of S. aureus strains, including the MRSA strain USA300. We have created fusion proteins that retain all three parental hydrolase activities. These fusions demonstrate increased molar specific activity as compared to the parental enzymes in multiple in vitro S. aureus lytic assays. We have demonstrated that these constructs are able to eliminate an innoculum of 3x103 cfu/ml in less than 90 minutes in heparinized whole rat blood. In addition, these fusions are able to lyse coagulase negative staphylococci (in many of which the peptidoglycan is a poor substrate for Lysostaphin’s endopeptidase domain). We predict that no bacterium can evade three simultaneous, unique lytic activities and thus propose that these triple-acting fusion antimicrobials will be refractory to resistance development.