Engineering MRSA antimicrobials that are refractory to resistance development

Authors: Becker, Stephen; Donovan, David; Foster Frey, Juli

Submitted to: BARC Poster Day
Publication Type: Abstract Only
Publication Acceptance Date: 4/2/2008
Publication Date: 4/23/2008
Citation: Becker, S.C., Donovan, D.M., Foster Frey, J.A. 2008. Engineering MRSA antimicrobials that are refractory to resistance development. BARC Poster Day.

Interpretive Summary:

Technical Abstract: Methicillin resistant Staphylococcus aureus (MRSA) is one of the most costly multi-drug resistant pathogens to both human animal health, with billions of dollars are spent annually to treat human infections. MRSA is also appearing in livestock (bovine, porcine, poultry) as well as companion animals (dogs and cats). Currently the most common treatment for MRSA is vancomycin, but resistance to this antibiotic is increasing with VRSA (vancomycin resistant) isolates already apparent. Novel antimicrobials for the treatment of these resistant pathogens are sorely needed. Phage lysins (peptidoglycan hydrolases) are a upcoming area for the development of antimicrobials causing cell lysis by degrading cell wall peptidoglycan. No bacteria have been identified that can evade lysis by their bacteriophage endolysin (despite efforts to identify them), presumably due to the phage having co-evolved with the host(s). Lysins are modular in structure, often with multiple lytic domains and a cell wall binding domain. It has been shown that fusing domains from multiple enzymes, generates chimeric molecules that maintain their parental specificities and lytic activities. We predict that by creating fusion lysins with multiple enzymatic domains, will decreases the probability of resistance development. LysK is a staphylococcal bacteriophage endolysin from the phage K. It is a peptidoglycan hydrolase enzyme containing two independent peptidoglycan degrading activities which can lyse many staphylococcal strains and thus is a potent antimicrobial against S. aureus, including MRSA. Lysostaphin is a bacteriocin secreted by S. simulans to kill S. aureus, and has been shown to also be a potent antimicrobial for many antibiotic resistant strains of S. aureus. This study describes a strategy to create triple acting antimicrobials against MRSA that we predict will be refractory to resistance development. Fusion proteins will be examined in multiple bactericidal assays, and for their efficacy against multiple pathogens. Importantly, we have identified two hydrolases that demonstrate synergy when used in combination. In an effort to further improve our engineered fusion constructs, we show that by fusing the enzymatic CHAP domain from Lambda-SA2, a Streptococcus agalactiae phage lysin with weak stapholytic activity, to the SH3b cell wall binding domain of the S. aureus bacteriocin lysostaphin, we can improve the Lambda Sa2 lysins ability to kill S. aureus, increasing its activity >10 fold over the wild type enzyme. By testing multiple lysins for optimal enzymatic and binding domains for different species we will be able to generate novel antimicrobials which are refractory to resistance.