Development of novel selective antimicrobials is gaining momentum due to increased bacterial drug resistance, microbiome dysbiosis caused by broad-spectrum antibiotics and the need to understand and precisely engineer human and environmental microbiomes. Cell lytic enzymes, including bacteriocins, autolysins and bacteriophage endolysins represent a unique, highly specific catalytic repertoire hydrolyzing bacterial peptidoglycan. Owing to their exquisite binding and/or catalytic specificity and lower probability of gaining microbial resistance, they are not only emerging as promising alternative to antibiotics for therapy and infrastructural resilience applications but also potentially for microbiome engineering and remodeling via selective pathogen elimination. We have assessed the ability of two different lytic enzymes, bacteriocin lysostaphin (Lst) and endolysin PlyPH targeting skin pathogens Staphylococcus aureus and Bacillus cereus, respectively, to control a community (mixture or co-culture) of S. aureus and B. cereus with a skin commensal Micrococcus luteus. Lst (100 ug/mL) was highly effective and selective, causing ~ 3-log reduction in S. aureus, however, 100 ug/mL PlyPH selective but less active, resulting in ~1.5-log reduction in B. cereus in an actively growing co-culture. Lst and PlyPH were limited by proteases secreted during growth, as suggested by their increased activity in culture supernatants in the presence of a protease inhibitor cocktail, highlighting effect of bacterial metabolism on enzyme activity. Since Lst and PlyPH activity is governed by cell wall binding, analysis of their binding is critical for elucidating activity in nutrient-rich environments mimicking natural microbiomes. Lst and PlyPH binding was measured in four increasingly complex media by evaluating binding isotherms and real-time binding kinetics of their isolated fluorescently-tagged binding domains using both fluorescence and Surface Plasmon Resonance. Slower binding kinetics was observed with increased medium complexity, causing limited killing in nutrient rich media. In conclusion, we demonstrate utility of lytic enzymes and fundamental considerations to enhance their robustness for microbiome engineering.

Towards microbiome engineering using highly selective bacteriocin lysostaphin and phage endolysin PlyPH

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.