Understanding cell wall lytic enzyme behavior in microbial consortium and bacterial growth supporting environments

Bhagwat, Amala Makarand
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Dordick, Jonathan S.
Collins, Cynthia H.
Koffas, Mattheos A. G.
Barquera, Blanca L.
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Chemical engineering
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Attribution-NonCommercial-NoDerivs 3.0 United States
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However, there was a reduction in the antimicrobial efficacy of both Lst and PlyPH in the actively growing consortium compared to non-growing conditions. Lst and PlyPH were found to be inhibited by proteases secreted during S. aureus and B. cereus growth in the co-culture. This work highlighted limitation of Lst and PlyPH activity in the presence of bacterial growth-supporting media. In order to investigate the cause for this limitation, we further studied the interdependence of Lst and PlyPH binding and killing activity in growth media. We hypothesized that the poor lytic enzyme activity in growth media results from compromised enzyme binding. Variation in the binding of isolated binding domains of Lst and PlyPH to S. aureus and B. cereus cells was quantified in the presence of three media with increasing nutrient complexity: a buffer, a defined and a complex growth medium. Evaluation of real-time kinetics and efficiency of the isolated domains to bind to their target cells was done using surface plasmon resonance (SPR) and flow cytometry. A reduction in the rate of enzyme association and a decrease in the cell population with bound Lst or PlyPH was observed with increase in medium complexity. Enzyme binding behavior was consistent with the catalytic behavior of Lst and PlyPH in the three media, thereby demonstrating the role of enzyme binding in determining its catalytic activity. This work suggests modulation of lytic enzyme binding properties as a potential avenue for enhancing lytic enzyme effectiveness in challenging bacterial growth-supporting environments and highlights the use of lytic enzymes for potential microbiome engineering applications.
Due to disruption of ecology of human microbiome by irrational use of broad-spectrum antibiotics and rapid emergence of antibiotic-resistant bacteria, there is an urgent need for narrow-spectrum antimicrobials. Modular bacterial cell wall lytic enzymes are highly potent narrow-spectrum antimicrobials owing to their selective target binding and peptidoglycan degradation. Lytic enzymes could be exploited for engineering a microbial consortium by selectively controlling its microbial population, for example, during a pathogenic bacterial infection. However, lytic enzyme behavior in a microbial community has not been studied exhaustively.
First, we aimed at using lytic enzymes for selective pathogen removal from a microbial consortium. To that end, lytic enzymes lysostaphin (Lst) and PlyPH were employed for targeting Gram-positive skin pathogens Staphylococcus aureus and Bacillus cereus respectively. We investigated Lst and PlyPH activity in a consortium of S. aureus and B. cereus under growth and non-growth conditions. Both the enzymes were highly selective in the consortium, suggesting the potential of lytic enzymes as tools for selective remodeling of a microbial community.
August 2020
School of Engineering
Dept. of Chemical and Biological Engineering
Rensselaer Polytechnic Institute, Troy, NY
Rensselaer Theses and Dissertations Online Collection
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