Improvement and functionalization of bacterial cell wall lytic enzymes for increased cell killing and reduction of bacteria on surfaces

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Authors
Mixon, Monica
Issue Date
2024-12
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Electronic thesis
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en_US
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Chemical engineering
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Abstract
Antibiotic resistant pathogenic bacteria are a rising global issue. Once successfully infecting an individual, these microbes are often extremely difficult to treat with commonly used antibiotics and may spread from person to person, especially in hospital settings. Clearly, new therapies are necessary to eliminate this threat. Enzymes are one alternative treatment that has proven efficacious to antibiotic susceptible as well as antibiotic resistant strains of bacteria. Many types of cell wall lytic enzymes have been studied for their antibacterial properties and have the advantage of being narrow-spectrum and possesses a lower chance of imparting resistance to their target. Lytic enzymes are naturally produced by bacteriophages, which perforate bacterial cell wall to infect and then escape a host bacterium, and by bacteria, which either employ lytic enzymes to help with cell growth and division or to eliminate competing bacteria. In my research, I have attempted to optimize the activity of two lytic enzymes, lysostaphin (Lst), whose target is Staphylococcus aureus, and PlyG, which targets Bacillus sp. Out of several optimization strategies, I have chosen to affect the electrostatic interactions between lytic enzyme and target cell wall by including a string of charged or neutral residues on the lytic enzymes. Because wall teichoic acids located on the bacterial cell surface render the cell wall largely negative in charge, I hypothesize that a lytic enzyme with a positively charged tag will be more drawn to the cell wall of bacteria, thus lysing a greater number of cells, while the opposite will hold true for a negatively charged construct. I have also sought to generate lytic enzymes that are especially active on various medical and non-medical surfaces, such as indwelling catheters and clothing. Peptide tags with affinity for silicone, polyethylene terephthalate (PET), polyvinyl chloride, and cellulose have been evaluated for their adherence to their respective surfaces. The surface binding peptides fused to Lst will provide a lytic enzyme that adheres to and inhibits bacterial growth on the surfaces. Because the lytic enzyme is not covalently attached, older enzymes can be removed and replaced by newer enzymes to enable efficient reuse of the surface. Finally, enzyme treatments for Corynebacteria species have been explored. Few lytic enzyme therapies exist to treat pathogenic species within this genus, most likely due to their unique cell wall architecture. Thus, Corynebacteria cell killing studies have been conducted in an attempt to further the literature.
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December2024
School of Engineering
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Rensselaer Polytechnic Institute, Troy, NY
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