Developing imaging tools to improve endolysin activity

Antibiotic resistant infections are one of the highest causes of morbidity and mortality around the world. The evolutionary rate of bacteria makes finding effective antibiotics to treat infections difficult. A promising alternative to traditional antibiotics is endolysins. These proteins lyse specific bacteria without inducing resistance. While effective, these enzymes suffer from low stability, limiting their use in clinical and agriculture settings. Additionally, there are a lack of tools available to assess their activity in a high throughput manner. Thus, new tools to improve endolysins are needed. The proposed project develops an engineered endolysin for improved activity. The model endolysin that will be used in the focus of this study is CHAPK. CHAPK demonstrates high lytic activity against Staphylococcus species but exhibits suboptimal activity at physiological conditions. In Aim 1, a panel of internally quenched substrates will be synthesized to assess CHAPK activity. These substrates will be characterized with recombinant enzyme and bacterial lysate to demonstrate its ability to report on hydrolysis events. Aim 2 will establish how these substrates can be used to evolve CHAPK for improved thermostability. A computation-guided approach will be used to select sites to mutate for improved thermostability. After generating the library, the synthetic substrate will be used to screen the mutants in a high-throughput manner. The top hits will be purified and further characterized. This proposal will expand the methods available to assess endolysin activity as well as provide a more guided approach to improving these potential therapeutics. The work proposed will be driven by undergraduate researchers and a postbaccalaureate student through summer and senior thesis research projects. Undergraduate students will also engage in this work through a course-based research experience. Funding from this project will increase participation from the next generation of scientists in the fields of chemistry and chemical biology. Additionally, the research environment at Scripps and Pitzer Colleges will be enhanced by increasing the number of research opportunities available for students, fostering new collaborations, and increasing the number of state-of-the-art instrumentations in the Department of Natural Sciences. Project Number: 1R15GM160937-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Sierra Williams | Institution: SCRIPPS COLLEGE, CLAREMONT, CA | Award Amount: $457,088 | Activity Code: R15 | Study Section: Special Emphasis Panel[ZRG1 MCST-U (85)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11221390