Developing imaging tools to improve endolysin activity - antibiotic resistant infections are one of the highest causes of morbidity and mortality around the world

Scripps College received a $457,088 Project Grant from the National Institute of General Medical Sciences (NIGMS) under the Biomedical Research and Research Training program (CFDA 93.859), effective September 2, 2025, through September 1, 2028. The award funds the development of imaging tools and engineered endolysins to address antibiotic-resistant bacterial infections. The project deliverables include: (1) synthesis and characterization of internally quenched fluorescent substrates to assess endolysin activity in a high-throughput manner, using CHAPK as a model endolysin; and (2) development of thermostability-enhanced endolysin variants through computational-guided mutagenesis and substrate-based screening of mutant libraries. The research will generate improved molecular tools for evaluating endolysin efficacy and provide more stable therapeutic candidates for clinical and agricultural applications.

This research initiative is substantially driven by undergraduate researchers and postbaccalaureate students through summer research and senior thesis projects, with additional student engagement through course-based research experiences. The project is conducted at Scripps College's facilities in California and directly supports NIGMS's mission to advance fundamental biomedical research while providing hands-on training and workforce development for early-career researchers in the life sciences.

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.