Project Summary Clostridioides (Clostridium) difficile infections of the colon strike close to 500,000 people a year in the United States, leading to nearly 30,000 deaths. The CDC has declared this organism an “urgent” threat to public health, the highest threat category. C. difficile infections are difficult to treat in large part because the organism forms dormant spores that survive antibiotic therapy and seed recolonization of the gut when antibiotics are withdrawn. This problem is exacerbated by the fact that the antibiotics used against C. difficile also kill many of the healthy gut bacteria, clearing the way for C. difficile to recolonize when spores germinate. Thus, there is a tremendous need for new drugs that target C. difficile without disrupting the healthy microbiota. The premise of this proposal is that a deeper understanding of cell envelope biogenesis can pave the way towards developing better ways to treat C. difficile infections. The cell envelope is a well-validated target for antibiotics, and in C. difficile the envelope has some unusual features that suggest its assembly requires novel proteins that could be exploited as targets of C. difficile-selective antibiotics. In Aim 1 we will use genetics, biochemistry and microscopy to understand the roles and regulation of enzymes that crosslink the peptidoglycan cell wall. These enzymes captured our attention because in C. difficile the cell wall contains an unusually high percentage of “3-3” crosslinks as compared to the “4-3” crosslinks that predominate in most bacteria. Our experiments will address the following questions: Which enzymes are responsible for 3-3 and 4-3 crosslink formation and do they operate during division, elongation or both? How is the ratio of 3-3 to 4-3 crosslinking regulated? How does C. difficile benefit from using primarily 3-3 crosslinks? In Aim 2 we will leverage a powerful new gene-silencing tool called CRISPR interference (CRISPRi) to assign a set of ~50 putatively essential envelope biogenesis genes to more specific functional pathways. These genes are intrinsically interesting and constitute potential new antibiotic targets. We will also undertake a detailed analysis of a novel transcriptional regulatory system uncovered in a pilot version of our proposed screen. Collectively, the lines of investigation to be pursued here will greatly advance our understanding of C. difficile biology by identifying new proteins involved in assembly of the cell envelope and revealing how their activities are coordinated to accomplish the complex processes of growth and division.