Project Summary Clostridioides (Clostridium) difficile causes nearly 500,000 infections a year in the United States, leading to nearly 30,000 deaths. The CDC has declared C. difficile an “urgent” threat to public health, the highest threat category. New treatments are sorely needed. Many of our most useful antibiotics target the cell envelope. However, little is known about cell envelope biogenesis or cell envelope stress response in C. difficile. These cell envelope stress response systems may play an important role in antibiotic resistance. By understanding how C. difficile responds to cell envelope stress we may uncover better treatment options for C. difficile infections. Surotomycin, a daptomycin derivative, which targets peptidoglycan synthesis showed promise as a potential treatment for C. difficile. However, conflicting phase 3 clinical trials have halted development of surotomycin as a treatment for C. difficile infections. We used multiple genetic approaches to identify key factors involved in daptomycin resistance. By isolating spontaneous daptomycin resistant mutants and performing Tn-seq we identified two different two component regulatory systems, DraRS and DapRS, that are involved in daptomycin resistance. We will pursue three specific aims to dissect the role of these regulators in controlling daptomycin resistance and cell envelope stress response. In Aim 1 we will define how the DraRS regulatory system contributes to antibiotic resistance. We will test both gain-of-function and loss-of-function draRS mutants for their effect on resistance to a number of cell envelope stresses and on cell envelope biogenesis. We will also define the DraR regulon and determine the contribution of individual genes to antibiotic resistance and cell envelope biogenesis and stress response. In Aim 2 we will dissect how activity of DraR is controlled in response to cell envelope stress. Our preliminary data suggest DraRS is activated by antibiotics disrupt the lipid-II cycle. We will use CRISPRi to test this model by genetically recapitulating the effects of antibiotics and determining the effect on DraR activation. We will also isolate mutants of DraS that are unable to respond to cell envelope stress. In Aim 3 we will define the role of DapRS and the DapRS-regulated genes hexSDF in cell envelope biogenesis. Our data suggest they are required for production of a unique glycolipid which makes up 16% of the polar lipids in the C. difficile membrane. We will define the DapR regulon and how individual regulon members contribute to daptomycin resistance, cell charge, and lipid content and cell envelope stress response. Together these aims will advance our understanding of C. difficile by defining how it resists cell-wall acting antibiotics like daptomycin and vancomycin.