Regulation of the C. difficile cell envelope by Two-component systems

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Project Summary Clostridioides (Clostridium) difficile infections 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. New treatments are sorely needed. Here we focus on the C. difficile cell envelope because it is a common target of antimicrobial therapy as evidenced by antibiotics including penicillin, vancomycin and bacitracin as well as components of the innate immune system. The C. difficile cell envelope has some unusual features that may prove to be unique targets for antibiotics. Lysozyme which kills cells by cleaving the peptidoglycan. We isolated mutants of C. difficile with increased resistance to lysozyme and we identified two regulatory systems that when constitutively active increase lysozyme resistance. The C. difficile genome contains a relatively large number of two component systems of which few have been studied. It is important to understand how C. difficile senses extracellular stresses and how these regulatory systems control biogenesis of the cell envelope. We will pursue two specific aims to understand the role of these regulators in controlling cell envelope biogenesis. In Aim 1 we will construct a series of mutants that have gain of function and loss of function mutations in these regulatory systems. These mutants will then be studied for their effect on resistance to a number of cell envelope stresses and effects on cell envelope biogenesis. This will provide a greater understanding of the phenotypic effects of these two-component systems. In Aim 2 we will map the promoters of some genes we have identified whose expression is dependent upon these regulators. We will also define the regulons of both response regulators using RNA-seq. Finally, we will use CRISPR interference and overexpression to identify individual genes required for lysozyme resistance and cell envelope maintenance. Together these aims will advance our understanding of C. difficile biology by defining the role of two different two-component system in controlling cell envelope maintenance.