Defining the Structure and Function of a New Family of Peptidoglycan Crosslinking Enzymes

Project Summary Clostridioides difficile is an opportunistic pathogen and a leading cause of hospital-acquired infectious diarrhea. The Centers for Disease Control has declared C. difficile an urgent threat to public health. The frontline antibiotic against C. difficile is the broad-spectrum antibiotic vancomycin. Although vancomycin is highly effective against C. difficile, it also targets the healthy microbiota. C. difficile spores survive antibiotic treatment, leading to unacceptably high rates of relapse when the course of vancomycin comes to an end. An antibiotic that kills C. difficile more selectively would presumably improve outcomes, but developing such a drug requires identifying targets uniquely important to C. difficile. One potential target is the enzymes that crosslink the peptidoglycan (PG) cell wall that protects C. difficile from lysis due to internal osmotic pressure. The PG is a bag-like molecule composed of glycan strands stitched together with peptide crosslinks. In most bacteria, the crosslinks are classified as 4-3 crosslinks and are created by enzymes called PBPs. C. difficile is unusual in that most of its crosslinks are classified as 3- 3 crosslinks and are created by enzymes called LDTs. We recently discovered that 3-3 crosslinks and LDTs are essential for viability in C. difficile. That makes C. difficile the first organism known to require 3-3 crosslinks for survival. We also found that LDTs are important for cell division and sporulation. In the course of those studies, we identified a new family of LDTs whose hallmark is a catalytic VanW domain. We propose that a deeper understanding of VanW-type LDTs will advance basic science and might lead to the development of antibiotics that kill C. difficile without disrupting the healthy microbiota needed to keep C. difficile infections from recurring. The goals of this proposal are to: (i) determine the structure of a VanW domain and how it catalyzes transpeptidation; (ii) determine how VanW domain LDTs are recruited to the site of cell division; and (iii) determine the role of LDTs during sporulation. In summary, the work proposed here will provide insight into the function of a novel family of LDTs present in C. difficile and other Gram-positive bacteria and provide foundational knowledge to advance exploiting LDTs as drug targets.