Identification of a family of peptidoglycan transpeptidases reveals that <i>Clostridioides difficile</i> requires noncanonical cross-links for viability

Most bacteria are surrounded by a cell wall that contains peptidoglycan (PG), a large polymer composed of glycan strands held together by short peptide cross-links. There are two major types of cross-links, termed 4-3 and 3-3 based on the amino acids involved. 4-3 cross-links are created by penicillin-binding proteins, while 3-3 cross-links are created by L,D-transpeptidases (LDTs). In most bacteria, the predominant mode of cross-linking is 4-3, and these cross-links are essential for viability, while 3-3 cross-links comprise only a minor fraction and are not essential. However, in the opportunistic intestinal pathogen <i>Clostridioides difficile,</i> about 70% of the cross-links are 3-3. We show here that 3-3 cross-links and LDTs are essential for viability in <i>C. difficile</i>. We also show that <i>C. difficile</i> has five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many gram-positive bacteria but scarce in other lineages. We tested seven non-<i>C. difficile</i> VanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a previously unrecognized family of PG cross-linking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 cross-linking is essential for viability in <i>C. difficile</i>, the first time this has been shown in any bacterial species. The essentiality of LDTs in <i>C. difficile</i> makes them potential targets for antibiotics that kill <i>C. difficile</i> selectively.