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

• 成果类型: Article
• 署名作者: Bollinger, Kevin W.; Muh, Ute; Ocius, Karl L.; Apostolos, Alexis J.; Pires, Marcos M.; Helm, Richard F.; Popham, David L.; Weiss, David S.; Ellermeier, Craig D.
• 署名单位: University of Iowa; University of Virginia; Virginia Polytechnic Institute & State University; Virginia Polytechnic Institute & State University; University of Iowa
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-14646
• DOI: 10.1073/pnas.2408540121
• 发表日期: 2024-08-20
• 关键词: penicillin-binding proteins; cell-wall; vancomycin resistance; enterococcus-faecium; crystal-structure; antibiotics; expression; mechanism; ldt(mt2); platform

摘要

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). Inmost 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 Clostridioides difficile, about 70% of the cross- links are 3- 3. We show here that 3- 3 cross- links and LDTs are essential for viability in C. difficile. We also show that C. difficilehas 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-C. difficileVanW 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 C. difficile, the first time this has been shown in any bacterial species. The essentiality of LDTs in C. difficile makes them potential targets for antibiotics that kill C. difficile selectively. Significance Clostridioides difficile is an opportunistic pathogen that can cause significant intestinal health issues when gut microflora populations are disrupted. As the C. difficile peptidoglycan (PG) cell wall has an unusually high abundance of 3-3 cross-links, the enzymes synthesizing these structures, L,D-transpeptidases (LDTs), are potential drug targets. While three LDTs were previously known, deleting these genes caused minimal disruption to the PG structure. Here, we report the identification of a family of LDTs that are distinct from previously known LDTs. We show that depleting C. difficile LDTs leads to loss of 3-3 cross-links and cell death. Our findings establish a new class of bacterial PG cross-linking enzymes and suggest that LDTs might be exploited as selective antibiotic targets for C. difficile.