Journal article
Identification of a family of peptidoglycan transpeptidases reveals that Clostridioides difficile requires noncanonical cross-links for viability
Proceedings of the National Academy of Sciences - PNAS, Vol.121(34), e2408540121
08/20/2024
DOI: 10.1073/pnas.2408540121
PMCID: PMC11348318
PMID: 39150786
Abstract
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 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. difficile 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-C. difficile 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 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.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 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. difficile 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-C. difficile 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 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.
Details
- Title: Subtitle
- Identification of a family of peptidoglycan transpeptidases reveals that Clostridioides difficile requires noncanonical cross-links for viability
- Creators
- Kevin W Bollinger - University of IowaUte Müh - University of IowaKarl L Ocius - University of VirginiaAlexis J Apostolos - University of VirginiaMarcos M Pires - University of VirginiaRichard F Helm - Virginia TechDavid L Popham - Virginia TechDavid S Weiss - University of IowaCraig D Ellermeier - University of Iowa
- Resource Type
- Journal article
- Publication Details
- Proceedings of the National Academy of Sciences - PNAS, Vol.121(34), e2408540121
- Publisher
- NATL ACAD SCIENCES
- DOI
- 10.1073/pnas.2408540121
- PMID
- 39150786
- PMCID
- PMC11348318
- ISSN
- 1091-6490
- eISSN
- 1091-6490
- Grant note
- Veteran's Administration Medical CenterGenomics Division of the Iowa Institute of Human GeneticsUniversity of Iowa Carver College of Medicine
The following reagent was obtained through the ATCC: Paraclostridium bifermentans ATCC 638. The following reagents were obtained through Biodefense and Emerging Infections Resources, NIAID, NIH as part of the Human Microbiome Project: Eubacterium sp., Strain AS15, HM-766; Ruminococcaceaesp., Strain D16, HM-79; and Lachnospiraceaesp., Strain 5_1_57FAA, HM-157. This work was supported by the NIH (R01AI155492 to D.S.W. and C.D.E., R01GM138630 to D.L.P., and R35GM124893-06 to M.M.P.) The LC-MS work was funded by GlycoMIP, a NSF Materials Innovation Platform funded through Cooperative Agreement DMR-1933525. Cell fluorescence was quantitated atthe Flow Cytometry Facility,a core research facility at the University of Iowa funded through user fees and the generous financial support of the Carver College of Medicine, Holden Comprehensive Cancer Center, and Iowa City Veteran's Administration Medical Center. RNA integrity was characterized by the Genomics Division of the Iowa Institute of Human Genetics, which is supported, in part, by the University of Iowa Carver College of Medicine. We thank Johann Peltier for sharing information prior to publication, Atsushi Yahashiri for sharing B. subtilis PG sacculi and members of the Ellermeier and Weiss laboratories for helpful discussions.
- Language
- English
- Date published
- 08/20/2024
- Academic Unit
- Microbiology and Immunology
- Record Identifier
- 9984697059702771
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