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Carboxypeptidase activity drives L,D-transpeptidase essentiality during vegetative growth and sporulation in Clostridioides difficile
Journal article   Open access   Peer reviewed

Carboxypeptidase activity drives L,D-transpeptidase essentiality during vegetative growth and sporulation in Clostridioides difficile

Kevin W. Bollinger, Ute Müh, Paige B. Brannen, David L. Popham, David S. Weiss and Craig D. Ellermeier
mBio, e0158026
10/06/2026
DOI: 10.1128/mbio.01580-26
PMID: 42836616
url
https://doi.org/10.1128/mbio.01580-26View
Published (Version of record) Open Access

Abstract

In most bacteria, peptidoglycan contains mainly 4-3 crosslinks formed by penicillin-binding proteins (PBPs). However, in the opportunistic pathogen Clostridioides difficile, 70% of the crosslinks are 3-3 crosslinks formed by L,D-transpeptidases (LDTs), and LDTs are essential for viability. PBPs and LDTs use different acyl donors for crosslinking; PBPs require a pentapeptide, while LDTs require a tetrapeptide. Here, we determined the source of the tetrapeptides in C. difficile and investigated the consequences of reengineering PG crosslinking from predominantly 3-3 to exclusively 4-3. We found that two D-alanyl-D-alanine carboxypeptidases (DD-CPase), DacA and DacC, supply LDTs with tetrapeptides during vegetative growth. Deleting these enzymes was sufficient to bypass the normal requirement for LDTs. The resulting mutant (ΔdacAC Δldt) was remarkably healthy despite the absence of 3-3 crosslinks. Its only major phenotypic defect was a 3- to 4-log decrease in sporulation, which could, however, be overcome by deleting a third DD-CPase, dacB. These findings fill gaps in our understanding of the pathway for LD-transpeptidation in C. difficile and imply that LDTs are not essential components of the elongasome or divisome, both of which function well in the complete absence of LDTs, provided there is sufficient pentapeptide to sustain crosslinking by PBPs. Thus, LDTs are essential for viability because C. difficile has intrinsically high levels of DD-CPase activity. Finally, we propose a model for how PBPs and LDTs work together during PG synthesis. In this model, PBPs construct a sparsely crosslinked PG sacculus that is subsequently strengthened with crosslinks introduced by LDTs.IMPORTANCESynthesis of peptidoglycan (PG) in the opportunistic gut pathogen Clostridioides difficile depends mostly on 3-3 crosslinks formed by L,D-transpeptidases (LDTs). Here, we converted C. difficile from an organism that relies primarily on 3-3 crosslinks made by LDTs to one that relies exclusively on 4-3 crosslinks made by an alternative family of crosslinking enzymes, the penicillin-binding proteins (PBPs). Our findings explain why 3-3 crosslinking predominates over 4-3 crosslinking, clarify why C. difficile does not compensate for the loss of LDTs by increasing PBP activity, and suggest a model for how PBPs and LDTs work together in this problematic pathogen.
 Cell Wall Biosynthesis Cell Wall Biosynthesis Mutants Cell Wall Remodeling Enzymes Cell Wall Structure Cell Wall Synthesis and Assembly Envelope Structure Microbial Pathogenesis and Immunology Microbial Physiology and Genetics Molecular and Cellular Biology Peptidoglycan Architecture Peptidoglycan Layer Peptidoglycan Synthesis Regulation of Cell Wall Biosynthesis Research Article

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