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Carboxypeptidase activity drives L,D-transpeptidase essentiality during vegetative growth and sporulation in Clostridioides difficile
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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
bioRxiv
Cold Spring Harbor Laboratory
07/01/2026
DOI: 10.64898/2026.06.30.735746
PMID: 42427775
url
https://doi.org/10.64898/2026.06.30.735746View
Preprint (Author's original) This preprint has not been evaluated by subject experts through peer review. Preprints may undergo extensive changes and/or become peer-reviewed journal articles. Open Access

Abstract

In most bacteria, peptidoglycan contains mainly 4-3 crosslinks formed by penicillin-binding proteins (PBPs). But 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. Importance Synthesis of peptidoglycan (PG) in the opportunistic gut pathogen Clostridioides difficile depends mostly on 3-3 crosslinks created 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, why C. difficile does not compensate for loss of LDTs by increasing PBP activity, and suggest a model for how PBPs and LDTs work together in this problematic pathogen.

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