A cortex-specific penicillin-binding protein contributes to heat resistance in Clostridioides difficile spores

Abstract

Background Sporulation is a complex cell differentiation programme shared by many members of the Firmicutes, the end result of which is a highly resistant, metabolically inert spore that can survive harsh environmental insults. Clostridioides difficile spores are essential for transmission of disease and are also required for recurrent infection. However, the molecular basis of sporulation is poorly understood, despite parallels with the well-studied Bacillus subtilis system. The spore envelope consists of multiple protective layers, one of which is a specialised layer of peptidoglycan, called the cortex, that is essential for the resistant properties of the spore. We set out to identify the enzymes required for synthesis of cortex peptidoglycan in C. difficile . Methods Bioinformatic analysis of the C. difficile genome to identify putative homologues of Bacillus subtilis spoVD was combined with directed mutagenesis and microscopy to identify and characterise cortex-specific PBP activity. Results Deletion of CDR20291_2544 (SpoVD Cd ) abrogated spore formation and this phenotype was completely restored by complementation in cis . Analysis of SpoVD Cd revealed a three domain structure, consisting of dimerization, transpeptidase and PASTA domains, very similar to B. subtilis SpoVD. Complementation with SpoVD Cd domain mutants demonstrated that the PASTA domain was dispensable for formation of morphologically normal spores. SpoVD Cd was also seen to localise to the developing spore by super-resolution confocal microscopy. Conclusions We have identified and characterised a cortex specific PBP in C. difficile . This is the first characterisation of a cortex-specific PBP in C. difficile and begins the process of unravelling cortex biogenesis in this important pathogen.

Diversification of division mechanisms in endospore-forming bacteria revealed by analyses of peptidoglycan synthesis in Clostridioides difficile

“…2g ). Moreover, in agreement with a previous study 33 , C. difficile SpoVD localizes to polar septa during asymmetric division (Supplementary Fig. 9 ).…”

“…2a, b ), indicating that C. difficile SpoVD and SpoVE are not involved in vegetative cell division. Consistent with prior work 32 , 33 , the C. difficile ∆ spoVD strain failed to make heat-resistant spores (Supplementary Fig. 3 ) or synthesize a cortex layer based on transmission electron microscopy (TEM) analyses (Supplementary Fig.…”

Selective purification and rapid quantitative detection of spores using a “stepped” magnetic flow device

“…Raman spectroscopy can determine the content of different chemical groups in biological tissues using the peak positions and peak intensity values, which meet the above requirements. 31–33…”

“…Raman spectroscopy can determine the content of different chemical groups in biological tissues using the peak positions and peak intensity values, which meet the above requirements. [31][32][33] In this study, the objective was to provide a new method for spore purication during the coexistence of the spores and vegetative cells, which is based on a "stepped" magnetic ow device combined with an antibiotic affinity strategy for directly purifying spores. The "stepped" internal structure increased the contact area and collision probability between the sample and the magnetic site, which could be used for spore purication in complex microbiological samples without blockage.…”

Design, Synthesis, and Evaluation of Cephamycin-Based Antisporulation Agents targeting Clostridioides difficile

“…Cd SpoVD is essential for spore production, as interruption of the CdspoVD gene results in no spores being formed . Furthermore, Cd SpoVD activity is necessary for the production of the spore cortex, where PBPs, like Cd SpoVD, cross-link glycan chains of peptidoglycan . Further research showed that cephamycins (cefotetan, cefoxitin, and cefmetazole) can induce an antispore effect in vitro as well as prevent CDI relapse in in vivo models of disease.…”