Abstract
BackgroundSporulation 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. MethodsBioinformatic 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. ResultsDeletion of CDR20291_2544 (SpoVDCd) abrogated spore formation and this phenotype was completely restored by complementation in cis. Analysis of SpoVDCd revealed a three domain structure, consisting of dimerization, transpeptidase and PASTA domains, very similar to B. subtilis SpoVD. Complementation with SpoVDCd domain mutants demonstrated that the PASTA domain was dispensable for formation of morphologically normal spores. SpoVDCd was also seen to localise to the developing spore by super-resolution confocal microscopy. ConclusionsWe 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.
Highlights
C. difficile is the most common cause of nosocomial antibioticassociated diarrhoea, with an estimated 453,000 infections and 29,300 deaths per year in the USA alone [1]
C. difficile produces a SpoVD homologue that is required for sporulation
Five of the penicillinbinding protein (PBP) were required for formation of heat-resistant spores, including two with homology to the B. subtilis cortex specific PBP SpoVD, CDR20291_1067 and 2544
Summary
C. difficile is the most common cause of nosocomial antibioticassociated diarrhoea, with an estimated 453,000 infections and 29,300 deaths per year in the USA alone [1]. Transposon insertions in 798 genes were found to significantly impact sporulation, many with no clear homology to previously characterised proteins. Very few of these 798 genes have been studied in C. difficile but many have homologues in the well-studied Bacillus subtilis sporulation pathway. 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. 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.
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