A cortex-specific penicillin-binding protein contributes to heat resistance in <i>Clostridioides difficile</i> spores

AbstractBackgroundSporulation 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. <i>Clostridioides difficile</i> 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 <i>C. difficile</i>.MethodsBioinformatic analysis of the <i>C. difficile</i> genome to identify putative homologues of <i>Bacillus subtilis</i><i> </i><i>spoVD</i> was combined with directed mutagenesis and microscopy to identify and characterise cortex-specific PBP activity.ResultsDeletion of CDR20291_2544 (SpoVD_Cd) abrogated spore formation and this phenotype was completely restored by complementation <i>in cis</i>. Analysis of SpoVD_Cd revealed a three domain structure, consisting of dimerization, transpeptidase and PASTA domains, very similar to <i>B. subtilis</i> 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.ConclusionsWe have identified and characterised a cortex specific PBP in <i>C. difficile</i>. This is the first characterisation of a cortex-specific PBP in <i>C. difficile</i> and begins the process of unravelling cortex biogenesis in this important pathogen.<br>