A cortex-specific penicillin-binding protein contributes to heat resistance in <i>Clostridioides difficile</i> spores
<h2>Abstract</h2><h3>Background</h3><p dir="ltr"><a href="https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/sporogenesis" target="_blank">Sporulation</a> is a complex <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/cellular-differentiation" target="_blank">cell differentiation</a> programme shared by many members of the <a href="https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/firmicutes" target="_blank">Firmicutes</a>, 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 <a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/bacillus-subtilis" target="_blank">Bacillus subtilis</a> system. The spore envelope consists of multiple protective layers, one of which is a specialised layer of <a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/peptidoglycan" target="_blank">peptidoglycan</a>, called the cortex, that is essential for the resistant properties of the spore. We set out to identify the <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/enzyme" target="_blank">enzymes</a> required for synthesis of cortex <a href="https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/peptidoglycan" target="_blank">peptidoglycan</a> in <i>C. difficile</i>.</p><h3>Methods</h3><p dir="ltr">Bioinformatic analysis of the <i>C. difficile</i> genome to identify putative homologues of <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/bacillus-subtilis" target="_blank"><i>Bacillus subtilis</i></a><i> </i><i>spoVD</i> was combined with directed <a href="https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/mutagenesis" target="_blank">mutagenesis</a> and microscopy to identify and characterise cortex-specific <a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/penicillin-binding-protein" target="_blank">PBP</a> activity.</p><h3>Results</h3><p dir="ltr">Deletion of CDR20291_2544 (SpoVD<sub>Cd</sub>) abrogated spore formation and this phenotype was completely restored by complementation <i>in cis</i>. Analysis of SpoVD<sub>Cd</sub> revealed a three domain structure, consisting of <a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/dimerization" target="_blank">dimerization</a>, <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/gamma-glutamyl-transpeptidase" target="_blank">transpeptidase</a> and <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/threonine" target="_blank">PASTA</a> domains, very similar to <i>B. subtilis</i> SpoVD. Complementation with SpoVD<sub>Cd</sub> domain mutants demonstrated that the <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/serine" target="_blank">PASTA</a> domain was dispensable for formation of morphologically normal spores. SpoVD<sub>Cd</sub> was also seen to localise to the developing spore by super-resolution <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/confocal-microscopy" target="_blank">confocal microscopy</a>.</p><h3>Conclusions</h3><p dir="ltr">We have identified and characterised a cortex specific <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/penicillin-binding-protein" target="_blank">PBP</a> in <i>C. difficile</i>. This is the first characterisation of a cortex-specific <a href="https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/binding-protein" target="_blank">PBP</a> in <i>C. difficile</i> and begins the process of unravelling cortex biogenesis in this important pathogen.</p><ul><li><a href="https://www.sciencedirect.com/science/article/pii/S1075996421000500" target="_blank"><br></a></li></ul><p></p>
