Peptidoglycan remodelling during Clostridioides difficile sporulation.

Abstract

Clostridioides difficile is a Gram-positive enteric human pathogen capable of colonising the gut, and causing C. difficile infection (CDI), which can be life threatening. With a shifting CDI demographic, increasing virulence and high re-infection rates, an increased understanding of basic C. difficile physiology is required to identify new intervention targets. As an anaerobic pathogen, C. difficile must be transmitted through the aerobic environment as specially adapted cells called endospores. The process of transitioning from vegetative cells, that are active in the gut, to the metabolically inactive endospores is known as sporulation and requires considerable remodelling of the mother cell. Firstly, the mother cell produces an asymmetric septum, designating a portion of its cytoplasm as the forespore, which will eventually become the mature endospore. The mother cell then engulfs the forespore to produce a cell-within-a-cell structure. The forespore matures before lysis of the mother cell releases the endospore into the environment. The sporulation process involves considerable changes in all cell components, including remodelling of the cell wall peptidoglycan. This work focuses on the remodelling of peptidoglycan throughout sporulation, with a particular focus on the engulfment mechanisms. C. difficile peptidoglycan was characterised at various stages of engulfment. Surprisingly, the muropeptides identified in vegetative cells were detected throughout early stages of engulfment. Importantly, the proportions of each muropeptide differ throughout the process, indicating varying organisation of the peptidoglycan even at this early stage of sporulation. In parallel, this work also aimed to identify and characterise peptidoglycan modifying enzymes involved in sporulation. SpoIID and SpoIIP are peptidoglycan hydrolases that have been implicated in Bacillus subtilis sporulation. These proteins were purified, characterised and their activities tested on various peptidoglycan types. SpoIIP is a bifunctional amidase and endopeptidase that produces the substrates for the lytic transglycosylase, SpoIID. SpoIID activity was shown to be impacted by zinc binding, and the catalytic residue was identified. C. difficile peptidoglycan is unusual in that it is predominantly deacetylated. In order to further probe peptidoglycan remodelling during sporulation, polysaccharide deacetylase genes in C. difficile were investigated. Ten putative genes were identified and 2 were cloned and recombinantly expressed. Both putative deacetylases were characterised and were shown to be active on acetylated peptidoglycan, implicating them in peptidoglycan remodelling. This work furthers current understanding of C. difficile peptidoglycan biology during sporulation, both in providing a detailed analysis of cell wall composition, and characterising key engulfasome enzymes. It further opens new research avenues by identifying potential peptidoglycan deacetylases in C. difficile. Together, this provides a deeper understanding of key mechanisms involved in the production of the infective agents in CDI that could be explored as novel therapeutic targets.