Novel insights into the molecular mechanisms of Clostridioides difficile spore engulfment

Abstract

Clostridioides difficile is an antibiotic resistant bacterial human pathogen that colonises the large intestine in the absence of a healthy competing gut microbiota, generally following treatment with broad spectrum antibiotics. Current treatments for C. difficile infection (CDI), the most common cause of healthcare-associated diarrhoea, entail the use of antibiotics that perpetuate gut dysbiosis and enable disease recurrence. The shifting disease profile of CDI and emergence of hypervirulent strains highlight an urgent requirement to further understand the fundamental biology of this pathogen so novel therapies can be sought. As an obligate anaerobe, C. difficile undergoes sporulation, forming robust, metabolically dormant endospores that enable survival and transmission in the aerobic environment. Sporulation begins with an asymmetrical cell division and entails substantial peptidoglycan remodelling as the smaller forespore is engulfed by the larger mother cell, prior to spore maturation. Despite the crucial role sporulation plays in the transmission and persistence of CDI, the process remains poorly understood and unexploited as a therapeutic target. In this work, key enzymes involved in the peptidoglycan remodelling process during engulfment were explored and further characterised. During engulfment, hydrolytic enzymes remodel peptidoglycan to alleviate its steric hinderance, enabling the mother cell membrane to advance without compromising cell integrity. In C. difficile, the sequential activities of the dual amidase and endopeptidase SpoIIP and the lytic transglycosylase SpoIID underpin this process. Prior work has demonstrated that SpoIIP is present in multiple truncated forms in vivo, however the reason for this remained unclear. In this work, the truncated variants of SpoIIP were generated and biophysically characterised, and their enzymatic activity tested using peptidoglycan digestion assays. The data show that cleavage of SpoIIP, recently found to be mediated by the SpoIVB2 protease, generates an inactive isoform, potentially serving as a mechanism of inactivating SpoIIP post-engulfment when its hydrolytic activity is no longer needed. Alongside this, the substrate specificities of SpoIIP and SpoIID were further explored. Mature vegetative C. difficile peptidoglycan is predominantly N-deacetylated, while newly synthesised C. difficile peptidoglycan is N-acetylated. N-deacetylase enzymes are proposed to be responsible for this transition in N-acetylation state. In this work, peptidoglycan digestion assays confirmed that SpoIIP and SpoIID preferentially digest N-acetylated peptidoglycan. Furthermore, fluorescence microscopy experiments revealed that deletion of the sporulation-associated N-deacetylase CD1319 impairs spore maturation. Taken together, these data suggest that the preference of SpoIID and SpoIIP for an N-acetylated substrate allows peptidoglycan remodelling to proceed during engulfment without the risk of inadvertently digesting N-deacetylated mother cell peptidoglycan and prematurely initiating cell lysis. Moreover, N-deacetylases such as CD1319 may ensure directionality of SpoIIP and SpoIID activity, preventing aberrant peptidoglycan digestion which could compromise the integrity of the mature spore. The asynchronous nature of C. difficile sporulation is a long-standing issue which results in heterogeneous cultures of cells at different morphological stages, limiting analysis of peptidoglycan composition at a given sporulation stage. This work sought to address this problem for the first time by exploring flow cytometry sample enrichment using transcriptional SNAP-tag fusions reporters in sporulation-stalled strains. The technique allowed for the separation of cells that had stalled at specific sporulation stages from vegetative and intermediate populations, paving the way to analyse more homogeneous sporulation samples in the future. Overall, this work deepens the understanding of the molecular details that underpin C. difficile sporulation and proposes a promising new tool to research this pathogen. These findings also contribute to the long-term goal of developing of much-needed novel therapeutic avenues that interrupt spore formation, thereby eliminating the CDI transmission route.