Abstract
Summary Clostridium difficile remains the leading cause of antibiotic‐associated diarrhoea in hospitals worldwide, linked to significant morbidity and mortality. As a strict anaerobe, it produces dormant cell forms – spores – which allow it to survive in the aerobic environment. Importantly, spores are the transmission agent of C. difficile infections. A key aspect of sporulation is the engulfment of the future spore by the mother cell and several proteins have been proposed to be involved. Here, we investigated the role of the SpoIID, SpoIIM and SpoIIP (DMP) machinery and its interplay with the SpoIIQ:SpoIIIAH (Q:AH) complex in C. difficile. We show that, surprisingly, SpoIIM, the proposed machinery anchor, is not required for efficient engulfment and sporulation. We demonstrate the requirement of DP for engulfment due to their sequential peptidoglycan degradation activity, both in vitro and in vivo. Finally, new interactions within DMP and between DMP and Q:AH suggest that both systems form a single engulfment machinery to keep the mother cell and forespore membranes together throughout engulfment. This work sheds new light upon the engulfment process and on how different sporeformers might use the same components in different ways to drive spore formation.
Highlights
Clostridium difficile, a spore forming Gram-positive strict anaerobe, is a major cause of human morbidity and mortality in hospitals and the main cause of hospital-acquired diarrhoea
We investigated the role of the SpoIID, SpoIIM and SpoIIP (DMP) machinery and its interplay with the SpoIIQ:SpoIIIAH (Q:AH) complex in C. difficile
We set out to investigate the role of spoIIDMP by creating null mutants in all three components of the putative DMP complex in C. difficile 630Δerm, an erythromycin-sensitive derivative of the sequenced clinical isolate 630 (Hussain et al, 2005) using AlleleCoupled Exchange (ACE) (Heap et al, 2012; Ng et al, 2013)
Summary
Clostridium difficile, a spore forming Gram-positive strict anaerobe, is a major cause of human morbidity and mortality in hospitals and the main cause of hospital-acquired diarrhoea. CDI results from gut dysbiosis, typically caused by antibiotic therapy disrupting the normal microbiota. Current therapy for acute disease involves the use of one of three antibiotics: vancomycin, metronidazole or fidaxomycin (Silva et al, 1981; Teasley et al, 1983; Louie et al, 2011) that further promote dysbiosis, leaving patients acutely sensitive to reinfection or disease relapse. The recent emergence of more virulent strains with greater antibiotic resistance, more serious disease symptoms, progression and higher relapse rates (Hunt and Ballard, 2013), highlights the urgent need to develop more targeted therapeutic approaches that have minimal impact on the normal gut microbiota. One of the main issues in combating CDI and developing targeted therapeutics is the still limited understanding of C. difficile pathogenicity, at the molecular level. Clinical symptoms are largely attributed to the much-studied toxins; other aspects of the unique C. difficile pathobiology remain understudied
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