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Characterization of an Endolysin Targeting Clostridioides difficile That Affects Spore Outgrowth.

ABSTRACT: Clostridioides difficile is a spore-forming enteric pathogen causing life-threatening diarrhoea and colitis. Microbial disruption caused by antibiotics has been linked with susceptibility to, and transmission and relapse of, C. difficile infection. Therefore, there is an urgent need for novel therapeutics that are effective in preventing C. difficile growth, spore germination, and outgrowth. In recent years bacteriophage-derived endolysins and their derivatives show promise as a novel class of antibacterial agents. In this study, we recombinantly expressed and characterized a cell wall hydrolase (CWH) lysin from C. difficile phage, phiMMP01. The full-length CWH displayed lytic activity against selected C. difficile strains. However, removing the N-terminal cell wall binding domain, creating CWH351-656, resulted in increased and/or an expanded lytic spectrum of activity. C. difficile specificity was retained versus commensal clostridia and other bacterial species. As expected, the putative cell wall binding domain, CWH1-350, was completely inactive. We also observe the effect of CWH351-656 on preventing C. difficile spore outgrowth. Our results suggest that CWH351-656 has therapeutic potential as an antimicrobial agent against C. difficile infection.

SUBMITTER: Mondal SI

PROVIDER: S-EPMC8199566 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Characterization of an Endolysin Targeting Clostridioides difficile That Affects Spore Outgrowth.

Mondal Shakhinur Islam SI Akter Arzuba A Draper Lorraine A LA Ross R Paul RP Hill Colin C

International journal of molecular sciences 20210526 11

Clostridioides difficile is a spore-forming enteric pathogen causing life-threatening diarrhoea and colitis. Microbial disruption caused by antibiotics has been linked with susceptibility to, and transmission and relapse of, C. difficile infection. Therefore, there is an urgent need for novel therapeutics that are effective in preventing C. difficile growth, spore germination, and outgrowth. In recent years bacteriophage-derived endolysins and their derivatives show promise ...[more]

PMID: 34073633

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Project description:Spores are the major infectious particle of the Gram-positive nosocomial pathogen Clostridioides difficile (formerly Clostridium difficile), but the molecular details of how this organism forms these metabolically dormant cells remain poorly characterized. The composition of the spore coat in C. difficile differs markedly from that defined in the well-studied organism Bacillus subtilis, with only 25% of the ∼70 spore coat proteins being conserved between the two organisms and with only 2 of 9 coat assembly (morphogenetic) proteins defined in B. subtilis having homologs in C. difficile We previously identified SipL as a clostridium-specific coat protein essential for functional spore formation. Heterologous expression analyses in Escherichia coli revealed that SipL directly interacts with C. difficile SpoIVA, a coat-morphogenetic protein conserved in all spore-forming organisms, through SipL's C-terminal LysM domain. In this study, we show that SpoIVA-SipL binding is essential for C. difficile spore formation and identify specific residues within the LysM domain that stabilize this interaction. Fluorescence microscopy analyses indicate that binding of SipL's LysM domain to SpoIVA is required for SipL to localize to the forespore while SpoIVA requires SipL to promote encasement of SpoIVA around the forespore. Since we also show that clostridial LysM domains are functionally interchangeable at least in C. difficile, the basic mechanism for SipL-dependent assembly of clostridial spore coats may be conserved.IMPORTANCE The metabolically dormant spore form of the major nosocomial pathogen Clostridioides difficile is its major infectious particle. However, the mechanisms controlling the formation of this resistant cell type are not well understood, particularly with respect to its outermost layer, the spore coat. We previously identified two spore-morphogenetic proteins in C. difficile: SpoIVA, which is conserved in all spore-forming organisms, and SipL, which is conserved only in the clostridia. Both SpoIVA and SipL are essential for heat-resistant spore formation and directly interact through SipL's C-terminal LysM domain. In this study, we demonstrate that the LysM domain is critical for SipL and SpoIVA function, likely by helping recruit SipL to the forespore during spore morphogenesis. We further identified residues within the LysM domain that are important for binding SpoIVA and, thus, functional spore formation. These findings provide important insight into the molecular mechanisms controlling the assembly of infectious C. difficile spores.

Dataset Information

Characterization of an Endolysin Targeting Clostridioides difficile That Affects Spore Outgrowth.

Publications

Characterization of an Endolysin Targeting <i>Clostridioides difficile</i> That Affects Spore Outgrowth.

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