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Differential requirements for conserved peptidoglycan remodeling enzymes during Clostridioides difficile spore formation.

ABSTRACT: Spore formation is essential for the bacterial pathogen and obligate anaerobe, Clostridioides (Clostridium) difficile, to transmit disease. Completion of this process depends on the mother cell engulfing the developing forespore, but little is known about how engulfment occurs in C. difficile. In Bacillus subtilis, engulfment is mediated by a peptidoglycan degradation complex consisting of SpoIID, SpoIIP and SpoIIM, which are all individually required for spore formation. Using genetic analyses, we determined the functions of these engulfment-related proteins along with the putative endopeptidase, SpoIIQ, during C. difficile sporulation. While SpoIID, SpoIIP and SpoIIQ were critical for engulfment, loss of SpoIIM minimally impacted C. difficile spore formation. Interestingly, a small percentage of ?spoIID and ?spoIIQ cells generated heat-resistant spores through the actions of SpoIIQ and SpoIID, respectively. Loss of SpoIID and SpoIIQ also led to unique morphological phenotypes: asymmetric engulfment and forespore distortions, respectively. Catalytic mutant complementation analyses revealed that these phenotypes depend on the enzymatic activities of SpoIIP and SpoIID, respectively. Lastly, engulfment mutants mislocalized polymerized coat even though the basement layer coat proteins, SpoIVA and SipL, remained associated with the forespore. Collectively, these findings advance our understanding of several stages during infectious C. difficile spore assembly.

SUBMITTER: Ribis JW

PROVIDER: S-EPMC6311989 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Differential requirements for conserved peptidoglycan remodeling enzymes during Clostridioides difficile spore formation.

Ribis John W JW Fimlaid Kelly A KA Shen Aimee A

Molecular microbiology 20181101 3

Spore formation is essential for the bacterial pathogen and obligate anaerobe, Clostridioides (Clostridium) difficile, to transmit disease. Completion of this process depends on the mother cell engulfing the developing forespore, but little is known about how engulfment occurs in C. difficile. In Bacillus subtilis, engulfment is mediated by a peptidoglycan degradation complex consisting of SpoIID, SpoIIP and SpoIIM, which are all individually required for spore formation. Using genetic analyses, ...[more]

PMID: 30066347

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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.

Project description:The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health care-associated infections in the United States. In order for this obligate anaerobe to transmit infection, it must form metabolically dormant spores prior to exiting the host. A key step during this process is the assembly of a protective, multilayered proteinaceous coat around the spore. Coat assembly depends on coat morphogenetic proteins recruiting distinct subsets of coat proteins to the developing spore. While 10 coat morphogenetic proteins have been identified in Bacillus subtilis, only two of these morphogenetic proteins have homologs in the Clostridia: SpoIVA and SpoVM. C. difficile SpoIVA is critical for proper coat assembly and functional spore formation, but the requirement for SpoVM during this process was unknown. Here, we show that SpoVM is largely dispensable for C. difficile spore formation, in contrast with B. subtilis. Loss of C. difficile SpoVM resulted in modest decreases (~3-fold) in heat- and chloroform-resistant spore formation, while morphological defects such as coat detachment from the forespore and abnormal cortex thickness were observed in ~30% of spoVM mutant cells. Biochemical analyses revealed that C. difficile SpoIVA and SpoVM directly interact, similarly to their B. subtilis counterparts. However, in contrast with B. subtilis, C. difficile SpoVM was not essential for SpoIVA to encase the forespore. Since C. difficile coat morphogenesis requires SpoIVA-interacting protein L (SipL), which is conserved exclusively in the Clostridia, but not the more broadly conserved SpoVM, our results reveal another key difference between C. difficile and B. subtilis spore assembly pathways. IMPORTANCE The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and transform into vegetative, toxin-secreting cells. During infection, C. difficile must also induce spore formation to survive exit from the host. Since spore formation is essential for transmission, understanding the basic mechanisms underlying sporulation in C. difficile could inform the development of therapeutic strategies targeting spores. In this study, we determine the requirement of the C. difficile homolog of SpoVM, a protein that is essential for spore formation in Bacillus subtilis due to its regulation of coat and cortex formation. We observed that SpoVM plays a minor role in C. difficile spore formation, in contrast with B. subtilis, indicating that this protein would not be a good target for inhibiting spore formation.

Project description:Clostridioides (Clostridium) difficile is a major cause of hospital-acquired infections leading to antibiotic-associated diarrhea. C. difficile exhibits a very high level of resistance to lysozyme. Bacteria commonly resist lysozyme through modification of the cell wall. In C. difficile, σV is required for lysozyme resistance, and σV is activated in response to lysozyme. Once activated, σV, encoded by csfV, directs transcription of genes necessary for lysozyme resistance. Here, we analyze the contribution of individual genes in the σV regulon to lysozyme resistance. Using CRISPR-Cas9-mediated mutagenesis we constructed in-frame deletions of single genes in the csfV operon. We find that pdaV, which encodes a peptidoglycan deacetylase, is partially responsible for lysozyme resistance. We then performed CRISPR inhibition (CRISPRi) to identify a second peptidoglycan deacetylase, encoded by pgdA, that is important for lysozyme resistance. Deletion of either pgdA or pdaV resulted in modest decreases in lysozyme resistance. However, deletion of both pgdA and pdaV resulted in a 1,000-fold decrease in lysozyme resistance. Further, muropeptide analysis revealed that loss of either PgdA or PdaV had modest effects on peptidoglycan deacetylation but that loss of both PgdA and PdaV resulted in almost complete loss of peptidoglycan deacetylation. This suggests that PgdA and PdaV are redundant peptidoglycan deacetylases. We also used CRISPRi to compare other lysozyme resistance mechanisms and conclude that peptidoglycan deacetylation is the major mechanism of lysozyme resistance in C. difficile IMPORTANCE Clostridioides difficile is the leading cause of hospital-acquired diarrhea. C. difficile is highly resistant to lysozyme. We previously showed that the csfV operon is required for lysozyme resistance. Here, we used CRISPR-Cas9 mediated mutagenesis and CRISPRi knockdown to show that peptidoglycan deacetylation is necessary for lysozyme resistance and is the major lysozyme resistance mechanism in C. difficile We show that two peptidoglycan deacetylases in C. difficile are partially redundant and are required for lysozyme resistance. PgdA provides an intrinsic level of deacetylation, and PdaV, encoded by a part of the csfV operon, provides lysozyme-induced peptidoglycan deacetylation.

Dataset Information

Differential requirements for conserved peptidoglycan remodeling enzymes during Clostridioides difficile spore formation.

Publications

Differential requirements for conserved peptidoglycan remodeling enzymes during Clostridioides difficile spore formation.

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