Peptidoglycan degradation machinery in <i>Clostridium difficile</i> forespore engulfment

Bacteria of the Firmicutes phylum are able to enter a developmental pathway that culminates with the formation of highly resistant, dormant endospores. Endospores allow environmental persistence, dissemination and for pathogens, are also infection vehicles. In both the model Bacillus subtilis, an aerobic organism, and in the intestinal pathogen Clostridioides difficile, an obligate anaerobe, sporulation mobilizes hundreds of genes. Their expression is coordinated between the forespore and the mother cell, the two cells that participate in the process, and is kept in close register with the course of morphogenesis. The evolutionary mechanisms by which sporulation emerged and evolved in these two species, and more broadly across Firmicutes, remain largely unknown. Here, we trace the origin and evolution of sporulation using the genes known to be involved in the process in B. subtilis and C. difficile, and estimating their gain-loss dynamics in a comprehensive bacterial macroevolutionary framework. We show that sporulation evolution was driven by two major gene gain events, the first at the base of the Firmicutes and the second at the base of the B. subtilis group and within the Peptostreptococcaceae family, which includes C. difficile. We also show that early and late sporulation regulons have been coevolving and that sporulation genes entail greater innovation in B. subtilis with many Bacilli lineage-restricted genes. In contrast, C. difficile more often recruits new sporulation genes by horizontal gene transfer, which reflects both its highly mobile genome, the complexity of the gut microbiota, and an adjustment of sporulation to the gut ecosystem.

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“…2016). These genes are conserved in C. difficile , although spoIIM is largely dispensable for engulfment (Dembek et al. 2018; Ribis et al.…”

Section: Resultsmentioning

confidence: 99%

From Root to Tips: Sporulation Evolution and Specialization inBacillus subtilisand the Intestinal PathogenClostridioides difficile

Ramos-Silva

Serrano

Henriques

2019

Molecular Biology and Evolution

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“…While most of these B. subtilis morphogenetic factors are conserved across spore-formers (14,15), analyses of spore assembly in C. difficile indicate that many of these factors have different functions or requirements in C. difficile. The conserved transmembrane protein, SpoIIM, is essential for engulfment in B. subtilis (16) but dispensable for this process in C. difficile (17,18), while the lytic transglycosylase, SpoIID, is essential for engulfment in B. subtilis (19) but only partially required in C. difficile (17,18).…”

Section: Introductionmentioning

confidence: 99%

Role of SpoIVA ATPase Motifs DuringClostridioides difficileSporulation

Puebla

Giacalone

Cooper³

et al. 2020

Preprint

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AbstractThe nosocomial pathogen, Clostridioides difficile, is a spore-forming obligate anaerobe that depends on its aerotolerant spore form to transmit infections. Functional spore formation depends on the assembly of a proteinaceous layer known as the coat around the developing spore. In C. difficile, coat assembly depends on the conserved coat protein, SpoIVA, and the clostridial-specific coat protein, SipL, which directly interact. Mutations that disrupt their interaction cause coat to mislocalize and decrease functional spore formation. In B. subtilis, SpoIVA is an ATPase that uses ATP hydrolysis to help drive its polymerization around the forespore. Loss of SpoIVA ATPase activity impairs B. subtilis SpoIVA encasement of the forespore and activates a quality control mechanism that eliminates these defective cells. Since this mechanism is lacking in C. difficile, we tested whether mutations in C. difficile’s SpoIVA ATPase motifs impair functional spore formation. Disrupting C. difficile SpoIVA ATPase motifs resulted in phenotypes that were typically >104 less severe than the equivalent mutations in B. subtilis. Interestingly, mutation of ATPase motif residues predicted to abrogate SpoIVA binding to ATP decreased SpoIVA-SipL interaction, whereas mutation of ATPase motif residues predicted to disrupt ATP hydrolysis but retain binding to ATP enhanced SpoIVA-SipL interaction. When a sipL mutation known to reduce binding to SpoIVA was combined with a spoIVA mutation predicted to prevent SpoIVA binding to ATP, spore formation was severely exacerbated. Since this phenotype is allele-specific, our data implies that SipL recognizes the ATP-bound form of SpoIVA and highlights the importance of this interaction for functional C. difficile spore formation.ImportanceThe aerotolerant spores formed by the major nosocomial pathogen Clostridioides difficile are its primary infectious particle. However, the mechanism by which this critical cell type is assembled remains poorly characterized, especially with respect to its protective coat layer. We previously showed that binding between the spore morphogenetic proteins, SpoIVA and SipL, regulates coat assembly around the forespore. SpoIVA is widely conserved among spore-forming bacteria, and its ATPase activity is essential for Bacillus subtilis to form functional spores. In this study, we determined that mutations in C. difficile SpoIVA’s ATPase motifs result in relatively minor defects in spore formation in contrast with B. subtilis. Nevertheless, our data suggest that SipL preferentially recognizes the ATP-bound form of SpoIVA and identify a specific residue in SipL’s C-terminal LysM domain that is critical for recognizing the ATP-bound form of SpoIVA. These findings advance our understanding of how SpoIVA-SipL interactions regulate C. difficile spore assembly.

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“…While most of these B. subtilis morphogenetic factors are conserved across spore formers ( 14 , 15 ), analyses of spore assembly in C. difficile indicate that many of these factors have different functions or requirements in C. difficile . The conserved transmembrane protein SpoIIM is essential for engulfment in B. subtilis ( 16 ) but dispensable for this process in C. difficile ( 17 , 18 ), while the lytic transglycosylase SpoIID is essential for engulfment in B. subtilis ( 19 ) but only partially required in C. difficile ( 17 , 18 ). Although components of a channel complex are required to maintain the integrity of the forespore in both B. subtilis and C. difficile ( 20 – 22 ), this complex is required for engulfment in C. difficile ( 21 , 22 ) and yet dispensable under some conditions in B. subtilis ( 23 ).…”

Section: Introductionmentioning

confidence: 99%

Role of SpoIVA ATPase Motifs during Clostridioides difficile Sporulation

et al. 2020

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The nosocomial pathogen, Clostridioides difficile, is a spore-forming obligate anaerobe that depends on its aerotolerant spore form to transmit infections. Functional spore formation depends on the assembly of a proteinaceous layer known as the coat around the developing spore. In C. difficile, coat assembly depends on the conserved spore protein, SpoIVA, and the clostridial-specific spore protein, SipL, which directly interact. Mutations that disrupt their interaction cause coat to mislocalize and impair spore formation. In B. subtilis, SpoIVA is an ATPase that uses ATP hydrolysis to drive its polymerization around the forespore. Loss of SpoIVA ATPase activity impairs B. subtilis SpoIVA encasement of the forespore and activates a quality control mechanism that eliminates these defective cells. Since this mechanism is lacking in C. difficile, we tested whether mutations in C. difficile's SpoIVA ATPase motifs impact functional spore formation. Disrupting C. difficile SpoIVA ATPase motifs resulted in phenotypes that were typically >104 less severe than the equivalent mutations in B. subtilis. Interestingly, mutation of ATPase motif residues predicted to abrogate SpoIVA binding to ATP decreased SpoIVA-SipL interaction, whereas mutation of ATPase motif residues predicted to disrupt ATP hydrolysis but maintain ATP binding enhanced SpoIVA-SipL interaction. When a sipL mutation known to reduce binding to SpoIVA was combined with a spoIVA mutation predicted to prevent SpoIVA binding to ATP, spore formation was severely exacerbated. Since this phenotype is allele-specific, our data implies that SipL recognizes the ATP-bound form of SpoIVA and highlights the importance of this interaction for functional C. difficile spore formation. Importance The major pathogen Clostridioides difficile depends on its spore form to transmit disease. However, the mechanism by which C. difficile assembles spores remains poorly characterized. We previously showed that binding between the spore morphogenetic proteins, SpoIVA and SipL, regulates assembly of the protective coat layer around the forespore. In this study, we determined that mutations in C. difficile SpoIVA's ATPase motifs result in relatively minor defects in spore formation in contrast with Bacillus subtilis. Nevertheless, our data suggest that SipL preferentially recognizes the ATP-bound form of SpoIVA and identify a specific residue in SipL's C-terminal LysM domain that is critical for recognizing the ATP-bound form of SpoIVA. These findings advance our understanding of how SpoIVA-SipL interactions regulate C. difficile spore assembly.

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Peptidoglycan degradation machinery in Clostridium difficile forespore engulfment

Cited by 21 publications

References 54 publications

From Root to Tips: Sporulation Evolution and Specialization inBacillus subtilisand the Intestinal PathogenClostridioides difficile

From Root to Tips: Sporulation Evolution and Specialization inBacillus subtilisand the Intestinal PathogenClostridioides difficile

Role of SpoIVA ATPase Motifs DuringClostridioides difficileSporulation

Role of SpoIVA ATPase Motifs during Clostridioides difficile Sporulation

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