SpoIVA-SipL complex formation is essential for<i>Clostridioides difficile</i>spore assembly

Touchette, Megan H.; Puebla, Hector Benito de la; Ravichandran, Priyanka; Shen, Aimee

doi:10.1101/522235

Cited by 7 publications

(35 citation statements)

References 51 publications

(82 reference statements)

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“…Phase-bright spores resembling wild-type spores were purified from the ATPase motif mutants carrying alanine mutations, although circular spores and spores with appendages were more frequently observed in the mutant strains relative to wild-type ( Figure S3). The small amount of K35E spores that could be purified were primarily phase-bright sporelets, which are smaller and more swollen than wild type spores (25,40). Taken together, our results suggest that the K35E mutant's heat resistance defect is mainly caused by a defect in spore assembly.…”

Section: Alanine Mutations In Spoiva Atpase Motifs Do Not Strongly Rementioning

confidence: 64%

“…Given that loss of SpoIVA causes SipL-mCherry to redistribute to the cytosol (40), our data suggest that the SpoIVA ATPase motif mutants retain the ability to bind and recruit SipL to the forespore. However, prior co-affinity purification analyses with SpoIVA and SipL in E. coli indicated that the SpoIVA K35E strongly impairs binding to SipL (37).…”

Section: Mutation Of the Spoiva Walker A Motif Decreases Spoiva Bindimentioning

confidence: 78%

“…Coat protein encasement of the forespore is reduced in SpoIVA ATPase motif mutants C. difficile SpoIVA is required to recruit SipL to the forespore (40), so we tested whether the ATPase motif mutations decreased SipL localization using a SipL-mCherry fluorescent protein fusion we previously constructed (40). Since this fusion protein encases the forespore more efficiently when SipL-mCherry is the only form of SipL present in the cell (40), we deleted the sipL gene from the spoIVA mutants encoding ATPase motif mutations in their native loci.…”

Section: Atpase Motif Mutations Decrease Spoiva Encasement Of the Formentioning

confidence: 99%

“…Since localization of B. subtilis SpoVM and SpoIVA to the forespore cooperatively enhances their encasement of the forespore by increasing their local concentrations (33), we considered the possibility that SpoIVA-SipL binding might be stabilized in the context of the forespore. To determine how the SpoIVA ATPase motif mutations affect binding to SipL during sporulation, we immunoprecipitated FLAG-tagged SipL from a previously constructed strain that expresses sipL-3xFLAG from the pyrE locus of ∆ sipL (40). We then measured the amount of untagged SpoIVA ATPase motif mutant variants that were also pulled down.…”

Section: Mutation Of the Spoiva Walker A Motif Decreases Spoiva Bindimentioning

confidence: 99%

“…C. difficile sipL mutants phenocopy C. difficile spoIVA mutants in that they exhibit similar defects in coat localization and heat-resistant spore formation despite making a cortex layer (37). We previously showed that SipL directly binds SpoIVA through SipL's C-terminal LysM domain (37) and that disrupting SipL LysM domain binding to SpoIVA via mutation of specific residues causes defects in coat assembly and heat-resistant spore formation (40).…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Alanine Mutations In Spoiva Atpase Motifs Do Not Strongly Rementioning

confidence: 64%

Section: Mutation Of the Spoiva Walker A Motif Decreases Spoiva Bindimentioning

confidence: 78%

Section: Atpase Motif Mutations Decrease Spoiva Encasement Of the Formentioning

confidence: 99%

Section: Mutation Of the Spoiva Walker A Motif Decreases Spoiva Bindimentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Role of SpoIVA ATPase Motifs DuringClostridioides difficileSporulation

Puebla

Giacalone

Cooper³

et al. 2020

Preprint

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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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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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Identification of a novel regulator ofClostridioides difficilecortex formation

Touchette

Puebla²,

Feliciano³

et al. 2021

Preprint

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Clostridioides difficile is a leading cause of healthcare-associated infections worldwide. C. difficile infections are transmitted by its metabolically dormant, aerotolerant spore form. Functional spore formation depends on the assembly of two protective layers: a thick layer of modified peptidoglycan known as the cortex layer and a multilayered proteinaceous meshwork known as the coat. We previously identified two spore morphogenetic proteins, SpoIVA and SipL, that are essential for recruiting coat proteins to the developing forespore and making functional spores. While SpoIVA and SipL directly interact, the identities of the proteins they recruit to the forespore remained unknown. We used mass spectrometry-based affinity proteomics to identify proteins that interact with the SpoIVA-SipL complex. These analyses identified the Peptostreptococcaceae family-specific, sporulation-induced bitopic membrane protein CD3457 (renamed SpoVQ) as a protein that interacts with SipL and SpoIVA. Loss of SpoVQ decreased heat-resistant spore formation by ~5-fold and reduced cortex thickness ~2-fold; the thinner cortex layer of ∆spoVQ spores correlated with higher levels of spontaneous germination (i.e., in the absence of germinant). Notably, loss of SpoVQ in either spoIVA or sipL mutants prevented cortex synthesis altogether and greatly impaired the localization of a SipL-mCherry fusion protein around the forespore. Thus, SpoVQ is a novel regulator of C. difficile cortex synthesis that appears to link cortex and coat formation. The identification of SpoVQ as a spore morphogenetic protein further highlights how Peptostreptococcaceae family-specific mechanisms control spore formation in C. difficile.

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SpoIVA-SipL complex formation is essential forClostridioides difficilespore assembly

Cited by 7 publications

References 51 publications

Role of SpoIVA ATPase Motifs DuringClostridioides difficileSporulation

Role of SpoIVA ATPase Motifs DuringClostridioides difficileSporulation

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

Identification of a novel regulator ofClostridioides difficilecortex formation

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