Role of SpoIVA ATPase Motifs During<i>Clostridioides difficile</i>Sporulation

Puebla, Benitor de la; Giacalone,; Cooper, Andrea M.; Shen,

doi:10.1101/2020.07.02.183343

Cited by 3 publications

(11 citation statements)

References 61 publications

(128 reference statements)

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“…Given that the growth data suggested a role for ClpPs in C. difficile transition to stationary phase and, potentially, sporulation, we wanted to determine the essentiality of ClpP1 and ClpP2 in C. difficile sporulation. To do this, we analyzed the apparent sporulation behavior of clpP mutants using phase-contrast microscopy and assessed the heat stability of spores produced by each mutant compared to WT using a Heat Resistance Assay (HRA) described previously (Putnam, Nock et al 2013, Fimlaid, Jensen et al 2015, Fimlaid, JEnsen et al 2015, Edwards and McBride 2016, de la Puebla, Giacalone et al 2020). Sporulation was visualized by phase contrast microscopy after ~22 h growth on 70:30 solid medium for all data presented in Figure 3 .…”

Section: Resultsmentioning

confidence: 99%

Loss of ClpP Function inClostridioides difficile630 Significantly Impacts Sporulation Systems

Shadid

Lavey

et al. 2021

Preprint

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The Gram-positive bacterium Clostridioides difficile is a primary cause of hospital-acquired diarrhea, threatening both immunocompromised and healthy individuals. An important aspect of elucidating mechanisms that drive C. difficile persistence and virulence relies on developing a more complete understanding of sporulation. C. difficile sporulation is the single determinant of transmission and complicates treatment and prevention due to the chemical and physical resilience of spores. Hence, the identification of potentially druggable targets that significantly attenuate sporulation is important. In this report, we describe the impact of the loss of caseinolytic protease P (ClpP) isoforms in C. difficile strain 630 on sporulation phenotypes. Using CRISPR-Cas9 nickase mediated genome editing, stop codons were inserted early in the coding sequence for clpP1 and clpP2 to generate C. difficile mutants that no longer produced ClpP1 or ClpP2. The data show that these genetic modifications lead to altered sporulation phenotypes, germination efficiencies, and cytotoxicity. Comparative proteome profiling of C. difficile 630 WT and clpP mutants reveals potential proteolytic targets of ClpP that are involved in sporulation. These analyses further reveal the potential for preferred co-chaperone interactions for each ClpP isoform. Taken together, our results demonstrate that ClpP, a promising target in other Gram-positive pathogens, holds promise as an anti-sporulation target in C. difficile.

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Section: Resultsmentioning

confidence: 99%

Loss of ClpP Function inClostridioides difficile630 Significantly Impacts Sporulation Systems

Shadid

Lavey

et al. 2021

Preprint

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“…Both SpoIVA and SipL are landmark proteins for coat morphogenesis. Loss of either of these proteins results in polymerized coat mislocalizing to the cytosol or failing to fully encase the forespore (20,23,25). Our previous work suggests that SpoIVA and SipL are recruited as a complex to the forespore (25) and that binding between these two proteins facilitates their encasement of the forespore (23).…”

Section: Identification Of Sipl-binding Proteins Using Co-immunoprecipitationmentioning

confidence: 97%

“…subtilis SpoVM and SpoIVA are both essential for cortex formation because defects in their localization around the forespore triggers a quality control pathway that leads to mother cell lysis (17). While this quality control pathway is absent in the Clostridia (22), SpoVM and SpoIVA nevertheless impact cortex synthesis in C. difficile because mutants lacking either of these proteins generate forespores with cortex abnormalities (18,23).…”

Section: Introductionmentioning

confidence: 99%

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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“…Experimental evidence shows that while both of these proteins are essential for coat assembly in Bacillus , in Clostridia the vital role of SpoVM is diminished and instead carried out by the functionally homologous SipL ( Stevens et al, 1992 ; Levin et al, 1993 ; Ribis et al, 2017 ). Intriguingly, deficiencies in these coat platform proteins also inhibit formation of the cortex ( Ebmeier et al, 2012 ; Benito de la Puebla et al, 2020 ). Additional spore coat morphogenesis proteins of Bacilli include SafA and CotE, which are essential for formation of the inner and outer coat layers, respectively ( Zheng et al, 1988 ; Takamatsu et al, 1999 ; Abhyankar et al, 2013 ; Stelder et al, 2018 ).…”

Section: Spore Maturationmentioning

confidence: 99%

“…In general, all sporulation-associated machinery, including those involved in cell division, DNA transport and compaction, cortex formation, the AA-AH complex, DPA synthesis, spore coat formation and germination, is conserved between all endospore formers ( Galperin et al, 2012 ; Abecasis et al, 2013 ; Ramos-Silva et al, 2019 ). However, minor differences are observed with occurrence of supplementary pathways (such as DPA synthesis by EtfA, and Csp-mediated triggering of germination in the Clostridia-like spore formers; Orsburn et al, 2010 ; Rohlfing et al, 2019 ), replacement of individual components by their functional homologues (assembly of the spore coat base via SpoVM in Bacillus and SipL in Clostridium ; Ribis et al, 2017 ), losses of intermediate components (modification of Spo0A via a phosphorelay system in Bacilli and a two-component system in Clostridia), and slight discrepancies in co-regulation and co-dependence of cellular processes (e.g., influence of deficiencies in spore coat assembly on cortex formation; Ebmeier et al, 2012 ; Benito de la Puebla et al, 2020 ).…”

Section: Genetic Differences and Evolutionmentioning

confidence: 99%

Structural, Metabolic and Evolutionary Comparison of Bacterial Endospore and Exospore Formation

et al. 2021

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Sporulation is a specialized developmental program employed by a diverse set of bacteria which culminates in the formation of dormant cells displaying increased resilience to stressors. This represents a major survival strategy for bacteria facing harsh environmental conditions, including nutrient limitation, heat, desiccation, and exposure to antimicrobial compounds. Through dispersal to new environments via biotic or abiotic factors, sporulation provides a means for disseminating genetic material and promotes encounters with preferable environments thus promoting environmental selection. Several types of bacterial sporulation have been characterized, each involving numerous morphological changes regulated and performed by non-homologous pathways. Despite their likely independent evolutionary origins, all known modes of sporulation are typically triggered by limited nutrients and require extensive membrane and peptidoglycan remodeling. While distinct modes of sporulation have been observed in diverse species, two major types are at the forefront of understanding the role of sporulation in human health, and microbial population dynamics and survival. Here, we outline endospore and exospore formation by members of the phyla Firmicutes and Actinobacteria, respectively. Using recent advances in molecular and structural biology, we point to the regulatory, genetic, and morphological differences unique to endo- and exospore formation, discuss shared characteristics that contribute to the enhanced environmental survival of spores and, finally, cover the evolutionary aspects of sporulation that contribute to bacterial species diversification.

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Role of SpoIVA ATPase Motifs DuringClostridioides difficileSporulation

Cited by 3 publications

References 61 publications

Loss of ClpP Function inClostridioides difficile630 Significantly Impacts Sporulation Systems

Loss of ClpP Function inClostridioides difficile630 Significantly Impacts Sporulation Systems

Identification of a novel regulator ofClostridioides difficilecortex formation

Structural, Metabolic and Evolutionary Comparison of Bacterial Endospore and Exospore Formation

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