The Spore Coat Protein CotE Facilitates Host Colonization by Clostridium difficile.
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ABSTRACT:
SUBMITTER: Hong HA
PROVIDER: S-EPMC5853579 | biostudies-literature | 2017 Dec
REPOSITORIES: biostudies-literature
Publications
The Spore Coat Protein CotE Facilitates Host Colonization by Clostridium difficile.
Hong Huynh A HA Ferreira William T WT Hosseini Siamand S Anwar Saba S Hitri Krisztina K Wilkinson Anthony J AJ Vahjen Wilfried W Zentek Jürgen J Soloviev Mikhail M Cutting Simon M SM
The Journal of infectious diseases 20171201 11
Clostridium difficile infection (CDI) is an important hospital-acquired infection resulting from the germination of spores in the intestine as a consequence of antibiotic-mediated dysbiosis of the gut microbiota. Key to this is CotE, a protein displayed on the spore surface and carrying 2 functional elements, an N-terminal peroxiredoxin and a C-terminal chitinase domain. Using isogenic mutants, we show in vitro and ex vivo that CotE enables binding of spores to mucus by direct interaction with m ...[more]
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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.
| S-EPMC5607322 | biostudies-literature
Project description:Spores of Clostridium difficile play a key role in the dissemination of this important human pathogen, and until recently little has been known of their functional characteristics. Genes encoding six spore coat proteins (cotA, cotB, cotCB, cotD, cotE, and sodA) were disrupted by ClosTron insertional mutagenesis. Mutation of one gene, cotA, presented a major structural defect in spore assembly, with a clear misassembly of the outermost layers of the spore coat. The CotA protein is most probably subject to posttranslational modification and could play a key role in stabilizing the spore coat. Surprisingly, mutation of the other spore coat genes did not affect the integrity of the spore, although for the cotD, cotE, and sodA mutants, enzyme activity was reduced or abolished. This could imply that these enzymatic proteins are located in the exosporium or alternatively that they are structurally redundant. Of the spore coat proteins predicted to carry enzymatic activity, three were confirmed to be enzymes using both in vivo and in vitro methods, the latter using recombinant expressed proteins. These were a manganese catalase, encoded by cotD, a superoxide dismutase (SOD), encoded by sodA, and a bifunctional enzyme with peroxiredoxin and chitinase activity, encoded by cotE. These enzymes being exposed on the spore surface would play a role in coat polymerization and detoxification of H2O2. Two additional proteins, CotF (a tyrosine-rich protein and potential substrate for SodA) and CotG (a putative manganese catalase) were shown to be located at the spore surface.
| S-EPMC3624542 | biostudies-literature
Project description:Germination of Clostridium difficile spores is a crucial early requirement for colonization of the gastrointestinal tract. Likewise, C. difficile cannot cause disease pathologies unless its spores germinate into metabolically active, toxin-producing cells. Recent advances in our understanding of C. difficile spore germination mechanisms indicate that this process is both complex and unique. This review defines unique aspects of the germination pathways of C. difficile and compares them to those of two other well-studied organisms, Bacillus anthracis and Clostridium perfringensC. difficile germination is unique, as C. difficile does not contain any orthologs of the traditional GerA-type germinant receptor complexes and is the only known sporeformer to require bile salts in order to germinate. While recent advances describing C. difficile germination mechanisms have been made on several fronts, major gaps in our understanding of C. difficile germination signaling remain. This review provides an updated, in-depth summary of advances in understanding of C. difficile germination and potential avenues for the development of therapeutics, and discusses the major discrepancies between current models of germination and areas of ongoing investigation.
| S-EPMC6060349 | biostudies-literature
Project description:The germination of Clostridium difficile spores is an important stage of the C. difficile life cycle. In other endospore-forming bacteria, the composition of the medium in which the spores are generated influences the abundance of germination-specific proteins, thereby influencing the sensitivity of the spores towards germinants. In C. difficile media composition on the spores has only been reported to influence the number of spores produced. One of the measures of spore germination is the analysis of the release of DPA from the spore core. To detect DPA release in real time, terbium chloride is often added to the germination conditions because Tb3+ complexes with the released DPA and this can be detected using fluorescence measurements. Although C. difficile spores germinate in response to TA and glycine, recently calcium was identified as an enhancer for spore germination. Here, we find that germination by spores prepared in peptone rich media, such as 70:30, is positively influenced by terbium. We hypothesize that, in these assays, Tb3+ functions similarly to calcium. Although the mechanism(s) causing increased sensitivity of the C. difficile spores that are prepared in peptone rich media to terbium is still unknown, we suggest that the TbCl3 concentration used in the analysis of C. difficile DPA release be carefully titrated so as not to misinterpret future findings.
| S-EPMC6697597 | biostudies-literature
Project description:UnlabelledBacterial spore germination is a process whereby a dormant spore returns to active, vegetative growth, and this process has largely been studied in the model organism Bacillus subtilis. In B. subtilis, the initiation of germinant receptor-mediated spore germination is divided into two genetically separable stages. Stage I is characterized by the release of dipicolinic acid (DPA) from the spore core. Stage II is characterized by cortex degradation, and stage II is activated by the DPA released during stage I. Thus, DPA release precedes cortex hydrolysis during B. subtilis spore germination. Here, we investigated the timing of DPA release and cortex hydrolysis during Clostridium difficile spore germination and found that cortex hydrolysis precedes DPA release. Inactivation of either the bile acid germinant receptor, cspC, or the cortex hydrolase, sleC, prevented both cortex hydrolysis and DPA release. Because both cortex hydrolysis and DPA release during C. difficile spore germination are dependent on the presence of the germinant receptor and the cortex hydrolase, the release of DPA from the core may rely on the osmotic swelling of the core upon cortex hydrolysis. These results have implications for the hypothesized glycine receptor and suggest that the initiation of germinant receptor-mediated C. difficile spore germination proceeds through a novel germination pathway.ImportanceClostridium difficile infects antibiotic-treated hosts and spreads between hosts as a dormant spore. In a host, spores germinate to the vegetative form that produces the toxins necessary for disease. C. difficile spore germination is stimulated by certain bile acids and glycine. We recently identified the bile acid germinant receptor as the germination-specific, protease-like CspC. CspC is likely cortex localized, where it can transmit the bile acid signal to the cortex hydrolase, SleC. Due to the differences in location of CspC compared to the Bacillus subtilis germinant receptors, we hypothesized that there are fundamental differences in the germination processes between the model organism and C. difficile. We found that C. difficile spore germination proceeds through a novel pathway.
| S-EPMC4524186 | biostudies-literature
Project description:The strict anaerobe Clostridium difficile is an important nosocomial enteropathogen that has become the most common cause of antibiotic-associated diarrhea. The oxygen-resistant C. difficile spores play a central role in the infectious cycle, contributing to transmission, infection and recurrence. The spore surface layers, coat and exosporium, enable resistance of the spores to extreme physical and chemical stresses. Despite the critical importance of the spore in C. difficile infection, little is known about the mechanisms that orchestrate the assembly of its external layers. In this study, we identified and characterized a new C. difficile spore protein, named CotL, which is required for the assembly of the spore surface layers. The cotL gene was expressed in the mother cell compartment under the dual control of σE and σK. The CotL protein was localized in the C. difficile spore coat and cotL mutant spores had a major morphologic defect at the level of the coat/exosporium layers, as observed by transmission electron microscopy. Accordingly, spores of the cotL mutant contained a reduced amount of several proteins of the coat and exosporium, including CotB, CotE and CdeC. Additionally, cotL inactivation resulted in a defect in localization of late spore coat proteins in sporulating cells. Finally, spores of the cotL mutant were more sensitive to lysozyme and were impaired in germination. These defects are most likely associated with the structurally altered coat. Collectively, these results strongly suggest that CotL is a morphogenetic protein essential for the assembly of the spore coat in C. difficile.
2019-10-16 | PXD010359 | Pride
Project description:Clostridium difficile is a spore-forming, anaerobic, Gram-positive organism that is the leading cause of antibiotic-associated infectious diarrhea, commonly known as C. difficile infection (CDI). C. difficile spores play an important role in the pathogenesis of CDI. Spore proteins, especially those that are surface-bound may play an essential role in the germination, colonization and persistence of C. difficile in the human gut. In our current study, we report the identification of two surface-bound spore proteins, CdeC and CdeM that may be utilized as immunization candidates against C. difficile. These spore proteins are immunogenic in mice and are able to protect mice against challenge with C. difficile UK1, a clinically-relevant 027/B1/NAP1 strain. These spore proteins are also able to afford high levels of protection against challenge with C. difficile 630?erm in golden Syrian hamsters. This unprecedented study shows the vaccination potential of C. difficile spore exosporium proteins.
| S-EPMC4770901 | biostudies-literature
Project description:Clostridium difficile is a major nosocomial pathogen whose infections are difficult to treat because of their frequent recurrence. The spores of C. difficile are responsible for these clinical features, as they resist common disinfectants and antibiotic treatment. Although spores are the major transmissive form of C. difficile, little is known about their composition or morphogenesis. Spore morphogenesis has been well characterized for Bacillus sp., but Bacillus sp. spore coat proteins are poorly conserved in Clostridium sp. Of the known spore morphogenetic proteins in Bacillus subtilis, SpoIVA is one of the mostly highly conserved in the Bacilli and the Clostridia. Using genetic analyses, we demonstrate that SpoIVA is required for proper spore morphogenesis in C. difficile. In particular, a spoIVA mutant exhibits defects in spore coat localization but not cortex formation. Our study also identifies SipL, a previously uncharacterized protein found in proteomic studies of C. difficile spores, as another critical spore morphogenetic protein, since a sipL mutant phenocopies a spoIVA mutant. Biochemical analyses and mutational analyses indicate that SpoIVA and SipL directly interact. This interaction depends on the Walker A ATP binding motif of SpoIVA and the LysM domain of SipL. Collectively, these results provide the first insights into spore morphogenesis in C. difficile.
| S-EPMC3592010 | biostudies-literature
Project description:BACKGROUND/AIMS:Clostridium difficile infection (CDI) frequently complicates ulcerative colitis (UC) and can mimic disease flare. Differentiating UC flare from CDI remains a clinical challenge, particularly due to C. difficile colonization. Procalcitonin (PCT) is a serum biomarker for bacterial infections. We hypothesized that PCT would differentiate acute CDI from UC flare and C. difficile colonization. METHODS:A single-center prospective cohort study was conducted from 2013 to 2016. All UC patients with a stool sample for C. difficile testing were eligible. A total of 117 patients were enrolled, while 20 were excluded. Chart review was performed. RESULTS:Among 27 patients with CDI, median PCT was 60.7 (range 26-560.6) pg/mL, while among 90 patients without CDI, median PCT was 56.7 (range 25.1-2,252) pg/mL (p = 0.9). It was found that 14 patients with CDI responded completely to C. difficile treatment (CDI-R), while 8 patients did not and were diagnosed with UC flare (CDI-NR). For CDI-R, median PCT was 104.5 (range 26.3-560.6), compared to 40.3 (range 26.0-116.3) for CDI-NR (p = 0.036). CONCLUSIONS:In UC patients presenting with diarrhea, serum PCT was not significantly higher in UC patients with positive C. difficile testing. However, PCT was significantly elevated in CDI-R versus CDI-NR, suggesting that PCT may have utility in making this discrimination.
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Project description:Endosporulation is an ancient bacterial developmental program that culminates with the differentiation of a highly resistant endospore. In the model organism Bacillus subtilis, gene expression in the forespore and in the mother cell, the two cells that participate in endospore development, is governed by cell type-specific RNA polymerase sigma subunits. ?(F) in the forespore, and ?(E) in the mother cell control early stages of development and are replaced, at later stages, by ?(G) and ?(K), respectively. Starting with ?(F), the activation of the sigma factors is sequential, requires the preceding factor, and involves cell-cell signaling pathways that operate at key morphological stages. Here, we have studied the function and regulation of the sporulation sigma factors in the intestinal pathogen Clostridium difficile, an obligate anaerobe in which the endospores are central to the infectious cycle. The morphological characterization of mutants for the sporulation sigma factors, in parallel with use of a fluorescence reporter for single cell analysis of gene expression, unraveled important deviations from the B. subtilis paradigm. While the main periods of activity of the sigma factors are conserved, we show that the activity of ?(E) is partially independent of ?(F), that ?(G) activity is not dependent on ?(E), and that the activity of ?(K) does not require ?(G). We also show that ?(K) is not strictly required for heat resistant spore formation. In all, our results indicate reduced temporal segregation between the activities of the early and late sigma factors, and reduced requirement for the ?(F)-to-?(E), ?(E)-to-?(G), and ?(G)-to-?(K) cell-cell signaling pathways. Nevertheless, our results support the view that the top level of the endosporulation network is conserved in evolution, with the sigma factors acting as the key regulators of the pathway, established some 2.5 billion years ago upon its emergence at the base of the Firmicutes Phylum.
| S-EPMC3789829 | biostudies-literature