Clostridioides difficile Sporulation.

Some members of the Firmicutes phylum, including many members of the human gut microbiota, are able to differentiate a dormant and highly resistant cell type, the endospore (hereinafter spore for simplicity). Spore-formers can colonize virtually any habitat and, because of their resistance to a wide variety of physical and chemical insults, spores can remain viable in the environment for long periods of time. In the anaerobic enteric pathogen Clostridioides difficile the aetiologic agent is the oxygen-resistant spore, while the toxins produced by actively growing cells are the main cause of the disease symptoms. Here, we review the regulatory circuits that govern entry into sporulation. We also cover the role of spores in the infectious cycle of C. difficile in relation to spore structure and function and the main control points along spore morphogenesis.

Redistribution of the Novel Clostridioides difficile Spore Adherence Receptor E-Cadherin by TcdA and TcdB Increases Spore Binding to Adherens Junctions.

Clostridioides difficile causes antibiotic-associated diseases in humans, ranging from mild diarrhea to severe pseudomembranous colitis and death. A major clinical challenge is the prevention of disease recurrence, which affects nearly ~20 to 30% of the patients with a primary C. difficile infection (CDI). During CDI, C. difficile forms metabolically dormant spores that are essential for recurrence of CDI (R-CDI). In prior studies, we have shown that C. difficile spores interact with intestinal epithelial cells (IECs), which contribute to R-CDI. However, this interaction remains poorly understood. Here, we provide evidence that C. difficile spores interact with E-cadherin, contributing to spore adherence and internalization into IECs. C. difficile toxins TcdA and TcdB lead to adherens junctions opening and increase spore adherence to IECs. Confocal micrographs demonstrate that C. difficile spores associate with accessible E-cadherin; spore-E-cadherin association increases upon TcdA and TcdB intoxication. The presence of anti-E-cadherin antibodies decreased spore adherence and entry into IECs. By enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and immunogold labeling, we observed that E-cadherin binds to C. difficile spores, specifically to the hairlike projections of the spore, reducing spore adherence to IECs. Overall, these results expand our knowledge of how C. difficile spores bind to IECs by providing evidence that E-cadherin acts as a spore adherence receptor to IECs and by revealing how toxin-mediated damage affects spore interactions with IECs.

Role of novel polysaccharide layers in assembly of the exosporium, the outermost protein layer of the Bacillus anthracis spore.

A fundamental question in cell biology is how cells assemble their outer layers. The bacterial endospore is a well-established model for cell layer assembly. However, the assembly of the exosporium, a complex protein shell comprising the outermost layer in the pathogen Bacillus anthracis, remains poorly understood. Exosporium assembly begins with the deposition of proteins at one side of the spore surface, followed by the progressive encirclement of the spore. We seek to resolve a major open question: the mechanism directing exosporium assembly to the spore, and then into a closed shell. We hypothesized that material directly underneath the exosporium (the interspace) directs exosporium assembly to the spore and drives encirclement. In support of this, we show that the interspace possesses at least two distinct layers of polysaccharide. Secondly, we show that putative polysaccharide biosynthetic genes are required for exosporium encirclement, suggesting a direct role for the interspace. These results not only significantly clarify the mechanism of assembly of the exosporium, an especially widespread bacterial outer layer, but also suggest a novel mechanism in which polysaccharide layers drive the assembly of a protein shell.

ExsY, CotY, and CotE Effects on Bacillus anthracis Outer Spore Layer Architecture.

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are the major pathogens of the spore-forming genus Bacillus and possess an outer spore layer, the exosporium, not found in many of the nonpathogenic species. The exosporium consists of a basal layer with the ExsY, CotY, and BxpB proteins being the major structural components and an exterior nap layer containing the BclA glycoprotein. During the assembly process, the nascent exosporium basal layer is attached to the spore coat by a protein linker that includes the CotO and CotE proteins. Using transmission electron microscopy, Western blotting, immunofluorescence, and fluorescent fusion protein approaches, we examined the impact of single, double, and triple mutants of the major exosporium proteins on exosporium protein content and distribution. Plasmid-based expression of exsY and cotE resulted in increased production of exosporium lacking spores, and the former also resulted in outer spore coat disruptions. The exosporium bottlecap produced by exsY null spores was found to be more stable than previously reported, and its spore association was partially dependent on CotE. Deletion mutants of five putative spore genes (bas1131, bas1142, bas1143, bas2277, and bas3594) were created and shown not to have obvious effects on spore morphology or BclA and BxpB content. The BclC collagen-like glycoprotein was found to be present in the spore and possibly localized to the interspace region. IMPORTANCE B. anthracis is an important zoonotic animal pathogen causing sporadic outbreaks of anthrax worldwide. Spores are the infectious form of the bacterium and can persist in soil for prolonged periods of time. The outermost B. anthracis spore layer is the exosporium, a protein shell that is the site of interactions with both the soil and with the innate immune system of infected hosts. Although much is known regarding the sporulation process among members of the genus Bacillus, significant gaps in our understanding of the exosporium assembly process exist. This study provides evidence for the properties of key exosporium basal layer structural proteins. The results of this work will guide future studies on exosporium protein-protein interactions during the assembly process.

Roles and Organization of BxpB (ExsFA) and ExsFB in the Exosporium Outer Basal Layer of Bacillus anthracis.

BxpB (also known as ExsFA) and ExsFB are an exosporium basal layer structural protein and a putative interspace protein of Bacillus anthracis that are known to be required for proper incorporation of the BclA collagen-like glycoprotein on the spore surface. Despite extensive similarity of the two proteins, their distribution in the spore is markedly different. We utilized a fluorescent fusion approach to examine features of the two genes that affect spore localization. The timing of expression of the bxpB and exsFB genes and their distinct N-terminal sequences were both found to be important for proper assembly into the exosporium basal layer. Results of this study provided evidence that the BclA nap glycoprotein is not covalently attached to BxpB protein despite the key role that the latter plays in BclA incorporation. Assembly of the BxpB- and ExsFB-containing outer basal layer appears not to be completely abolished in mutants lacking the ExsY and CotY basal layer structural proteins despite these spores lacking a visible exosporium. The BxpB and, to a lesser extent, the ExsFB proteins, were found to be capable of self-assembly in vitro into higher-molecular-weight forms that are stable to boiling in SDS under reducing conditions. IMPORTANCE The genus Bacillus consists of spore-forming bacteria. Some species of this genus, especially those that are pathogens of animals or insects, contain an outermost spore layer called the exosporium. The zoonotic pathogen B. anthracis is an example of this group. The exosporium likely contributes to virulence and environmental persistence of these pathogens. This work provides important new insights into the exosporium assembly process and the interplay between BclA and BxpB in this process.

Host-Endosymbiont Relationship Impacts the Retention of Bacteria-Containing Amoeba Spores in Porous Media.

Amoebae are protists that are commonly found in water, soil, and other habitats around the world and have complex interactions with other microorganisms. In this work, we investigated how host-endosymbiont interactions between amoebae and bacteria impacted the retention behavior of amoeba spores in porous media. A model amoeba species, Dictyostelium discoideum, and a representative bacterium, Burkholderia agricolaris B1qs70, were used to prepare amoeba spores that carried bacteria. After interacting with B. agricolaris, the retention of D. discoideum spores was enhanced compared to noninfected spores. Diverse proteins, especially proteins contributing to the looser exosporium structure and cell adhesion functionality, are secreted in higher quantities on the exosporium surface of infected spores compared to that of noninfected ones. Comprehensive examinations using a quartz crystal microbalance with dissipation (QCM-D), a parallel plate chamber, and a single-cell force microscope present coherent evidence that changes in the exosporium of D. discoideum spores due to infection by B. agricolaris enhance the connections between spores in the suspension and the spores that were previously deposited on the collector surface, thus resulting in more retention compared to the uninfected ones in porous media. This work provides novel insight into the retention of amoeba spores after bacterial infection in porous media and suggests that the host-endosymbiont relationship regulates the fate of biocolloids in drinking water systems, groundwater, and other porous environments.

Survival of Clostridioides difficile spores in thermal and chemo-thermal laundering processes and influence of the exosporium on their adherence to cotton bed sheets.

Clostridioides difficile spores were previously demonstrated to survive industrial laundering. Understanding interactions between heat, disinfectants and soiling (e.g. bodily fluids) affecting C. difficile spore survival could inform the optimization of healthcare laundry processes. Reducing spore attachment to linen could also enhance laundering efficacy. This study aimed to compare the sensitivity of C. difficile spores to heat and detergent, with and without soiling and to investigate adherence to cotton. Survival of C. difficile spores exposed to industrial laundering temperatures (71-90°C), reference detergent and industrial detergent was quantified with and without soiling. The adherence to cotton after 0 and 24 h air drying was determined with the exosporium of C. difficile spores partially or fully removed. Clostridioides difficile spores were stable at 71°C for 20 min (≤0·37 log10 reduction) while 90°C was sporicidal (3 log10 reduction); soiling exerted a protective effect. Industrial detergent was more effective at 71°C compared to 25°C (2·81 vs 0·84 log10 reductions), however, specifications for sporicidal activity (>3 log10 reduction) were not met. Clostridioides difficile spores increasingly adhered to cotton over time, with 49% adherence after 24 h. Removal of the exosporium increased adherence by 19-23% compared to untreated spores. Further understanding of the role of the exosporium in attachment to cotton could enhance spore removal and aid decontamination of linen.

Sporulation Strategies and Potential Role of the Exosporium in Survival and Persistence of Clostridium botulinum.

Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains are terrestrial, mesophilic, and produce highly heat-resistant spores, while Group II strains can be terrestrial (type B) or aquatic (type E) and are generally psychrotrophic and produce spores of moderate heat resistance. Group III strains are either terrestrial or aquatic, mesophilic or slightly thermophilic, and the heat resistance properties of their spores are poorly characterized. Here, we analyzed the sporulation dynamics in population, spore morphology, and other spore properties of 10 C. botulinum strains belonging to Groups I-III. We propose two distinct sporulation strategies used by C. botulinum Groups I-III strains, report their spore properties, and suggest a putative role for the exosporium in conferring high heat resistance. Strains within each physiological group produced spores with similar characteristics, likely reflecting adaptation to respective environmental habitats. Our work provides new information on the spores and on the population and single-cell level strategies in the sporulation of C. botulinum.

Insights into the Structure and Protein Composition of Moorella thermoacetica Spores Formed at Different Temperatures.

The bacterium Moorella thermoacetica produces the most heat-resistant spores of any spoilage-causing microorganism known in the food industry. Previous work by our group revealed that the resistance of these spores to wet heat and biocides was lower when spores were produced at a lower temperature than the optimal temperature. Here, we used electron microcopy to characterize the ultrastructure of the coat of the spores formed at different sporulation temperatures; we found that spores produced at 55 °C mainly exhibited a lamellar inner coat tightly associated with a diffuse outer coat, while spores produced at 45 °C showed an inner and an outer coat separated by a less electron-dense zone. Moreover, misarranged coat structures were more frequently observed when spores were produced at the lower temperature. We then analyzed the proteome of the spores obtained at either 45 °C or 55 °C with respect to proteins putatively involved in the spore coat, exosporium, or in spore resistance. Some putative spore coat proteins, such as CotSA, were only identified in spores produced at 55 °C; other putative exosporium and coat proteins were significantly less abundant in spores produced at 45 °C. Altogether, our results suggest that sporulation temperature affects the structure and protein composition of M. thermoacetica spores.

Spore-Associated Proteins Involved in c-di-GMP Synthesis and Degradation of Bacillus anthracis.

Bis-(3'-5')-cyclic-dimeric GMP (c-di-GMP) is an important bacterial regulatory signaling molecule affecting biofilm formation, toxin production, motility, and virulence. The genome of Bacillus anthracis, the causative agent of anthrax, is predicted to encode ten putative GGDEF/EAL/HD-GYP-domain containing proteins. Heterologous expression in Bacillus subtilis hosts indicated that there are five active GGDEF domain-containing proteins and four active EAL or HD-GYP domain-containing proteins. Using an mCherry gene fusion-Western blotting approach, the expression of the c-di-GMP-associated proteins was observed throughout the in vitro life cycle. Of the six c-di-GMP-associated proteins found to be present in sporulating cells, four (CdgA, CdgB, CdgD, and CdgG) contain active GGDEF domains. The six proteins expressed in sporulating cells are retained in spores in a CotE-independent manner and thus are not likely to be localized to the exosporium layer of the spores. Individual deletion mutations involving the nine GGDEF/EAL protein-encoding genes and one HD-GYP protein-encoding gene did not affect sporulation efficiency, the attachment of the exosporium glycoprotein BclA, or biofilm production. Notably, expression of anthrax toxin was not affected by deletion of any of the cdg determinants. Three determinants encoding proteins with active GGDEF domains were found to affect germination kinetics. This study reveals a spore association of cyclic-di-GMP regulatory proteins and a likely role for these proteins in the biology of the B. anthracis spore. IMPORTANCE The genus Bacillus is composed of Gram-positive, rod shaped, soil-dwelling bacteria. As a mechanism for survival in the harsh conditions in soil, the organisms undergo sporulation, and the resulting spores permit the organisms to survive harsh environmental conditions. Although most species are saprophytes, Bacillus cereus and Bacillus anthracis are human pathogens and Bacillus thuringiensis is an insect pathogen. The bacterial c-di-GMP regulatory system is an important control system affecting motility, biofilm formation, and toxin production. The role of c-di-GMP has been studied in the spore-forming bacilli Bacillus subtilis, Bacillus amyloliquefaciens, B. cereus, and B. thuringiensis. However, this regulatory system has not heretofore been examined in the high-consequence zoonotic pathogen of this genus, B. anthracis.

A putative exosporium lipoprotein GBAA0190 of Bacillus anthracis as a potential anthrax vaccine candidate.

BACKGROUND: Bacillus ancthracis causes cutaneous, pulmonary, or gastrointestinal forms of anthrax. B. anthracis is a pathogenic bacterium that is potentially to be used in bioterrorism because it can be produced in the form of spores. Currently, protective antigen (PA)-based vaccines are being used for the prevention of anthrax, but it is necessary to develop more safe and effective vaccines due to their prolonged immunization schedules and adverse reactions. METHODS: We selected the lipoprotein GBAA0190, a potent inducer of host immune response, present in anthrax spores as a novel potential vaccine candidate. Then, we evaluated its immune-stimulating activity in the bone marrow-derived macrophages (BMDMs) using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Protective efficacy of GBAA0190 was evaluated in the guinea pig (GP) model. RESULTS: The recombinant GBAA0190 (r0190) protein induced the expression of various inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) in the BMDMs. These immune responses were mediated through toll-like receptor 1/2 via activation of mitogen-activated protein (MAP) kinase and Nuclear factor-κB (NF-κB) pathways. We demonstrated that not only immunization of r0190 alone, but also combined immunization with r0190 and recombinant PA showed significant protective efficacy against B. anthracis spore challenges in the GP model. CONCLUSIONS: Our results suggest that r0190 may be a potential target for anthrax vaccine.

A Bacillus Spore-Based Display System for Bioremediation of Atrazine.

Owing to human activities, a large number of organic chemicals, including petroleum products, industrial solvents, pesticides, herbicides (including atrazine [ATR]), and pharmaceuticals, contaminate soil and aquatic environments. Remediation of these pollutants by conventional approaches is both technically and economically challenging. Bacillus endospores are highly resistant to most physical assaults and are capable of long-term persistence in soil. Spores can be engineered to express, on their surface, important enzymes for bioremediation purposes. We have developed a Bacillus thuringiensis spore platform system that can display a high density of proteins on the spore surface. The spore surface-tethered enzymes exhibit enhanced activity and stability relative to free enzymes in soil and water environments. In this study, we evaluated a B. thuringiensis spore display platform as a bioremediation tool against ATR. The Pseudomonas sp. strain ADP atzA determinant, an ATR chlorohydrolase important to the detoxification of ATR, was expressed as a fusion protein linked to the attachment domain of the BclA spore surface nap layer protein and expressed in B. thuringiensis Spores from this strain are decorated with AtzA N-terminally linked on the surface of the spores. The recombinant spores were assayed for ATR detoxification in liquid and soil environments, and enzyme kinetics and stability were assessed. We successfully demonstrated the utility of this spore-based enzyme display system to detoxify ATR in water and laboratory soil samples.IMPORTANCE Atrazine is one of the most widely applied herbicides in the U.S. midwestern states. The long environmental half-life of atrazine has contributed to the contamination of surface water and groundwater by atrazine and its chlorinated metabolites. The toxic properties of ATR have raised public health and ecological concerns. However, remediation of ATR by conventional approaches has proven to be costly and inefficient. We developed a novel B. thuringiensis spore platform system that is capable of long-term persistence in soil and can be engineered to surface express a high density of enzymes useful for bioremediation purposes. The enzymes are stably attached to the surface of the spore exosporium layer. The spore-based system will likely prove useful for remediation of other environmental pollutants as well.

Surface morphology differences in Clostridium difficile spores, based on different strains and methods of purification.

Infections linked to Clostridium difficile are a significant cause of suffering. In hospitals, the organism is primarily acquired through the faecal-oral route as spores excreted by infected patients contaminate the healthcare environment. We previously reported that members of the C. difficile group varied widely in their ability to adhere to stainless steel and proposed that these differences were a consequence of variations in spore architecture. In this study of clinical isolates and spore coat protein mutants of C. difficile we identified three distinct spore surfaces morphotypes; smooth, bag-like and "pineapple-like" using scanning electron microscopy (SEM). The frequency of each morphotype in a spore population derived from a single isolate varied depending on the host strain and the method used to produce and purify the spores. Our results suggest that the inclusion of a sonication step in the purification process had a marked effect on spore structure. In an attempt to link differences in spore appearance with key structural spore proteins we compared the morphology of spores of CD630 to those produced by CD630 variants lacking either CotE or BclA. While SEM images revealed no obvious structural differences between CD630 and its mutants we did observe significant differences (p < 0.001) in relative hydrophobicity suggesting that modifications had occurred but not at a level to be detectable by SEM. In conclusion, we observed significant variation in the spore morphology of clinical isolates of C. difficile due in part to the methods used to produce them. Sonication in particular can markedly change spore appearance and properties. The results of this study highlight the importance of adopting "standard" methods when attempting to compare results between studies and to understand the significance of their differences.

Nasal Immunization with the C-Terminal Domain of Bcla3 Induced Specific IgG Production and Attenuated Disease Symptoms in Mice Infected with Clostridioides difficile Spores.

Clostridioides difficile is a Gram-positive, spore-forming bacterium that causes a severe intestinal infection. Spores of this pathogen enter in the human body through the oral route, interact with intestinal epithelial cells and persist in the gut. Once germinated, the vegetative cells colonize the intestine and produce toxins that enhance an immune response that perpetuate the disease. Therefore, spores are major players of the infection and ideal targets for new therapies. In this context, spore surface proteins of C. difficile, are potential antigens for the development of vaccines targeting C. difficile spores. Here, we report that the C-terminal domain of the spore surface protein BclA3, BclA3CTD, was identified as an antigenic epitope, over-produced in Escherichia coli and tested as an immunogen in mice. To increase antigen stability and efficiency, BclA3CTD was also exposed on the surface of B. subtilis spores, a mucosal vaccine delivery system. In the experimental conditions used in this study, free BclA3CTD induced antibody production in mice and attenuated some C. difficile infection symptoms after a challenge with the pathogen, while the spore-displayed antigen resulted less effective. Although dose regimen and immunization routes need to be optimized, our results suggest BclA3CTD as a potentially effective antigen to develop a new vaccination strategy targeting C. difficile spores.

New insights for vaccine development against Clostridium difficile infections.

Increased antibiotic usage is the main risk factor for gut microbiota dysbiosis. In dysbiosis, there is an increased susceptibility to intestinal pathogens, such as Clostridium difficile infection, the leading cause of hospital-acquired infection worldwide. High-spectrum antibiotics, such as vancomycin or metronidazole, also increases the risk of developing CDI symptoms after the treatment. An impaired immune response could also be responsible for the high incidence of recurrence of CDI (R-CDI), suggesting that immune system stimulation could help eradicate the infection in patients suffering multiple episodes in CDI or prevent the infective course. Here, we discuss novel immunotherapeutic approaches that aid the immune system to target C. difficile and how these can be improved.

Glycosylation of BclA Glycoprotein from Bacillus cereus and Bacillus anthracis Exosporium Is Domain-specific.

The spores of the Bacillus cereus group (B. cereus, Bacillus anthracis, and Bacillus thuringiensis) are surrounded by a paracrystalline flexible yet resistant layer called exosporium that plays a major role in spore adhesion and virulence. The major constituent of its hairlike surface, the trimerized glycoprotein BclA, is attached to the basal layer through an N-terminal domain. It is then followed by a repetitive collagen-like neck bearing a globular head (C-terminal domain) that promotes glycoprotein trimerization. The collagen-like region of B. anthracis is known to be densely substituted by unusual O-glycans that may be used for developing species-specific diagnostics of B. anthracis spores and thus targeted therapeutic interventions. In the present study, we have explored the species and domain specificity of BclA glycosylation within the B. cereus group. First, we have established that the collagen-like regions of both B. anthracis and B. cereus are similarly substituted by short O-glycans that bear the species-specific deoxyhexose residues anthrose and the newly observed cereose, respectively. Second we have discovered that the C-terminal globular domains of BclA from both species are substituted by polysaccharide-like O-linked glycans whose structures are also species-specific. The presence of large carbohydrate polymers covering the surface of Bacillus spores may have a profound impact on the way that spores regulate their interactions with biotic and abiotic surfaces and represents potential new diagnostic targets.

Corynespora, Exosporium and Helminthosporium revisited - New species and generic reclassification.

Molecular phylogenetic analyses of a multigene matrix of partial nuSSU-ITS-LSU rDNA, rpb2 and tef1 sequences were performed to investigate the phylogenetic relationships of Corynespora, Exosporium and Helminthosporium species. Based on phylogenetic analyses and morphology, the genus Exosporium is synonymised with Helminthosporium, and the genus Corynespora is revealed as polyphyletic. Corynespora smithii is confirmed to be closely related to the generic type C. cassiicola and its morphology is described and illustrated. Exosporium tiliae, Corynespora caespitosa, C. endiandrae, C. leucadendri and C. olivacea are recognised in Helminthosporium, and Splanchnonema quercicola and S. kalakadense are combined in Helminthosporium. Based on pure culture studies and DNA sequence data, Massaria heterospora and Massarinula italica are shown to be the sexual morphs of Helminthosporium tiliae and H. microsorum, respectively. European accessions of Splanchnonema quercicola are recognised to differ from the North American type and are described as Helminthosporium quercinum. The sexual morph of H. oligosporum is recorded and described for the first time. The generic type of Helminthosporium, H. velutinum, is epitypified with a recent collection from the type host, Fagus sylvatica. Based on sequence data, Helminthosporium genistae is recognised as a distinct species. Several species for which subperidermal stromata have been reported are shown to be fungicolous on Diaporthales, the "stromata" representing aborted and transformed host stromata or conidiomata: H. caespitosum, H. microsorum, H. quercicola and H. quercinum on Coryneum spp.; H. hispanicum on conidiomata of Juglanconis juglandina; H. juglandinum on conidiomata of Diaporthe sp.; H. oligosporum and H. tiliae on Hercospora tiliae. The newly described H. austriacum is fungicolous on Amphisphaeria cf. millepunctata (Xylariales).

Mycosphaerellaceae - Chaos or clarity?

The Mycosphaerellaceae represent thousands of fungal species that are associated with diseases on a wide range of plant hosts. Understanding and stabilising the taxonomy of genera and species of Mycosphaerellaceae is therefore of the utmost importance given their impact on agriculture, horticulture and forestry. Based on previous molecular studies, several phylogenetic and morphologically distinct genera within the Mycosphaerellaceae have been delimited. In this study a multigene phylogenetic analysis (LSU, ITS and rpb2) was performed based on 415 isolates representing 297 taxa and incorporating ex-type strains where available. The main aim of this study was to resolve the phylogenetic relationships among the genera currently recognised within the family, and to clarify the position of the cercosporoid fungi among them. Based on these results many well-known genera are shown to be paraphyletic, with several synapomorphic characters that have evolved more than once within the family. As a consequence, several old generic names including Cercosporidium, Fulvia, Mycovellosiella, Phaeoramularia and Raghnildiana are resurrected, and 32 additional genera are described as new. Based on phylogenetic data 120 genera are now accepted within the family, but many currently accepted cercosporoid genera still remain unresolved pending fresh collections and DNA data. The present study provides a phylogenetic framework for future taxonomic work within the Mycosphaerellaceae.

Updates on Clostridium difficile spore biology.

Clostridium difficile is a Gram-positive, anaerobic spore former, and an important nosocomial pathogenic bacterium. C. difficile spores are the morphotype of transmission and recurrence of the disease. The formation of C. difficile spores and their subsequent germination are essential processes during the infection. Recent in vitro and in vivo work has shed light on how spores are formed and the timing of in vivo sporulation in a mouse model. Advances have also been made in our understanding of the machineries involved in spore germination, and how antibiotic-induced dysbiosis affects the metabolism of bile salts and thus impacts C. difficile germination in vivo. Studies have also attempted to identify how C. difficile spores interact with the host's intestinal mucosa. Spore resistance has also been revisited by several groups highlighting the extreme resistance of this morphotype to traditional food processing regimes and disinfectants used in clinical settings. Therefore, the aim of this review is to summarize recent advances on spore formation/germination in vitro and in vivo, spore-host interactions, and spore resistance that contribute to our knowledge of the role of C. difficile spores in the infectious process.

Immunogenicity and protective efficacy of Clostridium difficile spore proteins.

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 630Δerm in golden Syrian hamsters. This unprecedented study shows the vaccination potential of C. difficile spore exosporium proteins.