Molecular mechanism of germination of Clostridium perfringens spores

Clostridium perfringens is the causative agent of a wide variety of diseases in animals and humans. C. perfringens can produce more than 15 toxins. However, individual strains produce a subset of these toxins. Although a small percentage of C. perfringens isolates (mostly belonging to type A) produce C. perfringens enterotoxin (CPE), these are very important human gastrointestinal (GI) pathogens, causing C. perfringens type A food poisoning (FP) and nonfood-borne GI diseases (NFBGID). Due to its anaerobic nature and the ability to form extremely resistant spores found ubiquitously in the environment, to cause the wide array of C. perfringens-associated diseases (CPAD), these C. perfringens spores must germinate, release the nascent cell, grow and produce their toxins. Therefore, germination of C. perfringens spores is the initial and perhaps most important step for the progression of diseases in animals and humans. Although extensive research has been conducted on the mechanism of spore germination of Bacillus species, very few studies of spore germination have been conducted in Clostridium species mainly due to the lack of molecular genetic tools. Genomic comparisons reveal significant differences in the backbone of the germination apparatus between Bacillus and Clostridium species. Consequently, a detail understanding of the molecular mechanism of germination of C. perfringens spores is essential for the development of novel preventive strategies for CPAD as well as diseases caused by other pathogenic Clostridium species. The first focus of this work was to identify and characterize the germinants and the receptors involved in C. perfringens spore germination. Result from these studies found differential germination requirements between spores of FP and NFBGID isolates in that: (i) while a mixture of L-asparagine and KCl was a good germinant for spores of FP and NFBGID isolates, KCl and, to a lesser extent, L-asparagine triggered spore germination in FP isolates only; and ii) L-alanine and L-valine induced significant germination of spores of NFBGID but not FP isolates. In contrast to B. subtilis, C. perfrinegns genomes sequenced to date possess no tricistronic gerA-like operon, but has a monocistronic gerAA that is far from a gerK locus. The gerK locus contains a bicistronic gerKA-gerKC operon and a monocistronic gerKB upstream and in the opposite orientation to gerKA-gerKC. Consequently, through the construction of mutations into strain SM101, a C. perfringens FP isolate, the role of gerAA, gerKA-gerKC and gerKB genes in C. perfringens spore germination were investigated. Results indicated that KCl, L-asparagine and Ca-DPA required GerKA and/or GerKC receptors, while GerAA and GerKB played an auxiliary role in germination. Lack of GerKA and/or GerKC, and GerKB significantly reduced spores colony forming efficiency, indicating a role in spore viability. The fact that C. perfringens spores lacking the main germinant receptor(s) proteins, GerKA and/or GerKC, are still able to germinate albeit poorly compared to wild-type, and that C. perfringens spores germinate with K+ ions alone, raises the hypothesis that GrmA-like antiporters might also play some role in C. perfringens spore germination. Two putative GrmA-like antiporters (i.e., GerO and GerQ) are encoded in the genome of all C. perfringens sequenced to date. This study shows that gerO and gerQ genes are expressed uniquely during sporulation and the mother cell compartment of the sporulating cell. Complementation studies of K+ uptake and Na+ sensitive E. coli mutants indicate that while GerO is capable of translocating K+ and Na+, GerQ is only capable of translocating, to a small extent, Na+ ions. Spores lacking GerO had defective germination in rich medium, KCl, L-asparagine, and Ca-DPA, but not with dodecylamine, defect that might be prior to DPA release during germination. In contrast, loss of GerQ had a much smaller effect on spore germination. Two adjacent Asp residues, important in ion transcloation of the E. coli Na+/H+ antiporter NhaA were also present in GerO, but not GerQ, and replacement of these residues for Asn reduced the protein’s ability to complement gerO spores. Although results from this study indicate that putative antiporters have some role on C. perfringens spore germination, it is unclear whether their role is direct or during spore formation. C. perfringens type A FP spores are capable of germinating with K+ ions, an intrinsic mineral of meats commonly associated with FP. Inorganic phosphate (Pi) is also intrinsically found in meat products. Consequently, we hypothesized that FP spores are capable of germinating in presence of Pi. Results from this study show that spores of the majority of FP, but not NFBGID isolates, are able to germinate in presence of Pi. Pi-induced germination of FP spores is primarily through the GerKA and/or GerKC protein, while GerAA and to a much lesser extent, GerKB, play auxiliary roles. The putative Na+/K+-H+ antiporter, GerO, is also required for normal Pi-induced germination. These results suggest that the differential germination phenotypes between spores of FP and NFGID isolates is tightly regulated by their adaptation to different environmental niches. A second focus of this work was to investigate the mechanism of signal transduction between the germinant receptors and the downstream effectors. In B. subtilis, the SpoVA proteins have been associated with Ca-DPA uptake and subsequent release during sporulation and germination, respectively. In addition, Ca-DPA acts as a signal molecule for cortex hydrolysis during B. subtilis spore germination, activating the cortex lytic enzyme (CLE) CwlJ. Results from this study show that in contrast to B. subtilis spoVA mutants, where spores lyse quickly during purification, C. perfringens spoVA spores were stable and germinated similarly as wild-type spores. These results suggest major differences in the regulation of the germination pathway between C. perfringens and B. subtilis, and suggest that activation of CLEs in C. perfringens might be through a different pathway than the Ca-DPA pathway of B. subtilis. A third focus of this work was to investigate the in vivo role of the CLE involved in peptidoglycan (PG) spore cortex hydrolysis during C. perfringens spore germination. Two C. perfringens CLEs (i.e., SleC and SleM) degrade PG spore cortex hydrolysis in vitro, however, due to lack of genetic tools, their in vivo role in spore germination remains unclear. Results from this study show that C. perfringens sleC spores released their DPA slower than wild-type and were not able to germinate with nutrients and non-nutrient germinants. In contrast, sleM spores germinated similar as wild type in presence of nutrient and non-nutrient germinants, indicating that while SleC is essential for cortex hydrolysis and viability of C. perfringens spores, SleM although can degrade cortex PG in vitro, is not essential. A fourth focus of this work was to investigate the in vivo role of the Csp proteases in the initiation of cortex hydrolysis. In vitro work has shown that Csp proteins process the inactive proSleC into the mature enzyme, SleC. However, the in vivo role of the Csp proteins has not been established. In this study, spores a C. perfringens cspB mutant exhibited significantly less viability than wild-type spores, and were unable to germinate with either rich medium or Ca-DPA. Germination of cspB spores was blocked prior to DPA release and cortex hydrolysis. Results from this study indicate that CspB is essential to generate active SleC and allow cortex hydrolysis early in C. perfringens spore germination. In contrast to B. subtilis, Ca-DPA did not activate the CLEs during spore germination present in cspB spores supporting previous results that Ca-DPA acts trough the GerKA and/or GerKC receptor. A final focus of this work was to develop a strategy to inactivate C. perfringens spores in meat products. C. perfringens spores posses high heat and pressure resistance, however, they loss their resistance properties during early stages of germination. In contrast to B. subtilis spores, germination of C. perfringens spores could not be triggered with low pressures. However, they germinated efficiently when heat activated in presence of L-asparagine and KCl at temperatures lethal for vegetative cells, and these germinated spores were efficiently inactivated by subsequent treatment with pressure assisted thermal processing (586 MPa at 73ºC for 10 min). This study shows the feasibility of a novel strategy to inactivate C. perfringens spores in meat products formulated with germinants. Collectively, the present study contributes to the understanding of the mechanism of spore germination in the pathogenic bacterium C. perfringens, and developed an alternative and novel strategy to inactivate C. perfringens spores in meat products

Further Characterization of Clostridium perfringens Small Acid Soluble Protein-4 (Ssp4) Properties and Expression

Background: Clostridium perfringens type A food poisoning (FP) is usually caused by C. perfringens type A strains that carry a chromosomal enterotoxin gene (cpe) and produce spores with exceptional resistance against heat and nitrites. Previous studies showed that the extreme resistance of spores made by most FP strains is mediated, in large part, by a variant of small acid soluble protein 4 (Ssp4) that has Asp at residue 36; in contrast, the sensitive spores made by other C. perfringens type A isolates contain an Ssp4 variant with Gly at residue 36. Methodology/Principal Findings: The current study has further characterized Ssp4 properties and expression. Spores made by cpe-positive type C and D strains were found to contain the Ssp4 variant with Gly at residue 36 and were shown to be heat- and nitrite-sensitive; this finding may help to explain why cpe-positive type C and D isolates rarely cause food poisoning. Saturation mutagenesis indicated that both amino acid size and charge at Ssp4 residue 36 are important for DNA binding and for spore resistance. C. perfringens Ssp2 was shown to bind preferentially to GC-rich DNA on gel-shift assays, while Ssp4 preferred binding to AT-rich DNA sequences. Maximal spore heat and nitrite resistance required production of all four C. perfringens Ssps, indicating that these Ssps act cooperatively to protect the spore's DNA, perhaps by binding to different chromosomal sequences. The Ssp4 variant with Asp at residue 36 was also shown to facilitate exceptional spore survival at freezer and refrigerator temperatures. Finally, Ssp4 expression was shown to be dependent upon Spo0A, a master regulator. Conclusions/Significance: Collectively, these results provide additional support for the importance of Ssps, particularly the Ssp4 variant with Asp at residue 36, for the extreme spore resistance phenotype that likely contributes to C. perfringens type A food poisoning transmission. © 2009 Li et al

Characterization of Chicken IgY Specific to Clostridium difficile R20291 Spores and the Effect of Oral Administration in Mouse Models of Initiation and Recurrent Disease

Indexación: Web of Science; Scopus.Clostridium difficile infection (CDI) are the leading cause of world-wide nosocomial acquired diarrhea. The current main clinical challenge in CDI is the elevated rate of infection recurrence that may reach up to 30% of the patients, which has been associated to the formation of dormant spores during the infection. We sought to characterize the effects of oral administration of specific anti-spore IgY in mouse models of CDI and recurrent CDI. The specificity of anti-spore IgY was evaluated in vitro. In both, initiation mouse model and recurrence mouse model, we evaluated the prophylactic and therapeutic effect of anti-spore IgY, respectively. Our results demonstrate that anti-spore IgY exhibited high specificity and titers against C. difficile spores and reduced spore adherence to intestinal cells in vitro. Administration of anti-spore IgY to C57BL/6 mice prior and during CDI delayed the appearance of the diarrhea by 1.5 day, and spore adherence to the colonic mucosa by 90%. Notably, in the recurrence model, co-administration of anti-spore IgY coupled with vancomycin delayed the appearance of recurrent diarrhea by a median of 2 days. Collectively, these observations suggest that anti-spore IgY antibodies may be used as a novel prophylactic treatment for CDI, or in combination with antibiotics to treat CDI and prevent recurrence of the infection.https://www.frontiersin.org/articles/10.3389/fcimb.2017.00365/ful

Host serum factor triggers germination of Clostridium perfringens spores lacking the cortex hydrolysis machinery

Clostridium perfringens type A is the causative agent of a variety of histotoxic and enteric diseases. The ability of C. perfringens spores to germinate in vivo might be due to the presence of nutrient germinants in the host tissue and blood. In the current study, we investigated the ability of spores of C. perfringens wild-type and mutation strains to germinate in blood. Results indicate that spores of all three surveyed C. perfringens wild-type isolates germinated better in blood than in brain heart infusion (BHI) broth. However, as expected, spores lacking germinant receptor (GR) protein GerAA or GerKB germinated like wild-type spores in BHI broth and blood. Strikingly, while spores lacking GR proteins GerKA and GerKC showed significantly decreased germination in BHI broth, these spores germinated well in blood, suggesting that blood factor(s) can trigger spore germination through a GR-independent pathway. Using C. perfringens spores lacking cortex lytic enzymes (ΔcspB or ΔsleC ΔsleM), we were able to identify a host serum germination factor with peptidoglycan hydrolysing activity that (i) restored the colony-forming efficiencies of ΔcspB and ΔsleC ΔsleM spores up to ~5–20% of that of total colony-forming spores; (ii) increased the number of c.f.u. of decoated ΔcspB and ΔsleC ΔsleM spores to ~99% of that of colony-forming spores; (iii) and finally lost enzymic activity after heat inactivation, consistent with serum germination factor being an enzyme. Further characterization demonstrated that serum germination factor is very likely lysozyme, which can form a stable high molecular mass complex of ~120 kDa in serum. In conclusion, the current study indicates that a host serum germination factor with peptidoglycan hydrolysing activity is capable of triggering germination of C. perfringens spores by directly degrading the spore peptidoglycan cortex. Collectively, this study contributes to our understanding of the mechanism of in vivo germination of spores of C. perfringens

Transcriptional analysis of temporal gene expression in germinating Clostridium difficile 630 endospores.

Clostridium difficile is the leading cause of hospital acquired diarrhoea in industrialised countries. Under conditions that are not favourable for growth, the pathogen produces metabolically dormant endospores via asymmetric cell division. These are extremely resistant to both chemical and physical stress and provide the mechanism by which C. difficile can evade the potentially fatal consequences of exposure to heat, oxygen, alcohol, and certain disinfectants. Spores are the primary infective agent and must germinate to allow for vegetative cell growth and toxin production. While spore germination in Bacillus is well understood, little is known about C. difficile germination and outgrowth. Here we use genome-wide transcriptional analysis to elucidate the temporal gene expression patterns in C. difficile 630 endospore germination. We have optimized methods for large scale production and purification of spores. The germination characteristics of purified spores have been characterized and RNA extraction protocols have been optimized. Gene expression was highly dynamic during germination and outgrowth, and was found to involve a large number of genes. Using this genome-wide, microarray approach we have identified 511 genes that are significantly up- or down-regulated during C. difficile germination (p≤0.01). A number of functional groups of genes appeared to be co-regulated. These included transport, protein synthesis and secretion, motility and chemotaxis as well as cell wall biogenesis. These data give insight into how C. difficile re-establishes its metabolism, re-builds the basic structures of the vegetative cell and resumes growth

Characterization of the Adherence of Clostridium difficile Spores: The Integrity of the Outermost Layer Affects Adherence Properties of Spores of the Epidemic Strain R20291 to Components of the Intestinal Mucosa

Indexación: Web of Science.Clostridium difficile is the causative agent of the most frequently reported nosocomial diarrhea worldwide. The high incidence of recurrent infection is the main clinical challenge of C. difficile infections (CBI). Formation of C. difficile spores of the epidemic strain R20291 has been shown to be essential for recurrent infection and transmission of the disease in a mouse model. However, the underlying mechanisms of how these spores persist in the colonic environment remains unclear. In this work, we characterized the adherence properties of epidemic R20291 spores to components of the intestinal mucosa, and we assessed the role of the exosporium integrity in the adherence properties by using cdeC mutant spores with a defective exosporium layer. Our results showed that spores and vegetative cells of the epidemic R20291 strain adhered at high levels to monolayers of Caco-2 cells and mucin. Transmission electron micrographs of Caco-2 cells demonstrated that the hair-like projections on the surface of R20291 spores are in close proximity with the plasma membrane and microvilli of undifferentiated and differentiated monolayers of Caco-2 cells. Competitive-binding assay in differentiated Caco-2 cells suggests that spore-adherence is mediated by specific binding sites. By using spores of a cdeC mutant we demonstrated that the integrity of the exosporium layer determines the affinity of adherence of C. difficile spores to Caco-2 cells and mucin. Binding of fibronectin and vitronectin to the spore surface was concentration-dependent, and depending on the concentration, spore-adherence to Caco-2 cells was enhanced. In the presence of an aberrantly-assembled exosporium (cdeC spores), binding of fibronectin, but not vitronectin, was increased. Notably, independent of the exosporium integrity, only a fraction of the spores had fibronectin and vitronectin molecules binding to their surface. Collectively, these results demonstrate that the integrity of the exosporium layer of strain R20291 contributes to selective spore adherence to components of the intestinal mucosa.http://journal.frontiersin.org/article/10.3389/fcimb.2016.00099/ful

Clostridium difficile spores and its relevance in the persistence and transmission of the infection

Indexación: Web of Science; Scielo.Clostridium difficile es un patógeno anaerobio, formador de esporas y el agente etiológico más importante de las diarreas asociadas a antimicrobianos, tanto nosocomiales como adquiridas en la comunidad. Las infecciones asociadas a C. difficile poseen una elevada tasa de morbilidad en países desarrollados y en vías de desarrollo. Los dos factores de virulencia principales son TcdA y TcdB, toxinas que causan la remodelación del citoesqueleto lo cual desencadena los síntomas clínicos asociados a esta enfermedad infecciosa. A pesar que las esporas de C. difficile son el principal vehículo de infección, persistencia en el hospedero y de transmisión, pocos estudios se han enfocado sobre este clave aspecto. Es altamente probable que la espora juegue roles esenciales en los episodios de recurrencia y de transmisión horizontal de la infección por este microorganismo. Estudios recientes han revelado características únicas de las esporas de C. difficile que las hacen capaces de ser altamente transmisibles y persistir dentro del hospedero. Más aún, algunas de estas propiedades están relacionadas con la resistencia de sus esporas a los desinfectantes más comúnmente usados en los recintos hospitalarios. La presente revisión resume los conocimientos más relevantes en la biología de las esporas de C. difficile, con un énfasis en aquellos aspectos con implicancias clínicas, incluido el control de infecciones en el ambiente hospitalario.C. difficile is an anaerobic spore former pathogen and the most important etiologic agent of nosocomial and community acquired antibiotics associated diarrheas. C. difficile infections (CDI) are responsible for an elevated rate of morbidity in developed and developing countries. Although the major virulence factors responsible for clinical symptoms of CDI are the two toxins TcdA and TcdB, C. difficile spores are the main vehicle of infection, persistence and transmission of CDI. Recent work has unrevealed unique properties of C. difficile spores that make them remarkable morphotypes of persistence and transmission in the host, including their resistance to antibiotics, the host immune response and disinfectants. The present review summarizes relevant aspects of C. difficile spore biology that have major implications from a clinical and medical perspective.http://ref.scielo.org/3xfbk

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

Analysis of the Germination of Individual Clostridium sporogenes Spores with and without Germinant Receptors and Cortex-Lytic Enzymes

The Gram-positive spore-forming anaerobe Clostridium sporogenes is a significant cause of food spoilage, and it is also used as a surrogate for C. botulinum spores for testing the efficacy of commercial sterilization. C. sporogenes spores have also been proposed as a vector to deliver drugs to tumor cells for cancer treatments. Such an application of C. sporogenes spores requires their germination and return to life. In this study, Raman spectroscopy and differential interference contrast (DIC) microscopy were used to analyze the germination kinetics of multiple individual C. sporogenes wild-type and germination mutant spores. Most individual C. sporogenes spores germinated with L-alanine began slow leakage of ∼5% of their large Ca-dipicolinic acid (CaDPA) depot at T1, all transitioned to rapid CaDPA release at Tlag1, completed CaDPA release at Trelease, and finished peptidoglycan cortex hydrolysis at Tlys. T1, Tlag1, Trelease, and Tlys times for individual spores were heterogeneous, but ΔTrelease (Trelease – Tlag1) periods were relatively constant. However, variability in T1 (or Tlag1) times appeared to be the major reason for the heterogeneity between individual spores in their germination times. After Trelease, some spores also displayed another lag in rate of change in DIC image intensity before the start of a second obvious DIC image intensity decline of 25–30% at Tlag2 prior to Tlys. This has not been seen with spores of other species. Almost all C. sporogenes spores lacking the cortex-lytic enzyme (CLE) CwlJ spores exhibited a Tlag2 in L-alanine germination. Sublethal heat treatment potentiated C. sporogenes spore germination with L-alanine, primarily by shortening T1 times. Spores without the CLEs SleB or CwlJ exhibited greatly slowed germination with L-alanine, but spores lacking all germinant receptor proteins did not germinate with L-alanine. The absence of these various germination proteins also decreased but did not abolish germination with the non-GR-dependent germinants dodecylamine and CaDPA, but spores without CwlJ exhibited no germination with CaDPA. Finally, C. sporogenes spores displayed commitment in germination, but memory in GR-dependent germination was small, and less than the memory in Bacillus spore germination