Clostridioides difficile Flagella.

Clostridioides difficile is an important pathogen for humans with a lead in nosocomial infection, but it is also more and more common in communities. Our knowledge of the pathology has historically been focused on the toxins produced by the bacteria that remain its major virulence factors. But the dysbiosis of the intestinal microbiota creating the conditions for the colonization appears to be fundamental for our understanding of the disease. Colonization implies several steps for the bacteria that do or do not use their capacity of motility with the synthesis of flagella. In this review, we focus on the current understanding of different topics on the C. difficile flagellum, ranging from its genetic organization to the vaccinal interest in it.

Clostridioides difficile Flagellin Activates the Intracellular NLRC4 Inflammasome.

Clostridioides difficile (C. difficile), is a major cause of nosocomial diarrhea and colitis. C. difficile flagellin FliC contributes toxins to gut inflammation by interacting with the immune Toll-like receptor 5 (TLR5) to activate nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) signaling pathways. Flagella of intracellular pathogens can activate the NLR family CARD domain-containing protein 4 (NLRC4) inflammasome pathway. In this study, we assessed whether flagellin of the extracellular bacterium C. difficile internalizes into epithelial cells and activates the NLRC4 inflammasome. Confocal microscopy showed internalization of recombinant green fluorescent protein (GFP)-FliC into intestinal Caco-2/TC7 cell line. Full-length GFP-FliC activates NLRC4 in Caco-2/TC7 cells in contrast to truncated GFP-FliC lacking the C-terminal region recognized by the inflammasome. FliC induced cleavage of pro-caspase-1 into two subunits, p20 and p10 as well as gasdermin D (GSDMD), suggesting the caspase-1 and NLRC4 inflammasome activation. In addition, colocalization of GFP-FliC and pro-caspase-1 was observed, indicating the FliC-dependent NLRC4 inflammasome activation. Overexpression of the inflammasome-related interleukin (interleukin (IL)-1ß, IL-18, and IL-33) encoding genes as well as increasing of the IL-18 synthesis was detected after cell stimulation. Inhibition of I-kappa-B kinase alpha (IKK-α) decreased the FliC-dependent inflammasome interleukin gene expression suggesting a role of the NF-κB pathway in regulating inflammasome. Altogether, these results suggest that FliC internalizes into the Caco-2/TC7 cells and activates the intracellular NLRC4 inflammasome thus contributing to the inflammatory process of C. difficile infection.

Arr-cb Is a Rifampin Resistance Determinant Found Active or Cryptic in Clostridium bolteae Strains.

Clostridiumbolteae, which belongs to the Clostridium clostridioforme complex, is a member of the human gut microbiota. Recent analysis of seven genomes of Cbolteae revealed the presence of an arr-like gene. Among these strains, only 90A7 was found to be resistant to rifampin in the absence of alteration of RpoB. Cloning of arr-cb from 90A7 in Escherichia coli combined with directed mutagenesis demonstrated that Arr-cb was functional but that a Q127→R variant present in 90A9 and 90B3 was inactive. Quantitative reverse transcription-PCR analysis indicated that arr-cb was silent in the four remaining strains because of defective transcription. Thus, two independent mechanisms can make the probably intrinsic arr-cb gene of Cbolteae cryptic.

Comparative genomics of Clostridium bolteae and Clostridium clostridioforme reveals species-specific genomic properties and numerous putative antibiotic resistance determinants.

BACKGROUND: Clostridium bolteae and Clostridium clostridioforme, previously included in the complex C. clostridioforme in the group Clostridium XIVa, remain difficult to distinguish by phenotypic methods. These bacteria, prevailing in the human intestinal microbiota, are opportunistic pathogens with various drug susceptibility patterns. In order to better characterize the two species and to obtain information on their antibiotic resistance genes, we analyzed the genomes of six strains of C. bolteae and six strains of C. clostridioforme, isolated from human infection. RESULTS: The genome length of C. bolteae varied from 6159 to 6398 kb, and 5719 to 6059 CDSs were detected. The genomes of C. clostridioforme were smaller, between 5467 and 5927 kb, and contained 5231 to 5916 CDSs. The two species display different metabolic pathways. The genomes of C. bolteae contained lactose operons involving PTS system and complex regulation, which contribute to phenotypic differentiation from C. clostridioforme. The Acetyl-CoA pathway, similar to that of Faecalibacterium prausnitzii, a major butyrate producer in the human gut, was only found in C. clostridioforme. The two species have also developed diverse flagella mobility systems contributing to gut colonization. Their genomes harboured many CDSs involved in resistance to beta-lactams, glycopeptides, macrolides, chloramphenicol, lincosamides, rifampin, linezolid, bacitracin, aminoglycosides and tetracyclines. Overall antimicrobial resistance genes were similar within a species, but strain-specific resistance genes were found. We discovered a new group of genes coding for rifampin resistance in C. bolteae. C. bolteae 90B3 was resistant to phenicols and linezolide in producing a 23S rRNA methyltransferase. C. clostridioforme 90A8 contained the VanB-type Tn1549 operon conferring vancomycin resistance. We also detected numerous genes encoding proteins related to efflux pump systems. CONCLUSION: Genomic comparison of C. bolteae and C. clostridiofrome revealed functional differences in butyrate pathways and in flagellar systems, which play a critical role within human microbiota. Most of the resistance genes detected in both species were previously characterized in other bacterial species. A few of them were related to antibiotics inactive against Clostridium spp. Some were part of mobile genetic elements suggesting that these commensals of the human microbiota act as reservoir of antimicrobial resistances.

Clostridium difficile flagella predominantly activate TLR5-linked NF-κB pathway in epithelial cells.

Clostridium difficile has become the most common enteropathogen responsible for intestinal nosocomial post-antibiotic infections. This has coincided with the appearance of serious cases related to the emergence of hypervirulent strains. The toxins are the main virulence factors and elicit an inflammatory response during C. difficile infection. However, other bacterial components appear to be involved in the inflammatory process. In some pathogens, flagella play a role in pathogenesis through abnormal stimulation of the TLR5-mediated host immune response. To date, few studies have addressed this role for C. difficile flagella. In the current study, we confirm in two different epithelial cell models that C. difficile thanks to its FliC flagellin interacts with TLR5. In addition, thanks to inhibition and transcriptomic studies we demonstrate that the interaction of flagellin and TLR5 predominantly activates the NF-κB and, in a lesser degree, the MAPK pathways, via TLR5, leading to up-regulation of pro-inflammatory gene expression and synthesis of pro-inflammatory mediators. These results suggest a role for C. difficile flagella in contributing to inflammatory response in host intestinal cells.

Distribution of the vanG-like gene cluster in Clostridium difficile clinical isolates.

Treatment of Clostridium difficile infections generally requires cessation of their causative antibiotic and subsequent administration of metronidazole or vancomycin. Intriguingly, the genome of C. difficile 630 contains a cryptic gene cluster homologous to the vanG-type operon of Enterococcus faecalis BM4518. We detected this cluster by PCR in 35 out of 41 clinical isolates, confirming its large prevalence in this species. The cluster was found to be located in a unique locus. Comparison of this locus with that of strains devoid of the vanG-like cluster indicated that acquisition of the gene cluster occurred in a perfect 19-bp inverted repeat, in the absence of a detectable mobile structure.

Clostridium clostridioforme and Atopobium minutum clinical isolates with vanB-type resistance in France.

Acquired vancomycin resistance in Gram-positive anaerobes has been reported only in Australia and Canada from rare vanB-positive stool samples in the absence of vancomycin-resistant enterococci (VRE). We report the emergence of VanB-type resistance in Clostridium clostridioforme and Atopobium minutum involved in human infections in France.

Analysis of the mobilization functions of the vancomycin resistance transposon Tn1549, a member of a new family of conjugative elements.

Conjugative transfer from Clostridium symbiosum to enterococci of Tn1549, which confers VanB-type vancomycin resistance, has been reported. This indicates the presence of a transfer origin (oriT) in the element. Transcription analysis of Tn1549 indicated that orf29, orf28, orfz, and orf27 were cotranscribed. A pACYC184 derivative containing 250 bp intergenic to orf29-orf30 of Tn1549 was mobilized in Escherichia coli recA::RP4::Delta nic provided that orf28 and orf29 were delivered simultaneously. These open reading frame (ORF) genes were able to promote mobilization in trans, but a cis-acting preference was observed. On the basis of a mobilization assay, a minimal 28-bp oriT was delimited, although the frequency of transfer was significantly reduced compared to that of a 130-bp oriT fragment. The minimal oriT contained an inverted repeat and a core, which was homologous to the cleavage sequence found in certain Gram-positive rolling-circle replicating (RCR) plasmids. While Orf29 was a mobilization accessory component similar to MobC proteins, Orf28 was identified as a relaxase belonging to a new phyletic cluster of the MOB(p) superfamily. The nick site was identified within oriT by an oligonucleotide cleavage assay. Closely related oriTs linked to mobilization genes were detected in data banks; they were found in various integrative and conjugative elements (ICEs) originating mainly from anaerobes. These results support the notion that Tn1549 is a member of a MOB(p) clade. Interestingly, the Tn1549-derived constructs were mobilized by RP4 in E. coli, suggesting that a relaxosome resulting from DNA cleavage by Orf28 interacted with the coupling protein TraG. This demonstrates the capacity of Tn1549 to be mobilized by a heterologous transfer system.

Differential requirement for the translocation of clostridial binary toxins: iota toxin requires a membrane potential gradient.

Clostridial binary toxins, such as Clostridium perfringens Iota and Clostridium botulinum C2, are composed of a binding protein (Ib and C2-II, respectively) that recognizes distinct membrane receptors and mediates internalization of a catalytic protein (Ia and C2-I, respectively) with ADP-ribosyltransferase activity that depolymerizes the actin cytoskeleton. After internalization, it was found that C2 and Iota toxins were not routed to the Golgi apparatus and exhibited differential sensitivity to inhibitors of endosome acidification. While the C2-I component of C2 toxin was translocated into the cytosol from early endosomes, translocation of the Ia component of Iota toxin occurred between early and late endosomes, was dependent on more acidic conditions, and uniquely required a membrane potential gradient.

A cfr-like gene cfr(C) conferring linezolid resistance is common in Clostridium difficile.

Clostridium difficile T10 and Clostridium bolteae 90B3 were co-resistant to phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A (PhLOPSA) and harbored an unreported cfr-like determinant that may alter the 23S rRNA by m8A2503 methylation. The cfr-like cfr(C) gene was cloned in C. difficile 630Δerm in which it conferred PhLOPSA resistance. In C. bolteae 90B3: (i) qRT-PCR analysis indicated that cfr(C) was similarly expressed in the absence or presence of either chloramphenicol or clindamycin or linezolid; and (ii) cfr(C) was part of a putative 24 kb-transposon, which generated a detectable circular intermediate. An element differing by a single nucleotide was found in C. difficile DA00203 from GenBank data, consistent with a recent horizontal transfer. In silico analysis showed cfr(C) in 19 out of 274 C. difficile genomes. This gene was also detected by PCR analysis in 9 out of 80 C. difficile from our laboratory strain collection according to resistance to linezolid and florfenicol. The fact that cfr(C) was mainly confined in C. difficile within polymorphic environments indicates this microorganism is a reservoir for PhLOPSA resistance.

Clostridium difficile flagella induce a pro-inflammatory response in intestinal epithelium of mice in cooperation with toxins.

Clostridium difficile is the most important enteropathogen involved in gut nosocomial post-antibiotic infections. The emergence of hypervirulent strains has contributed to increased mortality and morbidity of CDI. The C. difficile toxins contribute directly to CDI-associated lesions of the gut, but other bacterial factors are needed for the bacteria to adhere and colonize the intestinal epithelium. The C. difficile flagella, which confer motility and chemotaxis for successful intestinal colonization, could play an additional role in bacterial pathogenesis by contributing to the inflammatory response of the host and mucosal injury. Indeed, by activating the TLR5, flagella can elicit activation of the MAPK and NF-κB cascades of cell signaling, leading to the secretion of pro-inflammatory cytokines. In the current study, we demonstrate, by using an animal model of CDI, a synergic effect of flagella and toxins in eliciting an inflammatory mucosal response. In this model, the absence of flagella dramatically decreases the degree of mucosal inflammation in mice and the sole presence of toxins without flagella was not enough to elicit epithelial lesions. These results highlight the important role of C. difficile flagella in eliciting mucosal lesions as long as the toxins exert their action on the epithelium.

Interaction between the two subdomains of the C-terminal part of the botulinum neurotoxin A is essential for the generation of protective antibodies.

The botulinum neurotoxin A C-terminal fragment (Hc), which mediates the binding of the toxin to neuronal cell surface receptors, comprises two subdomains, Hc-N (amino acids 873-1095) and Hc-C (amino acids 1096-1296). In order to define the minimal fragment of Hc carrying protective antigenic properties, Hc, Hc-N and Hc-C have been produced as recombinant proteins in Escherichia coli, and have been tested for their antigenicity in mouse protection assays. Hc, Hc-N and Hc-C induced similar antibody levels as shown by ELISA. However, a single immunization with Hc (10 microg) fully protected mice challenged with 10(3) mouse lethal dose 50 of toxin, whereas Hc-N, Hc-C, or Hc-N plus Hc-C did not give any protection. Triple immunizations with Hc-N or Hc-C were necessary to induce a higher level of protection. Circular dichroism and fluorescence studies showed that the isolated subdomains were folded and stable. However, an intense near-UV dichroic signal was only observed in the Hc spectrum, revealing a highly structured interface between both subdomains. Taken together, the results show that the generation of protective antibodies requires the whole Hc domain and especially the native structure of the interfacial region between Hc-N and Hc-C.

[Botulism: the agent, mode of action of the botulinum neurotoxins, forms of acquisition, treatment and prevention]. / Le botulisme: agent, mode d'action des neurotoxines botuliques, formes d'acquisition, traitement et prévention.

The botulinum neurotoxins are produced by anaerobic, spore-forming bacteria belonging to the Clostridium genus. They are synthesised as a single chain protein (150 kDa), which is not or weakly active. The active form results from a proteolysis cleaving the precursor in a light chain (about 50 kDa) and a heavy chain (about 100 kDa), which are linked by a disulfide bridge. The heavy chain is involved in the recognition of a specific neuronal surface receptor and mediates the internalization of the light chain into the cytosol. The light chain is responsible for the intracellular activity. It catalyses the proteolysis of SNARE proteins, which are involved in the exocytosis of synaptic vesicles containing acetylcholine. Hence, the release of acetylcholine at the neuromuscular junction is blocked, leading to a flaccid paralysis. Human botulism, usually type A, B or E, is associated with intoxination, ingestion of preformed toxin in food, with digestive toxi-infection, mainly in newborns (infant botulism), or with wound contamination (wound botulism). The treatment of botulism is usually symptomatic. The specific treatment is based on the serotherapy or on the use of purified specific antibodies. The vaccination against botulism is efficient. However, since the botulinum neurotoxins are widely used for the treatment of numerous dystonias, a generalised vaccination is not conceivable.

BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani.

Clostridium botulinum and Clostridium tetani, respectively, produce potent toxins, botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeTx), which are responsible for severe diseases, botulism and tetanus. Neurotoxin synthesis is a regulated process in Clostridium. The genes botR/A in C. botulinum A and tetR in C. tetani positively regulate expression of BoNT/A and associated non-toxic proteins (ANTPs), as well as TeTx respectively. The botR/A gene lies in close vicinity of the two operons which contain bont/A and antps genes in C. botulinum A, and tetR immediately precedes the tetX gene in C. tetani. We show that BotR/A and TetR function as specific alternative sigma factors rather than positive regulators based on the following results: (i) BotR/A and TetR associated with target DNAs only in the presence of the RNA polymerase core enzyme (Core), (ii) BotR/A and TetR directly bound with the core enzyme, (iii) BotR/A-Core recognized -35 and -10 regions of ntnh-bont/A promoter and (iv) BotR/A and TetR triggered in vitro transcription from the target promoters. In C. botulinum A, bont/A and antps genes are transcribed as bi- and tricistronic operons controlled by BotR/A. BotR/A and TetR are seemingly related to a new subgroup of the sigma70 family that includes TcdR and UviA, which, respectively, regulate production of toxins A and B in C. difficile and bacteriocin in C. perfringens. Sequences of -35 region are highly conserved in the promoter of target toxin genes in C. botulinum, C. tetani, C. difficile and C. perfringens. Overall, a common regulation mechanism probably controls toxin gene expression in these four toxigenic clostridial species.

Detergent-resistant membrane microdomains facilitate Ib oligomer formation and biological activity of Clostridium perfringens iota-toxin.

Clostridium perfringens iota-toxin consists of two separate proteins identified as a cell binding protein, iota b (Ib), which forms high-molecular-weight complexes on cells generating Na(+)/K(+)-permeable pores through which iota a (Ia), an ADP-ribosyltransferase, presumably enters the cytosol. Identity of the cell receptor and membrane domains involved in Ib binding, oligomer formation, and internalization is currently unknown. In this study, Vero (toxin-sensitive) and MRC-5 (toxin-resistant) cells were incubated with Ib, after which detergent-resistant membrane microdomains (DRMs) were extracted with cold Triton X-100. Western blotting revealed that Ib oligomers localized in DRMs extracted from Vero, but not MRC-5, cells while monomeric Ib was detected in the detergent-soluble fractions of both cell types. The Ib protoxin, previously shown to bind Vero cells but not form oligomers or induce cytotoxicity, was detected only in the soluble fractions. Vero cells pretreated with phosphatidylinositol-specific phospholipase C before addition of Ib indicated that glycosylphosphatidyl inositol-anchored proteins were minimally involved in Ib binding or oligomer formation. While pretreatment of Vero cells with filipin (which sequesters cholesterol) had no effect, methyl-beta-cyclodextrin (which extracts cholesterol) reduced Ib binding and oligomer formation and delayed iota-toxin cytotoxicity. These studies showed that iota-toxin exploits DRMs for oligomer formation to intoxicate cells.

Organization and regulation of the neurotoxin genes in Clostridium botulinum and Clostridium tetani.

Botulinum and tetanus neurotoxins are structurally and functionally related 150 kDa proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin associates with non-toxic proteins to form complexes of various sizes. The botulinum neurotoxin and non-toxic protein genes are clustered in a DNA segment called the botulinum locus. This locus is probably located on a mobile or degenerate mobile element, which accounts for the various genomic localizations (chromosome, plasmid, phage) in different Clostridium botulinum types. The botulinum neurotoxin and non-toxic protein genes are organized in two polycistronic operons (ntnh-bont and ha operons) transcribed in opposite orientations. The gene that separates the two operons of the botulinum locus in C. botulinum A encodes a 21 kDa protein BotR/A, which is a positive regulator of the expression of the botulinum locus genes. Similarly, in Clostridium tetani, the gene located immediately upstream of the tetanus toxin gene, encodes a positive regulatory protein, TetR. BotR and TetR are possibly alternative sigma factors related to TxeR and UviA, which regulate C. difficile toxin and C. perfringens bacteriocin production, respectively. TxeR and UviA define a new sub-group of the sigma(70) family of RNA polymerase initiation factors. In addition, the C. botulinum genome contains predicted two-component system genes, some of which are possibly involved in regulation of toxinogenesis.

Clostridium perfringens iota toxin: characterization of the cell-associated iota b complex.

Clostridium perfringens type E iota toxin consists of two unlinked proteins designated as iota a (Ia; molecular mass approximately 47 kDa), an ADP-ribosyltransferase and iota b (Ib; molecular mass approximately 81 kDa) which binds to the cell surface and facilitates Ia entry into the cytosol. By Western-blot analysis, Ib incubated with Vero cells at 37 degrees C generated a cell-associated, SDS-insoluble oligomer of Ib (molecular mass>220 kDa) within 15 s, which was still evident 110 min after washing cells. Ib oligomerization was temperature, but not pH, dependent and was facilitated by a cell-surface protein(s). Within 5 min at 37 degrees C, cell-bound Ib generated Na(+)/K(+) permeable channels that were blocked by Ia. However, Ib-induced channels or oligomers were not formed at 4 degrees C. Two monoclonal antibodies raised against Ib that recognize unique, neutralizing epitopes within residues 632-655 either inhibited Ib binding to cells and/or oligomerization, unlike a non-neutralizing monoclonal antibody that binds within Ib residues 28-66. The Ib protoxin (molecular mass approximately 98 kDa), which does not facilitate iota cytotoxicity but binds to Vero cells, did not oligomerize or form ion-permeable channels on cells, and neither trypsin nor chymotrypsin treatment of cell-bound Ib protoxin induced large complex formation. The link between Ib oligomers and iota toxicity was also apparent with a resistant cell line (MRC-5), which bound to Ib with no evidence of oligomerization. Overall, these studies revealed that the biological activity of iota toxin is dependent on a long-lived, cell-associated Ib complex that rapidly forms ion-permeable channels in cell membranes. These results further reveal the similarities of C. perfringens iota toxin with other bacterial binary toxins produced by Bacillus anthracis and C. botulinum.

Transcytosis of iota-toxin across polarized CaCo-2 cells.

Iota-toxin from Clostridium perfringens type E is a binary toxin consisting of two independent proteins, an enzymatic Ia and binding Ib component. Ia catalyses ADP-ribosylation of actin monomers, thus disrupting the actin cytoskeleton. In this report, we show that Ia plus Ib applied apically or basolaterally induce a rapid decrease in the transepithelial resistance (TER) of CaCo-2 cell monolayers and disorganization of actin filaments as well as the tight and adherens junctions. Ib alone, on the apical or basolateral side, slowly decreased the TER without affecting the actin cytoskeleton, possibly via pore formation. Interestingly, the two iota-toxin components inoculated separately on each cell surface induced cytopathic effects and a TER decrease. Anti-Ib sera, raised against the whole molecule or the Ia docking domain and applied to the opposite cell side versus Ib, neutralized the TER decrease. In addition, radioactive Ib incubated in the basolateral compartment was detected on the apical side by selective cell surface biotinylation. This argues for a transcytotic routing of Ib to mediate internalization of Ia from the opposite cell surface. Bafilomycin A1 also prevented the cytopathic effects of Ia and Ib applied separately to each cell side, possibly by blocking translocation of Ia into the cytosol and/or the intracellular transport of Ib. Ib is either routed into the cell independently of Ia, trans-cytosed and permanently exposed on the opposite cell surface or continuously recycled between an endosomal compartment and the cell surface.

Clostridium perfringens iota toxin. Mapping of the Ia domain involved in docking with Ib and cellular internalization.

Clostridium perfringens iota toxin consists of two unlinked proteins. The binding component (Ib) is required to internalize into cells an enzymatic component (Ia) that ADP-ribosylates G-actin. To characterize the Ia domain that interacts with Ib, fusion proteins were constructed between the C. botulinum C3 enzyme, which ADP-ribosylates Rho, and various truncated versions of Ia. These chimeric molecules retained the wild type ADP-ribosyltransferase activity specific for Rho and were recognized by antibodies against C3 enzyme and Ia. Internalization of each chimera into Vero cells was assessed by measuring the disorganization of the actin cytoskeleton and intracellular ADP-ribosylation of Rho. Fusion proteins containing C3 linked to the C terminus of Ia were transported most efficiently into cells like wild type Ia in an Ib-dependent manner that was blocked by bafilomycin A1. The minimal Ia fragment that promoted translocation of Ia-C3 chimeras into cells consisted of 128 central residues (129-257). These findings revealed that iota toxin is a suitable system for mediating the entry of heterologous proteins such as C3 into cells.