Jean-Christophe Marvaud

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: The interaction between model lipid membranes and the binding component (Ib) of the ADP-ribosylating iota-toxin of Clostridium perfringens was studied in detail. Ib had to be activated by trypsin to result in channel formation in artificial lipid bilayers. The channels formed readily by Ib had a small single-channel conductance of about 85 picosiemens in 1 m KCl. Channel function was blocked in single-channel and multichannel experiments by the enzymatic component Ia in a pH-dependent manner. The strong Ia-mediated channel block of Ib occurred only when the pH was at least lowered to pH 5.6. The single-channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific. Zero current membrane potential measurements suggested the Ib channel has an approximately 6-fold higher permeability for potassium ions than for chloride. The selectivity ratio changed for salts composed of cations and anions of different mobility in the aqueous phase, again suggesting that Ib formed a water-filled general diffusion pore. Asymmetric addition of activated Ib to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane. Titration experiments with chloroquine and different tetraalkylammonium ions suggested that the Ib channel was blocked by these compounds but had only a weak affinity to them. In vivo measurements using Vero cells demonstrate that chloroquine and related molecules also did not efficiently block intoxication of the cells by iota-toxin. The possible role of Ib in the translocation of iota-toxin across the target cell membrane is discussed.

Abstract: 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.

Abstract: 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.

Abstract: Botulinum neurotoxins are produced by anaerobic spore forming bacteria, Clostridiumbotulinum. They are synthesized as a single chain protein (150kDa) which is not or weakly active. The active form results from proteolysis that cleaves the precursor into a light chain (about 50kDa) and a heavy chain (about 100kDa) which are linked by a disulfide bridge. The heavy chain is involved in the recognition of a specific neurone surface receptor and mediates the internalization of the light chain into the cytosol. The light chain is responsible for the intracellular activity. It catalyzes 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. The tetanus neurotoxin shares with botulinum neurotoxins a common structure and mechanism of action. Tetanus neurotoxin blocks the release of neurotransmitters in the inhibitory interneurons leading to spastic paralysis. The paralytic properties of the botulinum neurotoxins are used to treat certain myoclonies such as blepharospasm, torticolis, hemifacial paralysis. Botulinum neurotoxins are thus efficient therapeutic agents helpful in avoiding surgery.

Abstract: Clostridium perfringens iota-toxin is a binary toxin consisting of iota a (Ia), an ADP-ribosyltransferase that modifies actin, and iota b (Ib), which binds to a cell surface protein and translocates Ia into a target cell. Fusion proteins of recombinant Ib and truncated variants were tested for binding to Vero cells and docking with Ia via fluorescence-activated cytometry and cytotoxicity experiments. C-terminal residues (656 to 665) of Ib were critical for cell surface binding, and truncated Ib variants containing > or = 200 amino acids of the C terminus were effective Ib competitors and prevented iota cytotoxicity. The N-terminal domain (residues 1 to 106) of Ib was important for Ia docking, yet this region was not an effective competitor of iota cytotoxicity. Further studies showed that Ib lacking just the N-terminal 27 residues did not facilitate Ia entry into a target cell and subsequent cytotoxicity. Five monoclonal antibodies against Ib were also tested with each truncated Ib variant for epitope and structural mapping by surface plasmon resonance and an enzyme-linked immunosorbent assay. Each antibody bound to a linear epitope within the N terminus (residues 28 to 66) or the C terminus (residues 632 to 655). Antibodies that target the C terminus neutralized in vitro cytotoxicity and delayed the lethal effects of iota-toxin in mice.

Abstract: The binding characteristics of iota toxin, a binary enterotoxin produced by Clostridium perfringens type E, were studied by fluorescence-activated cytometry. The proteolytically activated binding component of iota toxin, iota b (Ib), bound to various cell types when incubated at 4, 25, or 37 degrees C for 10 min. The binding of Ib was inhibited by antisera against C. perfringens type E or Clostridium spiroforme culture supernatants, but not C. perfringens types C or D. Pretreatment of Vero cells with glycosidases or lectins did not affect Ib interactions, while pronase effectively prevented Ib binding to the cell surface. The Ib protomer (Ibp) bound to the cell surface, but trypsinization of Ibp was necessary for docking of the ADP-ribosylating component, iota a (Ia). Ia attached to cell-bound Ib within 10 min at 37 degrees C, but surface levels of Ia decreased 90% after 30 min and were undetectable by 60 min. Detectable surface levels of Ib also diminished over time, and Western blot analysis suggested internalization or embedment of Ib into the membrane.

Abstract: Clostridium botulinum type A hemagglutinin-positive progenitor toxin consists of three distinct components: neurotoxin (NTX), hemagglutinin (HA), and non-toxic non-HA (NTNH). The HA consists of four subcomponents designated HA1, 2, 3a and 3b. By employing purified toxin and GST-fusion proteins of each HA subcomponent, we found that the HA-positive progenitor toxin, GST-HA1 and GST-HA3b bind to human erythrocytes and microvilli of guinea pig upper small intestinal sections. The HA-positive progenitor toxin and GST-HA1 bind via galactose moieties, GST-HA3b binds via sialic acid moieties. GST-2 and GST-3a showed no detectable binding.

Abstract:

Abstract: Mycoplasma fermentans lipoproteins (LAMPf) are capable of activating macrophages and inducing the secretion of proinflammatory cytokines. We have recently reported that mitogen-activated protein kinase (MAPK) pathways and NF-kappaB and activated protein 1 (AP-1) play a crucial role in the activation induced by this bacterial compound. To further elucidate the mechanisms by which LAMPf mediate the activation of macrophages, we assessed the effects of inhibiting small G proteins Rac, Cdc42, and Rho. The Rho-specific inhibitor C3 enzyme completely abolished the secretion of tumor necrosis factor alpha by macrophages stimulated with LAMPf and also inhibited the activation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 kinase. In addition, we have shown that LAMPf stimulate Cdc42 and that inhibition of Cdc42 or Rac by dominant negative mutants abrogates LAMPf-mediated activation of JNK and transactivation of NF-kappaB and AP-1 in the murine macrophage cell line RAW 264.7. These results indicate that small G proteins Rho, Cdc42, and Rac are involved in the cascade of events leading to the macrophage activation by mycoplasma lipoproteins.

Abstract: Lethal toxin (LT) from Clostridium sordellii has been shown in HeLa cells to glucosylate and inactivate Ras and Rac and, hence, to disorganize the actin cytoskeleton. In the present work, we demonstrate that LT treatment provokes the same effects in HL-60 cells. We show that guanosine 5'-O-(3-thiotriphosphate)-stimulated phospholipase D (PLD) activity is inhibited in a time- and dose-dependent manner after an overnight treatment with LT. A similar dose response to the toxin was found when PLD activity was stimulated by phorbol 12-myristate 13-acetate via the protein kinase C pathway. The toxin effect on actin organization seemed unlikely to account directly for PLD inhibition as cytochalasin D and iota toxin from Clostridium perfringens E disorganize the actin cytoskeleton without modifying PLD activity. However, the enzyme inhibition and actin cytoskeleton disorganization could both be related to a major decrease observed in phosphatidylinositol 4,5-bisphosphate (PtdIns(4, 5)P2). Likely in a relationship with this decrease, recombinant ADP-ribosylation factor, RhoA, Rac, and RalA were not able to reconstitute PLD activity in LT-treated cells permeabilized and depleted of cytosol. Studies of phosphoinositide kinase activities did not allow us to attribute the decrease in PtdIns(4,5)P2 to inactivation of PtdIns4P 5-kinase. LT was also found to provoke a major inhibition in phosphatidylinositol 3-kinase that could not account for the inhibition of PLD activity because wortmannin, at doses that fully inhibit phosphatidylinositol 3-kinase, had no effect on the phospholipase activity. Among the three small G-proteins, Ras, Rac, and RalA, inactivated by LT and involved in PLD regulation, inactivation of Ral proteins appeared to be responsible for PLD inhibition as LT toxin (strain 9048) unable to glucosylate Ral proteins did not modify PLD activity. In HL-60 cells, LT treatment appeared also to modify cytosol components in relationship with PLD inhibition as a cytosol prepared from LT-treated cells was less efficient than one from control HL-60 cells in stimulating PLD activity. Phosphatidylinositol transfer proteins involved in the regulation of polyphosphoinositides and ADP-ribosylation factor, a major cytosolic PLD activator in HL-60 cells, were unchanged, whereas the level of cytosolic protein kinase Calpha was decreased after LT treatment. We conclude that in HL-60 cells, lethal toxin from C. sordellii, in inactivating small G-proteins involved in PLD regulation, provokes major modifications at the membrane and the cytosol levels that participate in the inhibition of PLD activity. Although Ral appeared to play an essential role in PLD activity, we discuss the role of other small G-proteins inactivated by LT in the different modifications observed in HL-60 cells.

Abstract: Numerous bacterial toxins recognize the actin cytoskeleton as a target. The clostridial binary toxins (Iota and C2 families) ADP-ribosylate the actin monomers causing the dissociation of the actin filaments. The large clostridial toxins from Clostridium difficile, Clostridium sordellii and Clostridium novyi inactivate, by glucosylation, proteins from the Rho family that regulate actin polymerization. In contrast, the cytotoxic necrotic factor from Escherichia coli activates Rho by deamidation and increases the formation of actin filaments. The enterotoxin of Bacteroides fragilis is a protease specific for E-cadherin and it promotes the reorganization of the actin cytoskeleton. The bacterial toxins that modify the actin cytoskeleton induce various cell disfunctions including changes in cell barrier permeability and disruption of intercellular junctions.

Abstract: The genes of the botulinum neurotoxin A (BoNT) complex are clustered in a locus consisting of two divergent polycistronic operons, one containing the non-toxic, non-haemagglutinin (NTNH) component and bontA genes, the other containing the haemagglutinin (HA) component genes. The two operons are separated by a gene (botR/A, previously called orf21) encoding a 21 kDa protein. A recombinant Clostridium botulinum A strain that overexpresses botR/A was constructed by electroporating strain 62 with the vector pAT19 containing botR/A under the control of its own promoter. The transformed strain produced more BoNT/A and associated non-toxic proteins (ANTPs) and the corresponding mRNAs than the non-transformed strain. Partial inhibition of botR/A by antisense mRNA resulted in lower levels of BoNT/A, NTNH and HA70 and the levels of the corresponding mRNAs. Gel mobility shift assays and immunoprecipitations showed that BotR/A bound to the DNA promoter region upstream from the two BoNT/A complex operons. These results show that botR/A activated transcription of the genes encoding BoNT/A and ANTPs in C. botulinum A by interacting directly with the region promoter, and that the homologous genes in C. botulinum B, C and D presumably have the same function.

Abstract: The TetR gene immediately upstream from the tetanus toxin (TeTx) gene was characterized. It encodes a 21,562-Da protein which is related (50 to 65% identity) to the equivalent genes (botR) in Clostridium botulinum. TetR has the feature of a DNA binding protein with a basic pI (9.53). It contains a helix-turn-helix motif and shows 29% identity with other putative regulatory genes in Clostridium, i.e., uviA from C. perfringens and txeR from C. difficile. We report for the first time the transformation of C. tetani by electroporation, which permitted us to investigate the function of tetR. Overexpression of tetR in C. tetani induced an increase in TeTx production and in the level of the corresponding mRNA. This indicates that TetR is a transcriptional activator of the TeTx gene. Overexpression of botR/A (60% identity with TetR at the amino acid level) in C. tetani induced an increase in TeTx production comparable to that for overexpression of tetR. However, botR/C (50% identity with TetR at the amino acid level) was less efficient. This supports that TetR positively regulates the TeTx gene in C. tetani and that a conserved mechanism of regulation of the neurotoxin genes is involved in C. tetani and C. botulinum.

Abstract: The botulinal neurotoxins (BoNT) associate with non-toxic proteins (ANTP) by non-covalent bonds to form large complexes. In C. botulinum C, the BoNT/C1 locus consists of six genes which are organized in three clusters. Cluster 1 encompasses the genes of BoNT/C1 and ANTP/139 which could be involved in the resistance of the BoNT/C1 to the acidic pH and protease degradation. The second cluster consists of three genes which encode hemagglutinin components. The last gene encodes a DNA binding protein (Orf22) which might regulate the BoNT/C1 complex gene expression. BoNT and tetanus toxin (TeTx) display similar structure and mechanism of action at the molecular level. Their identity at the amino acid level range from 34 to 96.8%, indicating that the clostridial neurotoxin genes probably derive from a common ancestor. The fact that Clostridium other than C. botulinum such as C. butyricum and C. baratii can produce a BoNT suggests that the BoNT genes can be transferred between Clostridium strains. The toxigenic C. butyricum strains seem to derive from originally non-toxic strains by neurotoxin gene transfer from C. botulinum E, probably including a mobile DNA element. In C. botulinum C and D the gene encoding the exoenzyme C3 has been localized in a transposon-like element of 21.5 kbp. Transposons could be involved in BoNT gene transfer in C. botulinum.