Clostridial toxins are responsible for severe diseases in man and animals such as botulism, gangrenes and necrotic enteritis. Our laboratory is involved in the study of the regulation of the toxin synthesis in Clostridium botulinumand Clostridium tetanu, and of the mode of action of clostridial toxins such as lethal toxin from Clostridium sordellii.

The passage of botulinumneurotoxin type A BoNT/A trough the intestinal barrierwas investigated in polarized intestinal cell monolayers grown on filters. We have found that BoNT/A crosses intestinal cell monolayers via a receptor-mediated transcytosis and that BoNT/A passage is more efficient through the intestinal crypt cell line m-ICcl2, We used fluorescent BoNT/A C-terminal part of H chain (Hc) which mediates toxin binding to cell receptors, to monitor toxin entry into NG108-15 neuronal cells as well as into Caco-2 and m-ICcl2intestinal cells. BoNT/A Hc receptors were found to be distributed in membrane structures closely associated to cholesterol-enriched microdomains but distinct from detergent-resistant microdomains in both cell types. BoNT/A Hc was trapped into endocytic vesicles, which progressively migrated to a perinuclear area in NG108-15 cells, and in a more scattered manner in intestinal cells. In both cell types, BoNT/A Hc entered through a dynamin- and intersectin-dependent pathway, reached an early endosomal compartment labeled with EEA1. In neuronal cells, BoNT/A Hc entered mainly via a clathrin-dependent pathway, in contrast to intestinal cells where it followed a Cdc42-dependent pathway (Fig. 1), supporting a differential toxin routing in both cell types.

Using electronic microscopy, BoNT/A Hc was found to enter neuronal cells via a clathrin pathway and intestinal cells mainly via non-clathrin vesicles. Upon incubation at 4°C, BoNT/A Hc gold particles were mainly distributed to cell periphery, including coated pits, and cell extensions of NG108-15 and to a lower extent to m-ICcl2cells. A few coated vesicles close from the plasma membrane also contained gold particles in both cell types. After incubation at 37°C, BoNT/A Hc mostly transited in coated vesicles in NG108-15 cells, whereas in m-ICcl2cells toxin gold particles were localized in uncoated vesicles, which support the transcytotic passage of the toxin, and free in the cytosol probably resulting from an unspecific uptake.

Large clostridial toxinssuch as LT from C. sordelliiglucosylates various Rho and Ras GTPases. and alter the actin cytoskeleton and subsequently intercellular junctions. Inactivation of Rac seems to be the main target by which these toxins disturb the actin filaments. We observed that LT-82 induces a rapid dephosphorylation of paxillin, a protein regulating focal adhesion (FA), independently of inactivation of paxillin kinases such as Src, Fak and Pyk2. Amongst the small GTPases inactivated by this toxin, including Rac, Ras, Rap and Ral, we identified Rac1, as responsible for paxillin dephosphorylation using cells overexpressing dominant negative Rac1. Rac1 inactivation by LT82 modifies interactions between proteins from AJ and FA complexes as shown by pull down assays. We showed that in lipid rafts proteins from these complexes, namely E-cadherin, β-catenin, p120 catenin, and talin, are decreased upon LT82 intoxication, a treatment that also induces a rapid decrease in cell phosphoinositide content. Therefore, we propose that Rac inactivation by LT82 alters phosphoinositide metabolism leading to FA and AJ complex disorganization and actin depolymerization.

Characterization of the channel-forming domain of Clostridium perfringensepsilon toxin (ETX). Epsilon-toxin (ETX) is a potent toxin produced by Clostridiumperfringensstrains B and D. The bacteria are important pathogens in domestic animals and cause edema mediated by ETX. This toxin acts most likely by heptamer formation and rapid permeabilization of target cell membranes for monovalent anions and cations followed by a later entry of calcium. We have compared the primary structure of ETX with that of the channel-forming stretches of a variety of binding components of A B types of toxins such as Anthrax protective antigen (PA), C2II of C2-toxin and Ib of Iota-toxin and found a remarkable homology to amino acids 151 to 180 of ETX. Site-directed mutagenesis of amino acids within the putative channel-forming domain resulted in changes of cytotoxicity and effects on channel characteristics in lipid bilayer experiments including changes of selectivity and partial channel block by methanethiosulfonate-(MTS)-reagents and antibodies against His6-tags from the trans-side of the lipid bilayer membranes. Deletion of the predicted transmembrane domain from histidine-151 to alanine-181 totally abolished pore formation.

Clostridium septicumalpha-toxin forms pores and induces rapid cell necrosis.Alpha-toxin is the unique lethal virulent factor produced by Clostridium septicum, which causes traumatic and non-traumatic gas gangrene and necrotizing enterocolitis in humans. We have analyzed channel formation of septicumalpha-toxin and characterized its activity on living cells. Recombinant septicumalpha-toxin induces the formation of ion-permeable channels with a single-channel conductance of about 175 pS in 0.1 M KCl in lipid bilayers membranes, which is typical for a large diffusion pore. Septicumalpha-toxin channels remained mostly in the open configuration, displayed no lipid specificity, and exhibited slight anion-selectivity. Septicumalpha-toxin causes a rapid decrease in the transepithelial electrical resistance of MDCK cell monolayers grown on filters, and induces a rapid cell necrosis in a variety of cell lines, characterized by cell permeabilization to propidium iodide without DNA fragmentation and activation of caspase-3. Septicum alpha-toxin also induces a rapid K⁺efflux and ATP depletion. Incubation of the cells in K⁺-enriched medium delays cell death caused by septicumalpha-toxin or epsilon-toxin, another potent pore-forming toxin, suggesting that the rapid loss of intracellular K⁺ represents an early signal of pore-forming toxins-mediated cell necrosis.

Keywords: Anaerobes, toxins, botulism, Clostridium botulinum, Clostridium difficile, Clostridium sordellii. cytotoxicity, Rho-GTPases, transcytosis

- Knapp, O., Maier, E., Benz, R., Geny, B., Popoff, M. R. Identification of the channel-forming domain of Clostridium perfringensEpsilon-toxin (ETX). Biochim Biophys Acta 2009, 1788: 2584-2593.

- Couesnon A., Shimizu T., Popoff M. R. Differential entry of botulinum neurotoxin A into neuronal and intestinal cells. Cell. Microbiol. 2009, 11: 289-308.

- Geny B., Grassart A., Manich M., Chicanne G., Payrastre B., Sauvonnet N., Popoff M. R. Rac1 inactivation by lethal toxin from Clostridium sordellii modifies focal adhesion upstream actin depolymerization. Cell. Microbiol. 2010, 12: 217-232.

- Mazuet C., Dano J., Popoff M. R., Créminon C., Volland H. Characterization of botulinum neurotoin type A neutralizing antibodies and influence of their half-lives on therapeutic activity. PlosS One 2010, 5 (8) e12416.

- Knapp O., Maier E., Ben Mkaddem S., Bens M., Chenal A., Geny B., Vandewalle A., Popoff M. R. Clostridium septicumalpha-toxin forms pores and induces rapid cell necrosis. Toxicon 2010, 55: 61-72 Epub