Anaerobe Bacteria and Toxins  


  HEADPopoff M. R. / mpopoff@pasteur.fr
  MEMBERSBlandine Geny (Dr, INSERM) Philippe Bouvet (Dr, IP) Christelle Mazuet (Ingeneer, IP) Cecile Deneve (Dr, post doc) Manuela Lotierzo (Dr, postdoc) Oliver Knapp (Dr, postdoc) Deborah Allali (Master) Chloe Connan (Master) Maryse Gibert (technician) Marie Bedora (technician) Laetitia Belon (technician) Guylene K'Ouas (technician) Laure Moreau (technician) Nathalie Hatchi (secretary)


  Annual Report

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.

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.

In addition, LT exhibits Rho- Ras-GTPase-independent activity into cells. We showed that LT82 activates the three MAPK pathways (p42/.44, p38, and SAPK/JNK)but that only a permeable and specific JNK inhibitor, JNK inhibitor II, prevents toxin-dependent actin depolymerization and cell rounding. We showed that JNK activation is dependent on entry of the toxin N-terminal domain into the cytosol as bafilomycin A1, that prevents acidification of endocytic vesicle and subsequent cytosolic translocation of the toxin N-terminal domain, prevents JNK activation. Inhibition of JNK activity delays small GTPase glucosylation generated by N-terminal domain catalytic activity. Using a cell line mutant deficient in UDP-glucose, we observed that activation of JNK occurs even in the absence of small GTPase glucosylation and, thus, is independent of the toxin intrinsic catalytic activity. Facilitation of target glucosylation by JNK activation appeared to be restricted to LT82 and was not a general feature of large clostridial toxins. Indeed, it was not observed with Toxin B from Clostridium difficilealthough this toxin also activates JNK.

Characterization of Clostridium perfringensDelta toxin. Some C.perfringenstype B and C strains produce an additional toxin called Delta toxin, which has been found to be hemolytic and cytotoxic for cells expressing the ganglioside GM2in their membrane. We have reported the genetic characterization of Delta toxin and the production of active recombinant protein, and shows that its mode of action consists in pore formation in lipid bilayer. Although Delta toxin shows a significant homology with Beta toxin, both toxins recognize distinct cell surface receptors and differ by the properties of channels formed in lipid bilayers.

Characterization of Clostridiumdifficilevariants. We have previously evidenced the clonality of isolates: PCR ribotype 027, PCR ribotype 078, and toxin A-negative toxin B-positive isolates, that are frequently associated with human infections and food animals. Genome analysis shows that the epidemic 027 strain R20291 has five unique genetic regions, absent from both the non-epidemic 027 (CD196) and strain 630, which include a novel phage island, a two component regulatory system and transcriptional regulators. ToxB from the epidemic strain has a more potent activity in 8 cell lines than from the other strains. This might contributes to the hypervirulence of the epidemic strain.

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

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Figure 1. BoNT/A Hc entry into m-ICcl2is Cdc42-dependent, but not in neuronal cells NG108-15. Cells were transfected with plasmid encoding GFP-Cdc42T17N (green). Cells were incubated with Cy3-BoNT/A Hc (red) for 30 min at 4°C, then washed and incubated in HBSS/BSA for additional 5 min at 37°C, and processed for confocal microscopy observation. BoNT/A Hc accumulates at the periphery of cells transfected with plasmid encoding the negative dominant Cdc42, whereas the fluorescent protein is observed inside the transfected neuronal cells.



  Publications

– Manich M., Knapp O., Gibert M., Maier E., JoliveTeynaud C., Geny B., Benz R., Popoff M. R. ClostridiumperfringensDelta toxin is sequence related to Beta toxin, NetB, and Staphylococcuspore-forming toxins, but shows functional differences. PLos One2008, 3 (11) e3764.

- 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., Popoff M. R. Activation of a c-Jun-NH2-terminal kinase pathway by the lethal toxin from Clostridium sordellii, TcsL82, occurs independently of the toxin intrinsic enzymatic activity and facilitates small GTPase glucosylation. Cell. Microbiol. 2009,11: 1102-1113.

- Knapp, O., Maier, E., Benz, R., Geny, B., Popoff, M. R. Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX). Biochim Biophys Acta2009, 1788: 2584-2593.

- Stabler, R. A., He, M., Dawson, L., Martin, M., Valiente, E., Corton, C., Lawley, T. D., Sebaihia, M., Quail, M. A., Rose, G., Gerding, D. N., Gibert, M., Popoff, M. R.,Parkhill, J., Dougan, G., Wren, B. W. Comparative genome and phenotypic analysis of Clostridium difficile027 strains provides insight into the evolution of a hypervirulent bacterium. Genome Biol2009, 10: R102





Activity Reports 2009 - Institut Pasteur
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