Anaerobe Bacteria and Toxins


  HEADDr POPOFF Michel-Robert / mpopoff@pasteur.fr
  MEMBERSDr BOUVET Philippe / CARLIER Jean Philippe / Dr COUESNON Aurélie
Dr DAVID Gabriel / Dr GENY Blandine / Dr KNAPP Oliver / Dr PEREIRA Yannick


  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 botulinum and Clostridium tetani, and of the mode of action of clostridial toxins such as lethal toxin from Clostridium sordellii.

Regulation of botulinum toxin (BoNT) and tetanus toxin (TeNT) synthesis has been investigated in C. botulinum A and C. tetani. An alternative sigma factor, TetR and BotR, respectively, positively regulates the production of TeNT and BoNT as well as that of associated non-toxic protein (ANTP) forming BoNT complexes in C. botulinum. BotR and TetR are alternative sigma factors specific of the two operon promoters (Pntnh-bont/A and Pha34) of the botulinum locus, and of the tetx gene promoter (Ptetx), respectively. In C. botulinum A, the expression of BoNT and ANTP genes is strictly regulated at the transition phase between the late exponential growth and the beginning of the stationary phase, as monitored by quantitative reverse transcriptase-PCR. High temperature (44°C) does not control BoNT synthesis but activates a secreted C. botulinum protease witch degrades BoNT/A and ANTPs except hemagglutinins. Currently, we are studying whether the two component systems witch are present in the genome of C. botulinum strain Hall, are involved in the control of BoNT synthesis.

The passage of BoNT/A trough the intestinal barrier is investigated in polarized intestinal cell monolayers grown on filters. Our results confirm a receptor-mediated transcytosis mechanism. BoNT receptor on intestinal cells versus neuronal cells is analyzed by flow cytometry and immunocytochemistry, with fluorescent recombinant C-terminal BoNT (Hc fragment). Transport of BoNT/A is also analyzed in mouse intestinal loop with fluorescent Hc.

Specific tools based on heavy chain of botulinum neurotoxins or clostridial binary toxins are developed to selectively transport into neuronal cells protein of interest such as non-cleavable substrate of botulinum neurotoxins in order to rescue the function of neuronal cells intoxicated with botulinum toxins.

Large clostridial toxins such as LT from C. sordellii glucosylates various Rho and Ras GTPases. We have found that LT binds specifically to phosphatidylserine (PS) and that LT glucosylation activity is higher when the substrate Rac is linked to lipid membrane, preferentially enriched in PS. LT preferentially alters basolateral actin and E-cadherin intercellular junctions of polarized epithelial cells (Fig. 1), induces apoptosis in myeloid cell line, as well as degeneration and regeneration of skeletal neuromuscular tissue. Lethal activity of LT was investigated in mouse. LT induces a massive extravasation of blood fluid in the thoracic cage, resulting from an increase in lung vascular permeability, which generates profound modifications such as animal dehydration, increase in hematocrit, hypoxia (as assessed by the increase in serum erythropoietin), and, finally, cardio-respiratory distress. Immunohistochemical analysis demonstrates that in lung endothelial cells, VE-cadherin, a protein participating in intercellular adherens junctions, is redistributed from membrane to cytosol. No major sign of inflammation was induced by LT. Currently, the LT-dependent cellular pathways between inactivation of Rho/RasGTPases and actin depolymerization are investigated

popoff.jpg

Des cellules polarisées de rein de souris (MCCD) cultivées sur filtre et traitées avec LT82 10-8 M pendant 4h à 37°C ont été immunomarquées pour les protéines des jonctions serrées (occludine et ZO-1) ou de jonctions adhérentes (E-cadhérine et b-caténine). (A) Les molécules des jonctions serrées n'ont pas montré de modification après traitement par LT82 par rapport aux cellules contrôle sans toxine, (B) alors que cette toxine produit une redistribution marquée du complexe E-cadhérine-b-caténine de la membrane vers le cytosol.



  Publications

Publications 2006 of the unit on Pasteur's references database




Activity Reports 2006 - Institut Pasteur
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