Anaerobe Bacteria and Toxins  


  HEADPopoff M. R / mpopoff@pasteur.fr
  MEMBERSBlandine Geny (Dr, INSERM), Philippe Bouvet (Dr, IP), Jean Philippe Carlier (Ingeneer, IP), Aurélie Couesnon (Dr)
Christelle Mazuet (Ingeneer, IP), Yannick Pereira (Dr, postdoc), Manuela Lotierzo (Dr, postdoc), Oliver Knapp (Dr, postdoc)
Maryse Gibert (technician), Marie Bedora (technician), Guylene K'Ouas (technician), Maria Manich (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.

Regulation of botulinum toxin(BoNT) and tetanus toxin (TeNT) synthesis has been investigated in C. botulinum Aand 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/Aand Pha34) of the botulinum locus, and of the tetx gene promoter (Ptetx), respectively. In C. botulinumA, 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.botulinumprotease 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. botulinumstrain Hall, are involved in the control of BoNT synthesis.

The passage of BoNT/A trough the intestinal barrierwas investigated in polarized intestinal cell monolayers grown on filters. BoNT/A crosses intestinal cell monolayers via a receptor-mediated transcytosis, including a transport inhibition at 4°C and a passage at 37°C in a saturable manner within 30-60 min. BoNT/A passage rate was about 10-fold more efficient through the intestinal crypt cell line m-ICcl2, than through the carcinoma Caco-2 or T-84 cells, and was not increased when BoNT/A was associated with the non-toxic proteins (botulinum complex). Like for neuronal cells, BoNT/A binding to intestinal cells was mediated by the half C-terminal domain as tested by fluorescence activated cytometry and by transcytosis competition assay. Gangliosides of GD1band GT1bseries and recombinant intravesicular SV2-C domain partially impaired BoNT/A transcytosis, suggesting a putative role of gangliosides and SV2 or a related protein in BoNT/A transcytosis through Caco-2 and m-ICcl2cells.

Large clostridial toxinssuch as LT from C. sordelliiglucosylates 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, 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 (Fig. 1). 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.

Batox.jpg

Lung (A) and heart (B) vessel cryosections of control and TcsL-82-treated mice for 6 and 18 h were analyzed with an anti-mouse VE-cadherin detected with a relevant secondary antibody coupled to FITC, and with TRITC phalloidin. Note the irregular and more diffuse staining of VE-cadherin in lung vessels (arrows), indicating a redistribution of VE-cadherin and alteration of adherens junctions.



  Publications

Raffestin S., Dupuy B., Marvaud J. C., Popoff M. R. BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botullinumtype A and Clostridium tetani. Mol. Microbiol. 2005, 55:235-249.

Boehm C., Gibert M., Geny B., Popoff M. R., Rodriguez P. Modification of epithelial cell barrier permeability and intercellular junctions by Clostridium sordelliilethal toxin.  Cell. Microbiol.2006, 8: 1070-1085.

Geny B., Khun H., Fitting C., Zarantonelli L., Mazuet ., Cayet N., Szatanik M., Prevost M. C., Cavaillon J. M., Huerre M., Popoff M. R. Clostridiumsordelliilethal toxin kills mice by inducing a major increase in lung vascular permeability. Am. J. Pathol.2007, 170: 1003-1017.

Fehr D, Burr SE, Gibert M, d'Alayer J, Frey J, Popoff M. R. Aeromonas exoenzyme of aeromonas salmonicida is a bifunctional protein that targets the host cytoskeleton. J Biol Chem.2007, 282: 20843-20852.

Couesnon A., Pereira Y., Popoff M. R. Receptor-mediated transcytosis of botulinum neurotoxin A through intestinal cell monolayers. Cell. Microbiol.2007 in press.





Activity Reports 2007 - Institut Pasteur
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