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) Laetitia Belon (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 botulinum and Clostridium tetanu, and of the mode of action of clostridial toxins such as lethal toxin from Clostridium sordellii.

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 immunofluorescence (Fig. 1), 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. 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, 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.

Characterization of new toxins and Clostridiumdiffiicile variants.We have characterized a type III secreted toxin by Aeromonas salmonicida called AexT which alters the actin cytoskeleton. AexT catalyzes an ADP-ribosylation reaction with monomeric actin, as clostridial binary toxins do, and possesses a GTPase activity (GAP) towards Rho, Rac, and Cdc42, which are involved in the control of the assembly of actin filaments. AexT is the first toxin to be described that has a specific actin ADP-ribosylation activity and GAP activity towards Rho, Rac and Cdc42, both activities contributing to actin filament depolymerization.

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.

A triple-locus nucleotide sequence analysis based on toxin regulatory genes tcdC, tcdR,and cdtRwas investigated to characterize the genetic variability of C. difficileisolates. Five lineages have been distinguished, which correlate with the different types of deletion in tcdC. This study evidences 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.

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

Batox.jpg

Figure 1. Binding of Alexa488-labeled BoNT/A Hc to neuronal and intestinal cells. Cells grown on coverslips were incubated in HBSS/BSA containing labeled BoNT/A Hc (5 μg/ml) for 30 min at 4°C, then washed, fixed and mounted in Mowiol. In addition, the actin cytoskeleton of m-ICcl2 (mouse intestinal crypt cells) cell monolayers was stained with TRITC-phalloidin.



  Publications

Geny B., Khun H., Fitting C., Zarantonelli L., Mazuet C., 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.

Bouvet P., Popoff M. R. Genetic relatdness amongClostridiumdifficile isolates from various origins by triple-locus sequence analysis based on toxin regulatory genes tcdC, tcdR, and cdtR. J. Clin. Microbiol.2008, 46: 3703-3713.

Couesnon A., Pereira Y., Popoff M. R.Receptor-mediated transcytosis of botulinum neurotoxin A through intestinal cell monolayers. Cell. Microbiol.2008, 10: 375-387.

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.





Activity Reports 2009 - Institut Pasteur
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