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  Director : M. R. Popoff (mpopoff@pasteur.fr)


  abstract

 

Study of the toxinogenesis regulation in Clostridium botulinum and Clostridium tetani, investigation of the function of the regulatory proteins BotR and TetR, analysis of the two component systems of regulation. Study of large clostridial toxins (C. sordellii toxins) which glucosylate Rho and Ras GTPases and investigation of the signaling pathways involved in the control of the actin cytoskeleton and intercellular junctions. Study of the cell entry of clostridial binary toxins.



  report

cale

Research work on botulism

Study of the regulation of toxinogenesis in Clostridium botulinum

The botulinum neurotoxins (BoNT; 150 kDa) are associated non covalently with other non toxic proteins (associated non toxic proteins, ANTPs) to form large size botulinum comlexes (300 to 900 kDa). The genes endoding BoNT and ANTPs are clsutered on DNA fragment called botulinum locus. They are organized in two operons, one containing the genes of the botulinum neurotoxin and the non toxic non hemaglutinin protein (NTNH), and the other one contains the genes of the three hemaglutinins (HAs). Another gene encoding for a protein (BotR) which has the characteristics of DNA binding proteins, is localized in the 5' part of the botulinum locus in C. botulinum A and B. This gene is conserved in C. tetani (tetR). We have previously shown that BotR is a positive regulator of the expression of bont and antps genes by overexpressing botR gene in C. botulinum A or inhibiting it by an antisens mRNA construction. BotR/A from C. botulinum type A was produced as recombinant protein with a six His tag in E. coli and purified by affinity chromatography. Purified BotR/A and crude lysate from recombinant C. botulinum A which overproduces BotR/A were tested in gel shift assay with DNA fragments from the promoter regions of the two operons. The crude lysates from recombinant C. botulinum induced a gel shift, whereas purified BotR/A did not. However BotR/A in the presence of RNA polymerase core enzyme from E. coli caused a gel retardation of promoter dans. This indicates that BotR/A is probably an alternative sigma factor of the same family than TxeR from Clostridium difficile. The BotR/A-RNA polymerase core enzyme complex binds preferentially to the promoter DNAs from the two operons of the botulinum locus and weakly to the promoters of the other genes. This indicates that bont and antps genes are essentially transcribed as polycistronic genes.

Passage of the intestinal barrier by botulinum neurotoxin A

The intestinal cells CaCo-2 grown on filters form polarized cell monolayers with tight and adherent junctions. Purified BoNT/A or BoNT/A in complex form was inoculated on the apical side of CaCo-2 cells. The passage of the neurotoxin was monitored by measurement of the lethal activity on mice. The integrity of the cell monolayers checked by measurement of the electrical trans epithelial resistance and immunofluorescent staining of actin, apical junctions (ZO1), and adherens junctions (E-cadherin) was not modified by botulinum neurotoxin treatment. BoNT/A alone was able to cross CaCo-2 cell monolayers (yield of passage 1-5%), but the passage was increased when BoNT/A was associated to ANTPs. The BoNT/A passage was inhibited at 4°C. Moreover, the BoNT/A passage was saturable over a period of 20-30 min. This indicates that BoNT/A crosses CaCo-2 cell monlayers by a receptor mediated transcytosis mechanism.

Cell delivery system based on Clostridium perfringens iota toxin

Clostridium perfringens iota toxin is a binary toxin which consists in two independent proteins, one being the binding component (Ib) and the other one the enzymatic component (Ia). Ib recognizes a cell surface receptor and mediates the internalization of Ia which catalyzes the ADPribosylation of monomeric actin. The binary toxin s are efficient cell delivery system to internalize heterologous proteins such as antibodies against botulinum neurotoxin for therapeutic use.

The Ia domain which interacts with Ib was determined by means of fusion proteins between several Ia fragments and C3 enzyme. C. botulinum C3 enzyme, which has been characterized in our laboratory, ADPribosylates Rho protein but does not efficiently enter cells. The Ia fragments were produced from a Ia inactive mutant, in order that the fusion proteins only possess the C3 enzymatic activity. The chimeric proteins C3-Ia and Ia-C3 were tested for in vitro ADPribosylation of Rho, cytotoxicity (cell rounding, actin disorganization visualized with fluorescent phalloidin, in vivo ADPribosylation of Rho) in the presence of Ib, and binding activity to Vero cells preincubated with Ib as measured by flow cytometry. The minimal Ia fragment that promoted the binding of the Ia-C3 chimeras to Vero cell bound Ib, and their translocation into cells consists of 129 central residues (129-257). Thus, iota toxin is a suitable system for medaiting the entry of heterologous proteins such as C3 into cells .

The entry of iota toxin into cell was studied with polarized CaCo-2 cell monolayers. We showed that Ia plus Ib applied apically or basolaterally induce a rapid decrease in the transepithelial resistance (TER) of CaCo-2 cell monolayers and disorganization of actin filaments as well as the tight and adherens junctions. Ib alone, on the apical or basolateral side slowly decreased the TER without affecting the actin cytoskeleton, possibly via pore formation. . Interestingly, the two iota-toxin components inoculated separately on each cell surface induced cytopathic effects and a TER decrease. Anti-Ib serums raised against the whole molecule or the Ia docking domain and applied to the opposite cell side versus Ib, neutralized the TER decrease. In addition, radioactive Ib incubated in the basolateral compartment, was detected on the apical side by selective cell surface biotinylation. This argues for a transcytotic routing of Ib to mediate internalization of Ia from the opposite cell surface. Bafilomycin A1 also prevented the cytopathic effects of Ia and Ib applied separately to each cell side, possibly by blocking translocation of Ia into the cytosol and/or the intracellular transport of Ib. Ib is either routed into the cell independently of Ia, transcytosed and permanently exposed on the opposite cell surface, or Ib is continuously recycled between an endosomal compartment and the cell surface.

Clostridial toxins which modify the intestinal barrier

Some Clostridium are responsible for intestinal diseases by means of potent toxins which interact with epithelial intestinal cells.

Epsilon toxin produced by C. perfringens type D causes fatal enterotoxemia. We have studied the interaction of epsilon with kidney cells from dog (MDCK cells) which form impermeable cell barrier related to intestinal epithelium when grown on filters. We have found that epsilon toxin recognizes specifically a MDCK cell surface receptor and forms a large membrane complex corresponding to toxin oligomerization which is correlated with an enlargement of cell volume and loss of cell viability. The toxin is not internalized into the cytosol and acts on the membrane by forming a pore, leading to a perturbation of intracellular ions and small molecules. The epsilon toxin induced-channels have been characterized in MDCK cells and in lipid artificial bilayers. Epsilon toxin is very potent to decrease the trans-epithelial electrical resistance of polarized MDCK cells grown on filters without altering the organization of the junctional complexes. The dose-dependent decrease in trans-epithelial electrical resistance, more marked when the toxin was applied to the apical side than to the basal side of MDCK cells, was associated with a moderate increase of the paracellular permeability to low molecular weight compounds but not to macromolecules. Epsilon toxin probably acts by forming large membrane pores which permit the flux of ions and other molecules such as the entry of propidium iodide, and finally to the loss of cell viability.

The large clostridial toxins from C. difficile (ToxA, ToxB) and C. sordellii ((LT) glucosylate small GTPases of Rho and Ras family, and thus modify the actin cytoskeleton and subsequently the intercellular junctions. LT which only inactivate Rac among the Rho GTPases, disorganize the adherens junctions mediated by E-cadherin, whereas the apical tight junctions are weakly altered. LT also inhibits the neurotransmitter release in Aplysia which may result from a decrease of phosphoinositide production or a decrease of phospholipase D stimulation which are under the control of Rac. The GTPase-mediated cell signaling which is altered by LT is currently studied.

Photo: Actin cytoskeleton changes induced by wild type iota toxin, or chimeric iota-C3 proteins. Vero cells were treated with 10-7 M wild type iota toxin (Ia+Ib), or Ia-C3, Ia(129-257)-C3 in the presence of Ib for 2 h at 37°C. The cells were fixed and stained with FITC-phalloidin. Control cells show numerous actin stress fibers, whereas the actin cytoskeleton was completely depolymerized by wild type iota toxin. Ia-C3 constructs containing whole Ia or the minimal Ia(129-257) domain fused with C3 induce loss of stress fibers and wrinkled cortical actin which are characteristic of the C3 activity.

Keywords: Clostridium, toxin, botulism, actin, G proteins



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Ferrand Mireille Popoff, M. R. Marvaud Jean Christophe post doc jmarvaud@paster.fr

Boehm Catherine PhD studentcboehm@pasteur.fr

Raffestin Stephanie PhD studentsraff@pasteur.fr

Le Bourhis Anne Gaelle PhD student

Cerrato, Rosario, post doc

Knapp, Oliver, post doc
Carlier Jean Philippe, Ingeneer IPjcarlier@pasteur.fr

Bedora Marie technicianmbedora@pasteur.fr

Gibert Maryse technicianmgibert@pasteur.fr

K'Ouas Guylene techniciangkouas@pasteur.fr

Manich Maria technicianmmanich@pasteur.fr

Activity Reports 2002 - Institut Pasteur
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