Unit: Anaerobe Bacteria and Toxins

Director: Popoff Michel R.

Regulation of the toxinogenesis in Clostridium botulinum and Clostridium tetani, evidence that BotR and TetR are alternative sigma factors controlling the expression of toxin and associated protein genes. Study of large clostridial toxins (C. sordellii toxins) which glucosylate Rho and Ras GTPases and investigation of signaling pathways involved in the control of the actin cytoskeleton and intercellular junctions.

The National Reference Center for Anaerobes and Botulism is involved in the survey of botulism and identification of anaerobe isolates (see CNR for anaerobic bacteria and botulism.

BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani

Clostridium botulinum and Clostridium tetani produce potent toxins, botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeTx), which are responsible for severe deseases, botulism and tetanus, respectively. Neurotoxin synthesis is a regulated process in Clostridium. The genes botR/A in C. botulinum A and tetR in C. tetani, have been found to positively regulate the expression of BoNT/A and associated non toxic proteins (ANTPs), as well as TeTx genes, respectively. botR/A lies in the close vicinity of the two operons which contain bont/A and antps genes in C. botulinum A, and tetR immediately preceeds the tetx gene in C. tetani. Here we show that BotR/A 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 : i) BotR/A and TetR associated with target DNAs only in the presence of the RNA polymerase core enzyme (RNAP), ii) BotR/A and TetR bound to RNAP, iii) BotR/A-RNAP recognized -35 and -10 regions of Pntnh-bont/A by footprinting analysis, and iv) BotR/A and TetR triggered in vitro transcription fron the target promoters. In C. botulinum A, bont/A and antps genes are transcribed as bi-and tri-cistronic operon controled by BotR/A. BotR/A and TetR were functionally interchangeable in in vitro transcription, and are related to the alternative sigma factors TxeR and UviA, which regulate the production of toxins A and B in C. difficile and bacteriocin bcn in C. perfringens, respectively. Sequence of -35 region is highly conserved in the promoter of target toxin genes in C. botulinum, C. tetani, C. difficile and C. perfringens. An overall regulation mechanism probably controls toxin gene expression in these four toxigenic Clostridium.

Antibiotic-induced expression of a cryptic cpb2 (Clostridium perfringens Beta-2 toxin) gene in equine b2-toxigenic Clostridium perfringens.

The cpb2 gene of b2-toxigenic Clostridium perfringens isolated from horses, cattle, sheep, human and pigs was sequenced. The cpb2 gene of equine and other non-porcine isolates differed from porcine isolates by the absence of an adenine in a poly A tract immediately downstream of the start codon in all nonporcine C. perfringens strains. This deletion involved formation of a cryptic gene harbouring a premature stop codon after only nine amino acid codons, while the full b2-toxin protein consists of 265 amino acids. Immunoblots carried out with antibodies directed against a recombinant b2-toxin showed the absenceof expression of the b2-toxin in equine and the othernon-porcine strains under standard culture conditions. However, treatment of C. perfringens with the aminoglycosides gentamicin or streptomycin was able to induce expression of the cpb2 gene in a representative equine strain of this group, presumably by frameshifting. The presence of the b2-toxin was revealed by immunohistology in tissue samples of small and large intestine from horses with severe typhlocolitis that had been treated before with gentamicin. This result may explain the finding that antibiotic treatment of horses affected by b2-toxigenic C. perfringens leads to a more accentuated and fatal progression of equine typhlocolitis. Clinical observations show a reduced appearance of strong typhlocolitis in horses with intestinal complications admitted to hospital care since the standard use of gentamicin has been abandoned. This is the first report on expression of a bacterial toxin gene by antibiotic-induced ribosomal frameshifting.

Modification of epithelial cell barrier permeability and intercellular junctions by Clostridium sordellii lethal toxins

C. sordellii lethal toxin (LT) is a glucosyltransferase which inactivates small GTPases from the Rho and Ras families. In the present work, we studied the effects of two variants, LT82 and LT9048, on the integrity of epithelial cell barrier using polarized MCCD and MDCK cells. Our results demonstrate for the first time that LTs have very limited effects on tight junctions. In contrast, we show that both toxins modified the paracellular permeability within 2-4 hours. Concomitantly LT82 and LT9048 induced a disorganization of basolateral actin filaments, without modifying apical actin (Photo 1). Both toxins mainly altered adherens junctions by removing E-cadherin-catenin complexes from the membrane to the cytosol. Similar effects on adherens junctions have been observed with other toxins, which directly or indirectly depolymerize actin. Thereby, Rac, a commun substrate of both LTs, might play a central role in LT-dependent adherens junction alteration. Here, we show that adherens junction perturbation induced by LTs neither results from a direct effect of toxins on adherens junction proteins nor from an actin-independent Rac pathway, but rather from a Rac-dependent disorganization of basolateral actin cytoskeleton. This further supports that a dynamic equilibrium of cortical actin filaments is essential for functional E-cadherin organization in epithelia.

Use of clostridial toxins which target RhoGTPases or actin monomers to dissect cellular physiology processes involving regulation of the actin cytoskeleton such as cyclic AMP-dependent modulation of vascular smooth muscle cell morphology by inhibiting a Rac-independent signaling pathway, complementary effects of ERK2 and p38 in platelet adhesion, role of RhoGTPases (mainly Cdc42) involved in cortical actin dynamics in HIV-1 release, and participation to the characterization of a new GAP of RhoA and Cdc42 that controls of E-cadherin junctions and has a key role in Listeri monocytogenes entry.

Oribacterium sinus gen. nov., sp. nov., within the family. "Lachnospiraceae" (phylum Firmicutes) (Carlier JP, K'Ouas G, Bonne I, Lozniewski, Mory F. Int J Syst Evol Microbiol. 2004, 54: 1611-1615)

A hitherto unknown anaerobic bacillus isolated from sinus pus in a young child (strain AIP 354.02T) was characterized by using phenotypic and genotypic methods (Photo 2). 16S rRNA gene sequence analysis indicated that this strain was phylogenetically affiliated with several sequences of cloned 16S rRNA gene inserts previously deposited in the public databases. According to their 16S rRNA gene sequence similarities, these uncultivated bacteria, together with strain AIP354.02T, formed a separate subgroup belonging to the family ‘Lachnospiraceae' within the phylum Firmicutes. Oribacterium gen. nov. is proposed for this group of organisms and Oribacterium sinus gen. nov. sp. nov. for strain AIP354.02T (=CIP 107991T=CCUG 48084T).

Photos :

Photo 1. The two variants, LT82 and LT9048, of Clostridium sordellii lethal toxin preferentially disorganize the basolateral actin filaments whereas the architecture of apical actin of polarized MDCK cell monolayers is preserved.

Photo 2. Transmission electron micrograph of Oribacterium sinus gen. nov. sp. nov. AIP 354.02T. (a) general Morphology after negative staining; (b) ultrathin section showing the Gram-positive cell wall (CW) and the cytoplasmic membrane (CM). Bars, (a) 500 nm; (b) 100 nm

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