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. Immunogeniciy of botulinum toxin. 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. Structure of C. perfringens epsilon toxin.

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 respectively produce potent toxins, botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeTx), which are responsible for severe diseases, botulism and tetanus. Neurotoxin synthesis is a regulated process in Clostridium. The genes botR/A in C. botulinum A and tetR in C. tetani positively regulate expression of BoNT/A and associated non toxic proteins (ANTPs), as well as TeTx, respectively. The botR/A gene lies in close vicinity of the two operons which contain bont/A and antps genes in C. botulinum A, and tetR immediately precedes the tetX gene in C. tetani. We show that BotR/A and TetR function as specific alternative sigma factors rather than positive regulators based on the following results : i) BotR/A and TetR associated with target DNAs only in the presence of the RNA polymerase core enzyme (Core); ii) BotR/A and TetR directly bound with the core enzyme; iii) BotR/A-Core recognized -35 and -10 regions of ntnh-bont/A promoter; and iv) BotR/A and TetR triggered in vitro transcription from the target promoters. In C. botulinum A, bont/A and antps genes are transcribed as bi-and tri-cistronic operons controlled by BotR/A. BotR/A and TetR are seemingly related to a new subgroup of the &σ 70 family that includes TcdR and UviA, which respectively regulate production of toxins A and B in C. difficile and bacteriocin in C. perfringens. Sequences of -35 region are highly conserved in the promoter of target toxin genes in C. botulinum, C. tetani, C. difficile and C. perfringens. Overall, a common regulation mechanism probably controls toxin gene expression in these four toxigenic clostridial species.


Interaction between the two Subdomains of the C-terminal Part of the Botulinum Neurotoxin A is Essential for the Generation of Protective Antibodies

The botulinum neurotoxin A C-terminal fragment (Hc), which mediates the binding of the toxin to neuronal cell surface receptors, comprises two subdomains, Hc-N (amino acids 873-1095) and Hc-C (amino acids 1096-1296). In order to define the minimal fragment of Hc carrying protective antigenic properties, Hc, Hc-N and Hc-C have been produced as recombinant proteins in Escherichia coli, and have been tested for their antigenicity in mouse protection assays. Hc, Hc-N and Hc-C induced similar antibody levels a shown by ELISA. However, a single immunization with Hc (10 g) fully protected mice challenged with 103 mouse lethal dose 50 of toxin, whereas Hc-N, Hc-C, or Hc-N plus Hc-C did not give any protection. Triple immunizations with Hc-N or Hc-C were necessary to induce a higher level of protection. Circular dichroism and fluorescence studies showed that the isolated subdomains were folded and stable. However, an intense near-UV dichroic signal was only observed in the Hc spectrum, revealing a highly structured interface between both subdomains. Taken together, the results show that the generation of protective antibodies requires the whole Hc domain and especially the native structure of the interfacial region between Hc-N and Hc-C.

A phosphatidylserine-binding site in the cytosolic fragment of Clostridium sordellii lethal toxin facilitates glucosylation of membrane-bound Rac

Large clostridial toxins glucosylate some small G proteins on a threonine residue, thereby preventing their interaction with effector molecules and regulators. We show that the glucosyltransferase domain of lethal toxin from Clostridium sordellii (LTcyt; amino acids 1-546), which is released in the cytosol during cell infection, binds preferentially to liposomes containing phosphatidylserine as compared to other anionic lipids. The binding of LTcyt to phosphatidylserine increases by two-order of magnitude the rate of glucosylation of liposomebound geranyl-geranylated Rac-GDP. Limited proteolysis and deletion studies show that the binding site for phosphatidylserine lies within the first N terminal 18 residues of LTcyt. Deletion of these residues abolishes the effect of phosphatidylserine on the activity of LTcyt on liposome-bound geranyl-geranylated Rac-GDP and prevents the morphological effects induced by LTcyt microinjection into various cells, but does not affect the intrinsic activity of LTcyt on non-geranyl-geranylated Rac-GDP in solution. We conclude that the avidity of Ltcyt for phosphatidylserine facilitates its targeting to the cytosolic leaflet of cell membranes and notably the plasma membrane, where this anionic lipid is abundant and where several targets of lethal toxin reside.

Degeneration and Regeneration of Murine Skeletal Neuromuscular Junctions After Intramuscular Injection of a Sublethal Dose of Clostridium sordellii Lethal Toxin

Clostridium sordellii lethal toxin (LT), a 250 kDa protein which is the bacterium's major virulence factor, belongs to a family of large clostridial cytotoxins which glucosylate small GTP-binding proteins. Here, we report the results of our ex vivo analysis of the structure and function of skeletal neuromuscular tissue obtained from mice at various times after intramuscular injection of a sublethal dose of LT (0.25 ng/g body wt). The toxin caused, within 24 h, pronounced localized edema, inflammation, myofibril disassembly, and degeneration of skeletal muscle fibers in the injected area, and it glucosylated the muscle tissue's small GTPases. Regeneration of the damaged fibers was evident 6 to 9 days post-injury and was completed by 60 days. The expression of dystrophin, laminin, and fast- and neonatal-myosin in regenerating fibers, detected using immunofluorescence microscopy, confirmed that LT does not impair the high regenerative capacity of murine skeletal muscle fibers. Functional studies revealed that LT affects muscle contractility and neuromuscular transmission. However, partial recovery of nerve-evoked muscle twitches and tetanic contractions was observed by day 15 postinjection, and extensive remodeling of the neuromuscular junction's nerve terminals and clusters of muscle acetylcholine receptors was still evident 30 days postinjection. In conclusion, to the best of our knowledge, this is the first report to characterize the degeneration and regeneration of skeletal neuromuscular tissue after in vivo exposure to a large clostridial cytotoxin. In addition, our data may provide an explanation for the severe neuromuscular alterations accompanying wound infections caused by C. sordellii.

Clostridium perfringens epsilon toxin shows structural similarity to the pore-forming toxin aerolysin

Epsilon-Toxin from Clostridium perfringens is a lethal toxin. Recent studies suggest that the toxin acts via an unusually potent pore-forming mechanism. Here we report the crystal structure of epsilon-toxin, which reveals structural similarity to aerolysin from Aeromonas hydrophila. Pore-forming toxins can change conformation between soluble and transmembrane states. By comparing the two toxins, we have identified regions important for this transformation.

Photo 1

Representative transverse tibialis anterior muscle sections stained with hematoxylin and eosin, and at various times after mice received a sublethal, intramuscular injection of C. sordellii LT. Note the presence of only necrotic fibers (asterisks) and abundant inflammatory infiltrates (arrows) by 3 days post-injection. Regenerating small myocytes and central nuclei are evident (arrowheads) by 9 to 17 days post-injection, but necrotic fibers and connective tissue are not present, and skeletal muscles possess large regenerating fibers with central nuclei, by 17 to 30 days post-injection.

Photo 2

Structures of C. perfringens epsilon toxin and aerolysin with domains labeled. (a,b) Ribbon diagrams of ETX (a) and aerolysin (b) colored from red at the N terminus to blue at the C terminus. Extra helices and loops in aerolysin are gray and the region possibly inserted into the membrane is highlighted in purple. The coordinates of ETX have been deposited in the Protein Data Bank (accession code 1UYJ).


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