Tetanus Toxin Synthesis is Under the Control of A Complex Network of Regulatory Genes in Clostridium tetani.

Clostridium tetani produces a potent neurotoxin, the tetanus toxin (TeNT), which is responsible for an often-fatal neurological disease (tetanus) characterized by spastic paralysis. Prevention is efficiently acquired by vaccination with the TeNT toxoid, which is obtained by C.tetani fermentation and subsequent purification and chemical inactivation. C.tetani synthesizes TeNT in a regulated manner. Indeed, the TeNT gene (tent) is mainly expressed in the late exponential and early stationary growth phases. The gene tetR (tetanus regulatory gene), located immediately upstream of tent, encodes an alternative sigma factor which was previously identified as a positive regulator of tent. In addition, the genome of C.tetani encodes more than 127 putative regulators, including 30 two-component systems (TCSs). Here, we investigated the impact of 12 regulators on TeNT synthesis which were selected based on their homology with related regulatory elements involved in toxin production in other clostridial species. Among nine TCSs tested, three of them impact TeNT production, including two positive regulators that indirectly stimulate tent and tetR transcription. One negative regulator was identified that interacts with both tent and tetR promoters. Two other TCSs showed a moderate effect: one binds to the tent promoter and weakly increases the extracellular TeNT level, and another one has a weak inverse effect. In addition, CodY (control of dciA (decoyinine induced operon) Y) but not Spo0A (sporulation stage 0) or the DNA repair protein Mfd (mutation frequency decline) positively controls TeNT synthesis by interacting with the tent promoter. Moreover, we found that inorganic phosphate and carbonate are among the environmental factors that control TeNT production. Our data show that TeNT synthesis is under the control of a complex network of regulators that are largely distinct from those involved in the control of toxin production in Clostridium botulinum or Clostridium difficile.

The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation.

Clostridium sordellii lethal toxin (TcsL) is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP)-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat) into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1-93 domain) recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases.

Translocation and dissemination to target neurons of botulinum neurotoxin type B in the mouse intestinal wall.

Botulinum neurotoxins (BoNTs) are responsible for severe flaccid paralysis (botulism), which in most cases enter the organism via the digestive tract and then disseminate into the blood or lymph circulation to target autonomic and motor nerve endings. The passage way of BoNTs alone or in complex forms with associated nontoxic proteins through the epithelial barrier of the digestive tract still remains unclear. Here, we show using an in vivo model of mouse ligated intestinal loop that BoNT/B alone or the BoNT/B C-terminal domain of the heavy chain (HCcB), which interacts with cell surface receptors, translocates across the intestinal barrier. The BoNT/B or HCcB translocation through the intestinal barrier occurred via an endocytosis-dependent mechanism within 10-20 min, because Dynasore, a potent endocytosis inhibitor, significantly prevented BoNT/B as well as HCcB translocation. We also show that HCcB or BoNT/B specifically targets neuronal cells and neuronal extensions in the intestinal submucosa and musculosa expressing synaptotagmin, preferentially cholinergic neurons and to a lower extent other neuronal cell types, notably serotonergic neurons. Interestingly, rare intestinal epithelial cells accumulated HCcB suggesting that distinct cell types of the intestinal epithelium, still undefined, might mediate efficient translocation of BoNT/B.

The catalytic domains of Clostridium sordellii lethal toxin and related large clostridial glucosylating toxins specifically recognize the negatively charged phospholipids phosphatidylserine and phosphatidic acid.

Clostridium sordellii lethal toxin (TcsL) is a potent virulence factor belonging to the large clostridial glucosylating toxin family. TcsL enters target cells via receptor-mediated endocytosis and delivers the N-terminal catalytic domain (TcsL-cat) into the cytosol upon an autoproteolytic process. TcsL-cat inactivates small GTPases including Rac and Ras by glucosylation with uridine-diphosphate (UDP)-glucose as cofactor leading to drastic changes in cytoskeleton and cell viability. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to lipid membrane. We here report binding affinity measurements of TcsL-cat for brain PS-containing membranes by surface plasmon resonance and enzyme-linked immunosorbent assay (ELISA). In addition, TcsL-cat bound to phosphatidic acid (PA) and, to a lesser extent, to other anionic lipids, but not to neutral lipids, sphingolipids or sterol. We further show that the lipid unsaturation status influenced TcsL-cat binding to phospholipids, PS with unsaturated acyl chains and PA with saturated acyl chains being the preferred bindingsubstrates. Phospholipid binding site is localized at the N-terminal four helical bundle structure (1-93 domain). However, TcsL-1-93 bound to a broad range of substrates, whereas TcsL-cat, which is the active domain physiologically delivered into the cytosol, selectively bound to PS and PA. Similar findings were observed with the other large clostridial glucosylating toxins from C. difficile, C. novyi and C. perfringens.

CEMOVIS on a pathogen: analysis of Bacillus anthracis spores.

BACKGROUND INFORMATION: Under conditions of starvation, bacteria of Bacillus ssp. are able to form a highly structured cell type, the dormant spore. When the environment presents more favourable conditions, the spore starts to germinate, which will lead to the release of the vegetative form in the life cycle, the bacillus. For Bacillus anthracis, the aetiological agent of anthrax, germination is normally linked to host uptake and represents an important step in the onset of anthrax disease. Morphological studies analysing the organization of the spore and the changes during germination at the electron microscopy level were only previously performed with techniques relying on fixation with aldehydes and osmium, and subsequent dehydration, which can produce artefacts. RESULTS AND CONCLUSIONS: In the present study, we describe the morphology of dormant spores using CEMOVIS (Cryo-Electron Microscopy of Vitreous Sections). Biosafety measures do not permit freezing of native spores of B. anthracis without chemical fixation. To study the influence of aldehyde fixation on the ultrastructure of the spore, we chose to analyse spores of the closely related non-pathogen Bacillus cereus T. For none of the investigated structures could we find a difference in morphology induced by aldehyde fixation compared with the native preparations for CEMOVIS. This result legitimizes work with aldehyde-fixed spores from B. anthracis. Using CEMOVIS, we describe two new structures present in the spore: a rectangular structure, which connects the BclA filaments with the basal layer of the exosporium, and a repetitive structure, which can be found in the terminal layer of the coat. We studied the morphological changes of the spore during germination. After outgrowth of the bacillus, coat and exosporium stay associated, and the layered organization of the coat, as well as the repetitive structure within it, remain unchanged.