Clostridium botulinum C3 toxin

About: Clostridium botulinum C3 toxin is a research topic. Over the lifetime, 5 publications have been published within this topic receiving 256 citations.

cAMP-induced AQP2 translocation is associated with RhoA inhibition through RhoA phosphorylation and interaction with RhoGDI

Abstract: We have recently demonstrated that inhibition of Rho GTPase with Clostridium difficile toxin B, or with Clostridium botulinum C3 toxin, causes actin depolymerization and translocation of aquaporin 2 (AQP2) in renal CD8 cells in the absence of hormonal stimulation. Here we demonstrate that Rho inhibition is part of the signal transduction cascade activated by vasopressin leading to AQP2 insertion into the apical membrane. Quantitation of active RhoA (GTP-bound) by selective pull down experiments demonstrated that the amount of active RhoA decreased upon stimulation of CD8 cells with the cAMP-elevating agent forskolin. Consistent with this observation, forskolin treatment resulted in a decreased expression of membrane-associated (active) Rho, as assessed by cell fractionation followed by western blotting analysis. In addition, the abundance of the endogenous Rho GDP dissociation inhibitor (Rho-GDI) was found to have decreased in the membrane fraction after forskolin stimulation. Co-immunoprecipitation experiments revealed that, after forskolin stimulation, the amount of Rho-GDI complexed with RhoA increased, suggesting that Rho GTPase inhibition occurs through association of RhoA with Rho-GDI. Finally, forskolin stimulation was associated with an increase in Rho phosphorylation on a serine residue, a protein modification known to stabilize the inactive form of RhoA and to increase its interaction with Rho-GDI. Taken together, these data demonstrate that RhoA inhibition through Rho phosphorylation and interaction with Rho-GDI is a key event for cytoskeletal dynamics controlling cAMP-induced AQP2 translocation.

Involvement of Rho GTPases in calcium-regulated exocytosis from adrenal chromaffin cells.

Abstract: The Rho GTPase family, including Rho, Rac and Cdc42 proteins, is implicated in various cell functions requiring the reorganization of actin-based structures. In secretory cells, cytoskeletal rearrangements are a prerequisite for exocytosis. We previously described that, in chromaffin cells, the trimeric granule-bound Go protein controls peripheral actin and prevents exocytosis in resting cells through the regulation of RhoA. To provide further insight into the function of Rho proteins in exocytosis, we focus here on their intracellular distribution in chromaffin cells. By confocal immunofluorescence analysis, we found that Rac1 and Cdc42 are exclusively localized in the subplasmalemmal region in both resting and nicotine-stimulated cells. In contrast, RhoA is associated with the membrane of secretory granules. We then investigated the effects of clostridial toxins, which differentially impair the function of Rho GTPases, on the subplasmalemmal actin network and catecholamine secretion. Clostridium difficile toxin B, which inactivates Rho, Rac and Cdc42, markedly altered the distribution of peripheral actin filaments. Neither Clostridium botulinum C3 toxin, which selectively ADP-ribosylates Rho, nor Clostridium sordellii lethal toxin, which inactivates Rac, affected cortical actin, suggesting that Cdc42 plays a specific role in the organization of subplasmalemmal actin. Indeed, toxin B strongly reduced secretagogue-evoked catecholamine release. This effect on secretion was not observed in cells having their actin cytoskeleton depolymerized by cytochalasin E or Clostridium botulinum C2 toxin, suggesting that the inhibition of secretion by toxin B is entirely linked to the disorganization of actin. C. sordellii lethal toxin also inhibited catecholamine secretion, but this effect was not related to the actin cytoskeleton as seen in cells pretreated with cytochalasin E or C2 toxin. In contrast, C3 exoenzyme did not affect secretion. We propose that Cdc42 plays an active role in exocytosis by coupling the actin cytoskeleton to the sequential steps underlying membrane trafficking at the site of exocytosis.

Structure–function discrepancy in Clostridium botulinum C3 toxin for its rational prioritization as a subunit vaccine

Abstract: Clostridium botulinum is anaerobic pathogenic bacterium causing food-born botulism in human and animals by producing botulinum neurotoxins A-H, C2, and C3 cytotoxins. Physiological group III strains (type C and D) of this bacterium are capable of producing C2 and C3 toxins in cattle and avian. Herein, we have revealed the structure-function disparity of C3 toxins from two different C. botulinum type C phage (CboC) and type D phage (CboD) to design avirulent toxins rationally. Structure-function discrepancy of the both toxins was computationally evaluated from their homology models based on the conservation in sequence-structure-function relationships upon covariation and point mutations. It has shown that 8 avirulent mutants were generated from CboC of 34 mutants while 27 avirulent mutants resulted from CboD mutants. No major changes were found in tertiary structure of these toxins; however, some structural variations appeared in the coiled and loop regions. Correlated mutation on the first residue would disorder or revolutionize the hydrogen bonding pattern of the coevolved pairs. It suggested that the residues coupling in the local structural environments were compensated with coevolved pairs so as to preserve a pseudocatalytic function in the avirulent mutants. Avirulent mutants of C3 toxins have shown a stable structure with a common blue print of folding process and also attained a near-native backrub ensemble. Thus, we concluded that selecting the site-directed mutagenesis sites are very important criteria for designing avirulent toxins, in development of rational subunit vaccines, to cattle and avian, but the vaccine specificity can be determined by the C3 toxins of C. botulinum harboring phages.

Rho-inhibition and neuroprotective effect on rotenone-treated dopaminergic neurons in vitro.

Abstract: Mesencephalic cell cultures are a good model to study the vulnerability of dopaminergic neurons and reproduce, in vitro, experimental models of Parkinson’s disease. Rotenone associated as an environmental neurotoxin related to PD, is able to provoke dopaminergic neuron degeneration by inhibiting complex I of the mitochondrial respiratory chain and by inducing accumulation of α-synuclein. Recently, rotenone has been described to activate RhoA, a GTPase protein. In the present study we evaluated a possible neuroprotective effect of Rho-inhibitor molecules on rotenone-damaged dopaminergic (DA) neurons obtained from mouse primary mesencephalic cell culture. Our results showed that Clostridium Botulinum C3 toxin (C3) and simvastatin, as RhoA inhibitors, were able to protect DA neurons from rotenone damages. In fact, pretreatment with C3 or simvastatin significantly prevented the reduction of [3H]dopamine uptake, neurites injury and the expression patterns of proteins like α-syn, actin and connexin 43.