Molecular Evolutionary Constraints that Determine the Avirulence State of Clostridium botulinum C2 Toxin.

Clostridium botulinum (group-III) is an anaerobic bacterium producing C2 toxin along with botulinum neurotoxins. C2 toxin is belonged to binary toxin A family in bacterial ADP-ribosylation superfamily. A structural and functional diversity of binary toxin A family was inferred from different evolutionary constraints to determine the avirulence state of C2 toxin. Evolutionary genetic analyses revealed evidence of C2 toxin cluster evolution through horizontal gene transfer from the phage or plasmid origins, site-specific insertion by gene divergence, and homologous recombination event. It has also described that residue in conserved NAD-binding core, family-specific domain structure, and functional motifs found to predetermine its virulence state. Any mutational changes in these residues destabilized its structure-function relationship. Avirulent mutants of C2 toxin were screened and selected from a crucial site required for catalytic function of C2I and pore-forming function of C2II. We found coevolved amino acid pairs contributing an essential role in stabilization of its local structural environment. Avirulent toxins selected in this study were evaluated by detecting evolutionary constraints in stability of protein backbone structure, folding and conformational dynamic space, and antigenic peptides. We found 4 avirulent mutants of C2I and 5 mutants of C2II showing more stability in their local structural environment and backbone structure with rapid fold rate, and low conformational flexibility at mutated sites. Since, evolutionary constraints-free mutants with lack of catalytic and pore-forming function suggested as potential immunogenic candidates for treating C. botulinum infected poultry and veterinary animals. Single amino acid substitution in C2 toxin thus provides a major importance to understand its structure-function link, not only of a molecule but also of the pathogenesis.

Reduced gravitational loading does not account for the skeletal effect of botulinum toxin-induced muscle inhibition suggesting a direct effect of muscle on bone.

Intramuscular injection of botulinum toxin (botox) into rodent hindlimbs has developed as a useful model for exploring muscle-bone interactions. Botox-induced muscle inhibition rapidly induces muscle atrophy and subsequent bone loss, with the latter hypothesized to result from reduced muscular loading of the skeleton. However, botox-induced muscle inhibition also reduces gravitational loading (as evident by reduced ground reaction forces during gait) which may account for its negative skeletal effects. The aim of this study was to investigate the skeletal effect of botox-induced muscle inhibition in cage control and tail suspended mice, with tail suspension being used to control for the reduced gravitational loading associated with botox. Female C57BL/6J mice were injected unilaterally with botox and contralaterally with vehicle, and subsequently exposed to tail suspension or normal cage activities for 6 weeks. Botox-induced muscle inhibition combined with tail suspension had the largest detrimental effect on the skeleton, causing the least gains in midshaft tibial bone mass, cortical area and cortical thickness, greatest gains in midshaft tibial medullary area, and lowest proximal tibial trabecular bone volume fraction. These data indicate botox-induced muscle inhibition has skeletal effects over and above any effect it has in altering gravitational loading, suggesting that muscle has a direct effect on bone. This effect may be relevant in the development of strategies targeting musculoskeletal health.

[Botulism: structure and function of botulinum toxin and its clinical application].

Clostridium botulinum produces seven immunological distinct poisonous neurotoxins, A to G, with molecular masses of approximately 150kDa. In acidic foods and culture fluid, the neurotoxins associate with non-toxic components, and form large complexes designated progenitor toxins. The progenitor toxins are found in three forms named LL, L, and M. These neurotoxins and progenitor toxins were purified, and whole nucleotide sequences of their structure genes were determined. In this manuscript, the structure and function of these toxins, and the application of these toxins to clinical usage have been described.

Clostridial C3 proteins: recent approaches to improve neuronal growth and regeneration.

Bacterial C3 exoenzymes are widely used tools to investigate cellular events influenced by small GTPases of the Rho subfamily. In this respect they have gained increasing interest in addressing questions dealing with the neuronal morphogenic program during development and after lesion of the mature nervous system. Since central neurons display only very limited capacity to re-grow their axons after injury, successful strategies to improve regeneration are much sought-after. For a long time exclusively considered to be Rho-inhibiting exoenzymes, there is now accumulating evidence that C3 proteins of clostridial sources exhibit their often beneficial effects on neurite outgrowth by other means than ADP-ribosylation. The current review will outline previous attempts to foster neuronal cell growth by the use of C3 transferases and highlight the more recent approaches to improve regenerative axon outgrowth using enzyme-deficient C3 preparations.

Cyclophilin A facilitates translocation of the Clostridium botulinum C2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells.

The binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I, which mono-ADP-ribosylates actin in eukaryotic cells. Pore formation of C2IIa in early endosomal membranes facilitates translocation of unfolded C2I into the cytosol. We discovered earlier that translocation of C2I depends on the activity of the host cell chaperone heat shock protein Hsp90. Here, we demonstrate that cyclosporin A, which inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins, inhibited intoxication of cells with C2 toxin and prevented uptake of C2I into the cytosol. Cyclosporin A blocked the pH-dependent translocation of C2I activity across membranes of intact cells and of partially purified early endosomes. In vitro, the addition of cytosol to C2 toxin-loaded endosomes induced translocation of C2I activity into the cytosol, which was prevented by pretreatment of the cytosol with an antibody against cyclophilin A. Pull-down experiments with lysates from C2 toxin-treated cells revealed specific binding of cyclophilin A to the N-terminal domain of C2I. In conclusion, our results suggest an essential role of cyclophilin A for translocation of C2I across endosomal membranes during the uptake of C2 toxin into mammalian cells.

Inhibition of Rho-dependent pathways by Clostridium botulinum C3 protein induces a proinflammatory profile in microglia.

Successful regeneration in the central nervous system crucially depends on the adequate environment. Microglia as brain immune-competent cells importantly contribute to this task by producing pro- and anti-inflammatory mediators. Any environmental change transforms these cells towards an activated phenotype, leading to major morphological, transcriptional and functional alterations. Rho GTPases affect multiple cellular properties, including the cytoskeleton, and C3 proteins are widely used to study their involvement. Especially C3bot from Clostridium botulinum has been considered to promote neuronal regeneration by changing Rho activity. Yet C3bot may exert cellular influences through alternative mechanisms. To determine the role of Rho-dependent pathways in microglia we investigated the influence of C3bot on functional properties of cultivated primary mouse microglial cells. Nanomolar concentrations of C3bot transformed microglia towards an activated phenotype and triggered the release of nitric oxide and several proinflammatory cyto- and chemokines. These inductions were not mediated by the ROCK-kinase pathway, since its selective inhibitors Y27632 and H1152 had no effect. C3-induced and Rho-mediated NO release was instead found to be under the control of NFkappaB, as revealed by treatment with the NFkappaB inhibitor PDTC. Thus, C3bot induces a proinflammatory response in microglia resembling the classical proinflammatory phenotype elicited by bacterial LPS. The findings are relevant for the use of C3bot in regenerative approaches.

Presynaptic enzymatic neurotoxins.

Botulinum neurotoxins produced by anaerobic bacteria of the genus Clostridium are the most toxic proteins known, with mouse LD50 values in the 1-5 ng/kg range, and are solely responsible for the pathophysiology of botulism. These metalloproteinases enter peripheral cholinergic nerve terminals and cleave proteins of the neuroexocytosis apparatus, causing a persistent, but reversible, inhibition of neurotransmitter release. They are used in the therapy of many human syndromes caused by hyperactive nerve terminals. Snake presynaptic PLA2 neurotoxins block nerve terminals by binding to the nerve membrane and catalyzing phospholipid hydrolysis with production of lysophospholipids and fatty acids. These compounds change the membrane conformation, causing enhanced fusion of synaptic vesicle via hemifusion intermediate with release of neurotransmitter and, at the same time, inhibition of vesicle fission and recycling. It is possible to envisage clinical applications of the lysophospholipid/fatty acid mixture to inhibit hyperactive superficial nerve terminals.