Rational design of botulinum neurotoxin A1 mutants with improved oxidative stability.

Botulinum neurotoxins (BoNTs) are the most potent toxic proteins to mankind known but applied in low doses trigger a localized muscle paralysis that is beneficial for the therapy of several neurological disorders and aesthetic treatment. The paralytic effect is generated by the enzymatic activity of the light chain (LC) that cleaves specifically one of the SNARE proteins responsible for neurotransmitter exocytosis. The activity of the LC in a BoNT-containing therapeutic can be compromised by denaturing agents present during manufacturing and/or in the cell.

Design of modified botulinum neurotoxin A1 variants with a shorter persistence of paralysis and duration of action.

Botulinum neurotoxins (BoNTs) are classified by their antigenic properties into seven serotypes (A-G) and in addition by their corresponding subtypes. They are further characterized by divergent onset and duration of effect. Injections of low doses of botulinum neurotoxins cause localized muscle paralysis that is beneficial for the treatment of several medical disorders and aesthetic indications.

C3 peptide enhances recovery from spinal cord injury by improved regenerative growth of descending fiber tracts.

Functional recovery and regeneration of corticospinal tract (CST) fibers following spinal cord injury by compression or dorsal hemisection in mice was monitored after application of the enzyme-deficient Clostridium botulinum C3-protein-derived 29-amino-acid fragment C3bot(154-182). This peptide significantly improved locomotor restoration in both injury models as assessed by the open-field Basso Mouse Scale for locomotion test and Rotarod treadmill experiments. These data were supported by tracing studies showing an enhanced regenerative growth of CST fibers in treated animals as visualized by anterograde tracing.

Actin re-organization induced by Chlamydia trachomatis serovar D--evidence for a critical role of the effector protein CT166 targeting Rac.

The intracellular bacterium Chlamydia trachomatis causes infections of urogenital tract, eyes or lungs. Alignment reveals homology of CT166, a putative effector protein of urogenital C. trachomatis serovars, with the N-terminal glucosyltransferase domain of clostridial glucosylating toxins (CGTs).

Killing of rat basophilic leukemia cells by lethal toxin from Clostridium sordellii: critical role of phosphatidylinositide 3'-OH kinase/Akt signaling.

Clostridium sordellii lethal toxin (TcsL) belongs to the family of clostridial glucosylating toxins. TcsL exhibits glucosyltransferase activity to inactivate Rho and Ras proteins. On cultured cells, TcsL causes actin reorganization ("cytopathic effect") and apoptotic cell death ("cytotoxic effect").

A 29-amino acid fragment of Clostridium botulinum C3 protein enhances neuronal outgrowth, connectivity, and reinnervation.

Small GTPases of the Rho family play versatile roles in the formation and development of axons and dendrites, effects often studied by the Rho-inactivating C3 transferase (C3bot) from Clostridium botulinum. Recently, we reported that transferase-deficient C3bot also exerted axonotrophic activity. Using overlapping peptides from the C3bot sequence, we identified a small peptide of 29 amino acids (covering residues 154-182) from the C-terminal region of C3bot that promotes both axonal and dendritic growth, as well as branching of hippocampal neurons, at submicromolar concentrations.

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.

Glutamate uptake and release by astrocytes are enhanced by Clostridium botulinum C3 protein.

Inhibition of Rho activity by Clostridium botulinum C3 transferase (C3bot) versatily changes functional properties of neural cells. Using cultivated mouse astrocytes, we show here that C3bot increases both uptake and secretion of glutamate. The enhanced glutamate uptake is initiated by an NFkappaB-dependent up-regulation of the glial glutamate transporter 1 that is efficaciously sorted to the plasma membrane.

Neogenin-RGMa signaling at the growth cone is bone morphogenetic protein-independent and involves RhoA, ROCK, and PKC.

The repulsive guidance molecule RGMa has been shown to induce outgrowth inhibition of neurites by interacting with the transmembrane receptor neogenin. Here we show that RGMa-induced growth cone collapse is mediated by activation of the small GTPase RhoA, its downstream effector Rho kinase and PKC. In contrast to DRG cultures from neogenin-/- mice, in which no RGMa-mediated growth cone collapse and activation of RhoA occurred, treatment of wild type DRG neurites with soluble RGMa led to a marked activation of RhoA within 3 min followed by collapse, but left Rac1 and Cdc42 unaffected.

Cellular stability of Rho-GTPases glucosylated by Clostridium difficile toxin B.

Mono-glucosylation of Rho, Rac, and Cdc42 by Clostridium difficile toxin B (TcdB) induces changes of actin dynamics and apoptosis. When fibroblasts were treated with TcdB, an apparent decrease of the cellular Rac1 level was observed when applying anti-Rac1(Mab 102). This decrease was not based on degradation as inhibition of the proteasome by lactacystin did not stabilise cellular Rac1 levels.

Role of Rho GTPase in astrocyte morphology and migratory response during in vitro wound healing.

Small Rho GTPases are key regulators of the cytoskeleton in a great variety of cells. Rho function mediates morphological changes as well as locomotor activity. Using astrocyte cultures established from neonatal mice we investigated the role of Rho in process formation during astrocyte stellation.

Sphingosine-1-phosphate mediates migration of mature dendritic cells.

Sphingosine-1-phosphate (S1P) represents a potent modulator of diverse cellular activities, including lymphocyte trafficking and maintenance of lymphocyte homeostasis. The five known receptors for S1P (S1P(1-5)) belong to the family of G protein-coupled receptors. Upon binding S1P, they act downstream via heterotrimeric G proteins on members of the small GTPase family (Cdc42/Rac/Rho), evoking a S1P receptor-dependent activation pattern of Cdc42, Rac, and Rho, respectively.

Comparison of wild type with recombinant Clostridium difficile toxin A.

Toxins A and B from Clostridium difficile are single-chain proteins of 308,000 and 270,000 Da, respectively. They possess transferase activity to monoglucosylate proteins of the Rho GTPase family whereby Rho, Rac, and Cdc42 are the canonical substrates. For application of these toxins as specific Rho GTPase inhibitors the highest possible purity is of crucial interest.

Experimental generation of a tissue-engineered functional and vascularized trachea.

Objective: We sought to grow in vitro functional smooth muscle cells, chondrocytes, and respiratory epithelium on a biologic, directly vascularized matrix as a scaffold for tracheal tissue engineering.

Methods: Ten- to 15-cm-long free jejunal segments with their own vascular pedicle were harvested and acellularized from donor pigs (n = 10) and used as a vascular matrix. Autologous costal chondrocytes, smooth muscle cells, and respiratory epithelium and endothelial progenitor cells were first cultured in vitro and then disseminated on the previously acellularized vascular matrix.

Expression of recombinant Clostridium difficile toxin A using the Bacillus megaterium system.

Pathogenic Clostridium difficile produces two major protein toxins, toxin A and toxin B. We used the Bacillus megaterium expression system for expression of recombinant toxin A. The construct for the toxin A gene was obtained by the following cloning strategy: the gene for toxin A was generated in three parts, each of them ligated into a cloning vector.

Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis.

Neutrophils exposed to chemoattractants polarize and accumulate polymerized actin at the leading edge. In neutrophil-like HL-60 cells, this asymmetry depends on a positive feedback loop in which accumulation of a membrane lipid, phosphatidylinositol (PI) 3,4,5-trisphosphate (PI[3,4,5]P3), leads to activation of Rac and/or Cdc42, and vice versa. We now report that Rac and Cdc42 play distinct roles in regulating this asymmetry.

The complete receptor-binding domain of Clostridium difficile toxin A is required for endocytosis.

Clostridium difficile toxin A, the chief pathogenicity factor of the antibiotic-associated pseudomembranous colitis, is an intracellular acting cytotoxin that reaches its targets, the Rho GTPases, after receptor-mediated endocytosis. The C-terminal part, constructed of repetitive peptide elements, is thought to bind to a lot of carbohydrate containing receptor molecules to induce clustering and endocytosis. To study which part of the receptor-binding domain is in charge of addressing toxin A into the target cells, we studied the functional, i.