Hyaline fibromatosis syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors.

Hyaline Fibromatosis Syndrome (HFS) is a human genetic disease caused by mutations in the anthrax toxin receptor 2 (or cmg2) gene, which encodes a membrane protein thought to be involved in the homeostasis of the extracellular matrix. Little is known about the structure and function of the protein or the genotype-phenotype relationship of the disease. Through the analysis of four patients, we identify three novel mutants and determine their effects at the cellular level. Altogether, we show that missense mutations that map to the extracellular von Willebrand domain or the here characterized Ig-like domain of CMG2 lead to folding defects and thereby to retention of the mutated protein in the endoplasmic reticulum (ER). Mutations in the Ig-like domain prevent proper disulphide bond formation and are more efficiently targeted to ER-associated degradation. Finally, we show that mutant CMG2 can be rescued in fibroblasts of some patients by treatment with proteasome inhibitors and that CMG2 is then properly transported to the plasma membrane and signalling competent, identifying the ER folding and degradation pathway components as promising drug targets for HFS.

Botulinum toxin A in non-dystonic tremors.

The present pilot study evaluated the effect of botulinum toxin A on primarily non-dystonic tremors using accelerometry in a single-blind, placebo-controlled design. Resting, postural, intention, or head tremor were assessed before and approximately 1 month after intramuscular saline and botulinum toxin A (25-50 U) respectively. Half of the patients showed > or = 30% placebo effect. Tremor in 10 of 17 patients (60%) studied improved further after botulinum toxin A (range 30-95%), exceeding the placebo effect by > or = 30%. Nine patients demonstrated clinically significant focal weakness in the extensor muscles after botulinum toxin A which interfered with fine movements. Patients were subdivided into PD-like and ET-like tremor(s). Both groups experienced large placebo effects for resting tremor, with little or no further improvement after botulinum toxin A. The improvement in postural tremor after botulinum toxin A, of 40% in the PD-like and 57% in the ET-like groups, however, was approximately twice that of placebo. In conclusion, botulinum toxin A exerts a modest tremorlytic effect, however the dose, and its distribution over the sites injected, need to be optimised to minimise focal weakness.

Control and host-dependent activation of insect toxin expression in a root-associated biocontrol pseudomonad.

Pseudomonas fluorescens CHA0 is a root-associated biocontrol agent that suppresses soil-borne fungal diseases of crops. Remarkably, the pseudomonad is also endowed with systemic and oral activity against pest insects which depends on the production of the insecticidal Fit toxin. The toxin gene (fitD) is part of a virulence cassette encoding three regulators (FitF, FitG, FitH) and a type I secretion system (FitABC-E). Immunoassays with a toxin-specific antibody and transcriptional analyses involving fitG and fitH deletion and overexpression mutants identified LysR family regulator FitG and response regulator FitH as activator and repressor, respectively, of Fit toxin and transporter expression. To visualize and quantify toxin expression in single live cells by fluorescence microscopy, we developed reporters which in lieu of the native toxin protein express a fusion of the Fit toxin with red fluorescent mCherry. In a wild-type background, expression of the mCherry-tagged Fit toxin was activated at high levels in insect hosts, i.e. when needed, yet not on plant roots or in batch culture. By contrast, a derepressed fitH mutant expressed the toxin in all conditions. P. fluorescens hence can actively induce insect toxin production in response to the host environment, and FitH and FitG are key regulators in this mechanism.

A pilot study of delayed versus immediate serial casting after botulinum toxin injection for partially reducible spastic equinus.

BACKGROUND: Serial casting is often prescribed after botulinum toxin injections to improve joint ranges of motion and to potentiate the decrease in hypertonia. The aim of this study was to compare delayed versus immediate serial casting as an adjunct to botulinum toxin therapy for partially reducible spastic equinus. METHODS: Twelve children who presented spastic equinus associated with mild gastrosoleus contracture took part. Five of them had a diagnosis of spastic diplegia, whereas 7 had a diagnosis of congenital hemiplegia. Children were randomized to immediate serial casting (same day) or delayed serial casting (4 weeks later) after botulinum toxin injection to their gastrosolei. Casts were replaced weekly for 3 weeks. RESULTS: Three children complained of pain that required recasting in the immediate casting group versus none in the delayed casting group (P = 0.08). At 3 months, there was a 27-degree improvement in the fast dorsiflexion angle (Tardieu R1) in the delayed casting group versus 17 degrees in the immediate casting group (P = 0.029). At 6 months, a 19-degree improvement persisted in the delayed group compared with 11 degrees in the immediate group (P = 0.010). CONCLUSIONS: There is a clear benefit in delaying serial casting after the injection of botulinum toxin in the recurrence of spasticity at the gastrosoleus that may also offer an advantage regarding the incidence of painful episodes associated with casting. Most importantly, reducing the recurrence of spasticity by delayed serial casting may offer the possibility of decreasing the frequency of botulinum toxin reinjections.

Polymeric IgA is superior to monomeric IgA and IgG carrying the same variable domain in preventing Clostridium difficile toxin A damaging of T84 monolayers.

The two exotoxins A and B produced by Clostridium difficile are responsible for antibiotic-associated enterocolitis in human and animals. When added apically to human colonic carcinoma-derived T84 cell monolayers, toxin A, but not toxin B, abolished the transepithelial electrical resistance and altered the morphological integrity. Apical addition of suboptimal concentration of toxin A made the cell monolayer sensitive to toxin B. Both toxins induced drastic and rapid epithelial alterations when applied basolaterally with a complete disorganization of tight junctions and vacuolization of the cells. Toxin A-specific IgG2a from hybridoma PCG-4 added apically with toxin A alone or in combination with toxin B abolished the toxin-induced epithelial alterations for up to 8 h. The Ab neutralized basolateral toxin A for 4 h, but not the mixture of the two toxins. Using an identical Ab:Ag ratio, we found that recombinant polymeric IgA (IgAd/p) with the same Fv fragments extended protection against toxin A for at least 24 h in both compartments. In contrast, the recombinant monomeric IgA counterpart behaved as the PCG-4 IgG2a Ab. The direct comparison between different Ig isotype and molecular forms, but of unique specificity, demonstrates that IgAd/p Ab is more efficient in neutralizing toxin A than monomeric IgG and IgA. We conclude that immune protection against C. difficile toxins requires toxin A-specific secretory Abs in the intestinal lumen and IgAd/p specific for both toxins in the lamina propria.

The Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth and induces encystment in Acanthamoeba castellanii.

L-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a toxic antimetabolite produced by the opportunistic pathogen Pseudomonas aeruginosa. To evaluate its importance as a potential virulence factor, we tested the host response towards AMB using an Acanthamoeba castellanii cell model. We found that AMB (at concentrations ≥ 0.5 mM) caused amoebal encystment in salt buffer, while inhibiting amoebal growth in rich medium in a dose-dependent manner. However, no difference in amoebal plaque formation was observed on bacterial lawns of wild type and AMB-negative P. aeruginosa strains. We thereby conclude that AMB may eventually act as a virulence factor, but only at relatively high concentrations.

An anti-CD19 antibody coupled to a tetanus toxin peptide induces efficient Fas ligand (FasL)-mediated cytotoxicity of a transformed human B cell line by specific CD4+ T cells.

Treatment of B cell lymphoma patients with MoAbs specific for the common B cell marker (CD20) has shown a good overall response rate, but the number of complete remissions is still very low. The use of MoAbs coupled to radioisotopes can improve the results, but induces undesirable myelodepression. As an alternative, we proposed to combine the specificity of MoAbs with the immunogenicity of T cell epitopes. We have previously shown that an anti-Ig lambda MoAb coupled to an MHC class II-restricted universal T cell epitope peptide P2 derived from tetanus toxin induces efficient lysis of a human B cell lymphoma by a specific CD4+ T cell line. Here we demonstrate that the antigen presentation properties of the MoAb peptide conjugate are maintained using a MoAb directed against a common B cell marker, CD19, which is known to be co-internalized with the B cell immunoglobulin receptor. In addition, we provide evidence that B cell lysis is mediated by the Fas apoptosis pathway, since Fas (CD95), but not tumour necrosis factor receptor (TNFr) or TNF-related receptors, is expressed by the target B cells, and FasL, but not perforin, is expressed by the effector T cells. These results show that B cell lymphomas can be 'foreignized' by MoAb-peptide P2 conjugates directed against the common B cell marker CD19 and eliminated by peptide P2-specific CD4+ T cells, via the ubiquitous Fas receptor. This approach, which bridges the specificity of passive antibody therapy with an active T cell immune response, may be complementary to and more efficient than the present therapy results with unconjugated chimeric anti-CD20 MoAbs.

Inflammasome activation by adenylate cyclase toxin directs Th17 responses and protection against Bordetella pertussis.

Inflammasome-mediated IL-1beta production is central to the innate immune defects that give rise to certain autoinflammatory diseases and may also be associated with the generation of IL-17-producing CD4(+) T (Th17) cells that mediate autoimmunity. However, the role of the inflammasome in driving adaptive immunity to infection has not been addressed. In this article, we demonstrate that inflammasome-mediated IL-1beta plays a critical role in promoting Ag-specific Th17 cells and in generating protective immunity against Bordetella pertussis infection. Using a murine respiratory challenge model, we demonstrated that the course of B. pertussis infection was significantly exacerbated in IL-1R type I-defective (IL-1RI(-/-)) mice. We found that adenylate cyclase toxin (CyaA), a key virulence factor secreted by B. pertussis, induced robust IL-1beta production by dendritic cells through activation of caspase-1 and the NALP3-containing inflammasome complex. Using mutant toxins, we demonstrate that CyaA-mediated activation of caspase-1 was not dependent on adenylate cyclase enzyme activity but was dependent on the pore-forming capacity of CyaA. In addition, CyaA promoted the induction of Ag-specific Th17 cells in wild-type but not IL-1RI(-/-) mice. Furthermore, the bacterial load was enhanced in IL-17-defective mice. Our findings demonstrate that CyaA, a virulence factor from B. pertussis, promotes innate IL-1beta production via activation of the NALP3 inflammasome and, thereby, polarizes T cell responses toward the Th17 subtype. In addition to its known role in subverting host immunity, our findings suggest that CyaA can promote IL-1beta-mediated Th17 cells, which promote clearance of the bacteria from the respiratory tract.

Regulation of the expression of Fit insect toxin locus genes in the root-associated biocontrol pseudomonad CHA0

The root-colonizing Pseudomonas fluorescens strain CHA0 is a biocontrol agent of soil-borne plant diseases caused by fungal and oomycete pathogens. Remarkably, this plant-beneficial pseudomonad is also endowed with potent insecticidal activity that depends on the production of a large protein toxin termed Fit (for P. fluorescens insecticidal toxin). In our present work, the genomic locus encoding the P. fluorescens insect toxin is subjected to a detailed molecular analysis. The Fit toxin gene fitD is flanked upstream by the fitABC genes and downstream by the fitE gene that encode the ABC transporter, membrane fusion, and outer membrane efflux components of a type I protein secretion system predicted to function in toxin export. The fitF, fitG, and fitH genes located downstream of fitE code for regulatory proteins having domain structures typical of signal transduction histidine kinases, LysR-type transcriptional regulators, and response regulators, respectively. The role of these insect toxin locus-associated control elements is being investigated with mutants defective for the regulatory genes and with GFP-based reporter fusions to putative promoter regions upstream of the transporter genes fitA and fitE, the toxin gene fitD, and the regulatory genes fitF and fitH. Our preliminary findings suggest that the three regulators interact with known global regulators of biocontrol factor expression to control Fit toxin expression and secretion.

Occurrence and molecular diversity of the Fit insect toxin locus in plant-beneficial pseudomonads

The application of plant-beneficial pseudomonads provides a promising alternative to chemical pest management in agriculture. The fact that Pseudomonas fluorescens CHA0 and Pf-5, both well-known biocontrol agents of fungal root diseases, exhibit also potent insecticidal activity is of particular interest, as these plant-beneficial bacteria naturally colonize the rhizosphere of important crop plants. Insecticidal activity in these strains depends on a novel locus encoding the production of a protein toxin termed Fit (for P. fluorescens insecticidal toxin). To gain a better understanding of the ecological relevance of the Pseudomonas anti-insect activity, we have begun to investigate the occurrence and molecular diversity of the Fit toxin genes among root-associated pseudomonads. To this end, we have screened a large world-wide collection of fluorescent Pseudomonas sp. isolated from the roots of different plant species using molecular fingerprinting techniques. The strains are already well characterized for exoproduct patterns and disease-suppressive ability and are currently being tested for insecticidal activity in a greater wax moth larvae assay system.

Did cholera toxin finally get caught?

To orchestrate immune responses, pathogen-recognition receptors have evolved sophisticated strategies to monitor pathogenic processes. In this issue of Cell Host & Microbe, a study by Cho et al. reveals a mechanism of immune recognition that relies on the sensing of cholera toxin within the endoplasmic reticulum.

Intranasal administration of the synthetic polypeptide from the C-terminus of the circumsporozoite protein of Plasmodium berghei with the modified heat-labile toxin of Escherichia coli (LTK63) induces a complete protection against malaria challenge.

Needle-free procedures are very attractive ways to deliver vaccines because they diminish the risk of contamination and may reduce local reactions, pain or pain fear especially in young children with a consequence of increasing the vaccination coverage for the whole population. For this purpose, the possible development of a mucosal malaria vaccine was investigated. Intranasal immunization was performed in BALB/c mice using a well-studied Plasmodium berghei model antigen derived from the circumsporozoite protein with the modified heat-labile toxin of Escherichia coli (LTK63), which is devoid of any enzymatic activity compared to the wild type form. Here, we show that intranasal administration of the two compounds activates the T and B cell immune response locally and systemically. In addition, a total protection of mice is obtained upon a challenge with live sporozoites.

Le facteur D du complément: toxine urémique [Factor D of complement: Uremic toxin?].

Factor D is an essential enzyme of the alternative pathway of complement. Its catabolism is mainly renal. The concentration of factor D increases with renal failure, and is approximately 10-fold higher in patients with end-stage renal disease. The accumulation of factor D is responsible for an enhancement of alternative pathway activation. Whether this excess of factor D has pathophysiological consequences remains to be determined, however, complement activation might participate in adverse effects during hemodialysis and in the progression of renal injury.

Crystallization and preliminary X-ray analysis of monalysin, a novel β-pore-forming toxin from the entomopathogen Pseudomonas entomophila.

Monalysin was recently described as a novel pore-forming toxin (PFT) secreted by the Drosophila pathogen Pseudomonas entomophila. Recombinant monalysin is multimeric in solution, whereas PFTs are supposed to be monomeric until target membrane association. Monalysin crystals were obtained by the hanging-drop vapour-diffusion method using PEG 8000 as precipitant. Preliminary X-ray diffraction analysis revealed that monalysin crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 162.4, b = 146.2, c = 144.4 Å, β = 122.8°, and diffracted to 2.85 Å resolution using synchrotron radiation. Patterson self-rotation analysis and Matthews coefficient calculation indicate that the asymmetric unit contains nine copies of monalysin. Heavy-atom derivative data were collected and a Ta6Br14 cluster derivative data set confirmed the presence of ninefold noncrystallographic symmetry.