Domenico Azarnia Tehran

Thioredoxin and Its Reductase Are Present on Synaptic Vesicles, and Their Inhibition Prevents the Paralysis Induced by Botulinum Neurotoxins

Botulinum neurotoxins consist of a metalloprotease linked via a conserved interchain disulfide bond to a heavy chain responsible for neurospecific binding and translocation of the enzymatic domain in the nerve terminal cytosol. The metalloprotease activity is enabled upon disulfide reduction and causes neuroparalysis by cleaving the SNARE proteins. Here, we show that the thioredoxin reductase-thioredoxin protein disulfide-reducing system is present on synaptic vesicles and that it is functional and responsible for the reduction of the interchain disulfide of botulinum neurotoxin serotypes A, C, and E. Specific inhibitors of thioredoxin reductase or thioredoxin prevent intoxication of cultured neurons in a dose-dependent manner and are also very effective inhibitors of the paralysis of the neuromuscular junction. We found that this group of inhibitors of botulinum neurotoxins is very effective in vivo. Most of them are nontoxic and are good candidates as preventive and therapeutic drugs for human botulism.

The first non Clostridial botulinum-like toxin cleaves VAMP within the juxtamembrane domain.

The genome of Weissella oryzae SG25T was recently sequenced and a botulinum neurotoxin (BoNT) like gene was identified by bioinformatics methods. The typical three-domains organization of BoNTs with a N-terminal metalloprotease domain, a translocation and a cell binding domains could be identified. The BoNT family of neurotoxins is rapidly growing, but this was the first indication of the possible expression of a BoNT toxin outside the Clostridium genus. We performed molecular modeling and dynamics simulations showing that the 50 kDa N-terminal domain folds very similarly to the metalloprotease domain of BoNT/B, whilst the binding part is different. However, neither the recombinant metalloprotease nor the binding domains showed cross-reactivity with the standard antisera that define the seven serotypes of BoNTs. We found that the purified Weissella metalloprotease cleaves VAMP at a single site untouched by the other VAMP-specific BoNTs. This site is a unique Trp-Trp peptide bond located within the juxtamembrane segment of VAMP which is essential for neurotransmitter release. Therefore, the present study identifies the first non-Clostridial BoNT-like metalloprotease that cleaves VAMP at a novel and relevant site and we propose to label it BoNT/Wo.

Genomic characterization of Italian Clostridium botulinum group I strains.

Clostridium botulinum is a gram-positive bacterium capable of producing the botulinum neurotoxin, a powerful poison that causes botulism, a severe neuroparalytic disease. Its genome has been sequenced entirely and its gene content has been analyzed. To date, 19 full genomes and 64 draft genomes are available. The geographical origin of these genomes is predominantly from the US. In the present study, 10 Italian genomes of C. botulinum group I were analyzed and compared with previously sequenced group I genomes, in order to genetically characterize the Italian population of C. botulinum group I and to investigate the phylogenetic relationships among different lineages. Using the suites of software ClonalFrame and ClonalOrigin to perform genomic analysis, we demonstrated that Italian C. botulinum group I population is phylogenetically heterogeneous encompassing different and distant lineages including overseas strains, too. Moreover, a high recombination rate was demonstrated in the evolution of C. botulinum group I species. Finally, genome sequencing of the strain 357 led us to identify a novel botulinum neurotoxin subtype, F8.

A Novel Inhibitor Prevents the Peripheral Neuroparalysis of Botulinum Neurotoxins.

Botulinum neurotoxins (BoNTs) form a large class of potent and deadly neurotoxins. Given their growing number, it is of paramount importance to discover novel inhibitors targeting common steps of their intoxication process. Recently, EGA was shown to inhibit the action of bacterial toxins and viruses exhibiting a pH-dependent translocation step in mammalian cells, by interfering with their entry route. As BoNTs act in the cytosol of nerve terminals, the entry into an appropriate compartment wherefrom they translocate the catalytic moiety is essential for toxicity. Herein we propose an optimized procedure to synthesize EGA and we show that, in vitro, it prevents the neurotoxicity of different BoNT serotypes by interfering with their trafficking. Furthermore, in mice, EGA mitigates botulism symptoms induced by BoNT/A and significantly decreases the lethality of BoNT/B and BoNT/D. This opens the possibility of using EGA as a lead compound to develop novel inhibitors of botulinum neurotoxins.

Novel Botulinum Neurotoxins: Exploring Underneath the Iceberg Tip

Botulinum neurotoxins (BoNTs), the etiological agents of botulism, are the deadliest toxins known to humans. Yet, thanks to their biological and toxicological features, BoNTs have become sophisticated tools to study neuronal physiology and valuable therapeutics for an increasing number of human disorders. BoNTs are produced by multiple bacteria of the genus Clostridium and, on the basis of their different immunological properties, were classified as seven distinct types of toxin. BoNT classification remained stagnant for the last 50 years until, via bioinformatics and high-throughput sequencing techniques, dozens of BoNT variants, novel serotypes as well as BoNT-like toxins within non-clostridial species have been discovered. Here, we discuss how the now “booming field” of botulinum neurotoxin may shed light on their evolutionary origin and open exciting avenues for future therapeutic applications.

EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin

The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins.

Semicarbazone EGA Inhibits Uptake of Diphtheria Toxin into Human Cells and Protects Cells from Intoxication

Diphtheria toxin is a single-chain protein toxin that invades human cells by receptor-mediated endocytosis. In acidic endosomes, its translocation domain inserts into endosomal membranes and facilitates the transport of the catalytic domain (DTA) from endosomal lumen into the host cell cytosol. Here, DTA ADP-ribosylates elongation factor 2 inhibits protein synthesis and leads to cell death. The compound 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA) has been previously shown to protect cells from various bacterial protein toxins which deliver their enzymatic subunits from acidic endosomes to the cytosol, including Bacillus anthracis lethal toxin and the binary clostridial actin ADP-ribosylating toxins C2, iota and Clostridium difficile binary toxin (CDT). Here, we demonstrate that EGA also protects human cells from diphtheria toxin by inhibiting the pH-dependent translocation of DTA across cell membranes. The results suggest that EGA might serve for treatment and/or prevention of the severe disease diphtheria.

Multiple-locus variable number of tandem repeat analysis as a tool for molecular epidemiology of botulism: The Italian experience

Clostridium botulinum is the bacterial agent of botulism, a rare but severe neuro-paralytic disease. Because of its high impact, in Italy botulism is monitored by an ad hoc surveillance system. The National Reference Centre for Botulism, as part of this system, collects and analyzes all demographic, epidemiologic, microbiological, and molecular data recovered during cases and/or outbreaks occurred in Italy. A panel of 312 C. botulinum strains belonging to group I were submitted to MLVA sub-typing. Strains, isolated from clinical specimens, food and environmental samples collected during the surveillance activities, were representative of all forms of botulism from all Italian regions. Through clustering analysis isolates were grouped into 12 main clusters. No regional or temporal clustering was detected, demonstrating the high heterogeneity of strains circulating in Italy. This study confirmed that MLVA is capable of sub-typing C. botulinum strains. Moreover, MLVA is effective at tracing and tracking the source of contamination and is helpful for the surveillance system in terms of planning and upgrading of procedures, activities and data collection forms.