An acute and highly reproducible motor axon terminal degeneration followed by complete regeneration is induced by some animal presynaptic neurotoxins, representing an appropriate and controlled system to dissect the molecular mechanisms underlying degeneration and regeneration of peripheral nerve terminals. We have previously shown that nerve terminals exposed to spider or snake presynaptic neurotoxins degenerate as a result of calcium overload and mitochondrial failure. Here we show that toxin-treated primary neurons release signaling molecules derived from mitochondria: hydrogen peroxide, mitochondrial DNA, and cytochrome c. These molecules activate isolated primary Schwann cells, Schwann cells cocultured with neurons and at neuromuscular junction in vivo through the MAPK pathway. We propose that this inter-and intracellular signaling is involved in triggering the regeneration of peripheral nerve terminals affected by other forms of neurodegenerative diseases.motor axon degeneration | presynaptic neurotoxins | mitochondrial alarmins | Schwann cells

AIP56 (apoptosis-inducing protein of 56 kDa) is a major virulence factor of Photobacterium damselae piscicida (Phdp), a Gram-negative pathogen that causes septicemic infections, which are among the most threatening diseases in mariculture. The toxin triggers apoptosis of host macrophages and neutrophils through a process that, in vivo, culminates with secondary necrosis of the apoptotic cells contributing to the necrotic lesions observed in the diseased animals. Here, we show that AIP56 is a NF-κB p65-cleaving zinc-metalloprotease whose catalytic activity is required for the apoptogenic effect. Most of the bacterial effectors known to target NF-κB are type III secreted effectors. In contrast, we demonstrate that AIP56 is an A-B toxin capable of acting at distance, without requiring contact of the bacteria with the target cell. We also show that the N-terminal domain cleaves NF-κB at the Cys39-Glu40 peptide bond and that the C-terminal domain is involved in binding and internalization into the cytosol.

The entry and enzymatic activity of the anthrax edema factor (EF) in different cell types was studied by monitoring EF-induced changes in intracellular cAMP with biochemical and microscopic methods. cAMP was imaged in live cells, transfected with a fluorescence resonance energy transfer biosensor based on the protein kinase A regulatory and catalytic subunits fused to CFP and YFP, respectively. The cAMP biosensor was located either in the cytosol or was membrane-bound owing to the addition of a tag determining its myristoylation/palmitoylation. Real-time imaging of cells expressing the cAMP biosensors provided the time course of EF catalytic activity and an indication of its subcellular localization. Bafilomycin A1, an inhibitor of the vacuolar ATPase proton pump, completely prevented EF activity, even when added long after the toxin. The time course of appearance of the adenylate cyclase activity and of bafilomycin A1 action suggests that EF enters the cytosol from late endosomes. EF remains associated to these compartments and its activity shows a perinuclear localization generating intracellular cAMP concentration gradients from the cell centre to the periphery.

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.

SummaryTo investigate the cell entry and intracellular trafficking of anthrax oedema factor (EF) and lethal factor (LF), they were C-terminally fused to the enhanced green fluorescent protein (EGFP) and monomeric Cherry (mCherry) fluorescent proteins. Both chimeras bound to the surface of BHK cells treated with protective antigen (PA) in a patchy mode. Binding was followed by rapid internalization, and the two anthrax factors were found to traffic along the same endocytic route and with identical kinetics, indicating that their intracellular path is essentially dictated by PA. Colocalization studies indicated that anthrax toxins enter caveolin-1 containing compartments and then endosomes marked by phoshatidylinositol 3-phoshate and Rab5, but not by early endosome antigen 1 and transferrin. After 40 min, both EF and LF chimeras were observed to localize within late compartments. Eventually, LF and EF appeared in the cytosol with a time-course consistent with translocation from late endosomes. Only the EGFP derivatives reached the cytosol because they are translocated by the PA channel, while the mCherry derivatives are not. This difference is attributed to a higher resistance of mCherry to unfolding. After translocation, LF disperses in the cytosol, while EF localizes on the cytosolic face of late endosomes.