Mal-dependent generation of extracellular vesicles, a new role of epsilon toxin from clostridium perfringens

Resumen

Clostridium perfringens produces the third most lethal bacterial toxin known (Epsilon toxin), among other toxins. Epsilon toxin (Etx) has been classified into the aerolysin-like β-pore forming toxins. The toxin has been associated to a possible environmental trigger for Multiple Sclerosis (MS), as MS patients were immunoreactive against the toxin, whereas controls were not reactive. The toxin has been described to cause an ATP release in MDCK and MOLT4 cells, where the Myelin and Lymphocyte (MAL) protein is highly expressed. Moreover, MAL has been stated as the cellular receptor for the toxin. Our results describe a new association between the Etx-MAL complex with TMEM16 protein family. TMEM16 protein, also known as Anoctamin proteins are a family of protein with 10 transmembrane segments and act as dimers. They have a divergence in functionality, where TMEM16A and TMEM16B are classical calcium activated chloride channels and the rest of the family mostly act as scramblase proteins. The scramblase proteins translocate phospholipids from the internal leaflet to the outer leaflet of the plasma membrane and vice versa without the consumption of ATP. We studied the action of Etx through the unicellular model of Xenopus laevis oocytes. We proceeded with electrophysiological recording, imaging and histological analysis to undercover the effect of the toxin. The expression of human MAL was necessary for the action of the toxin, which mainly caused a release of ATP, an activation of current of TMEM16 channels and the generation of extracellular microvesicles (EVs). The action of the toxin involved an increase of intracellular calcium concentration originating from intracellular stores. The blockage of the calcium concentration increase leaded to an abolishment of the action of the toxin. Hence, MAL expression and an increment in calcium concentrations were required for the toxin to have an effect. Due to the action of the toxin we observe a generation of EVs from X. laevis and MOLT4 cells treated with Etx. We demonstrate that TMEM16A is involved in the current activation observed in oocytes treated with the toxin and also in the generation of EVs, as a scramblase activity is triggered. Hence we conclude that Etx causes a functional conversion to TMEM16A, where the protein has a dual function of anion channel and scramblase protein. Leading to the generation of EVs and the consequent and coincident release of ATP. In parallel we also attempted to generate a mouse model of MS, Etx-injected mice showed a decrement in movement compared to PBS injected mice. In conclusion, Etx activates TMEM16A channel and triggers the generation of EVs from the plasma membrane in X. Laevis oocytes and MOLT-4 cells. These EVs contain the Etx oligomer as well as MAL and TMEM16A. The generation of EVs is coincident with the release of ATP, which is dependent on the expression of MAL.