Sticholysin II (StII) is a pore-forming toxin of biomedical interest that belongs to the actinoporin protein family. Sticholysins are currently under examination as an active immunomodulating component of a vaccinal platform against tumoral cells and as a key element of a nucleic acids delivery system to cell cytosol. These proteins form pores in the plasma membrane leading to ion imbalance and cell lysis. However, the intracellular mechanisms triggered by actinoporins upon binding to membranes and its consequences for cell death are barely understood. Here, we have examined the cytotoxicity and intracellular responses induced by StII upon binding to human B-cell lymphoma Raji in vitro. StII cytotoxicity involves a functional actin cytoskeleton, induces cellular swelling, lysis and the concomitant release of cytosol content. In addition, StII induces calcium release mainly from the Endoplasmic Reticulum, activates Mitogen-Activated Protein Kinase ERK and impairs mitochondrial membrane potential. Furthermore, StII stimulates the expression of receptor interacting protein kinase 1 (RIP1), normally related to different forms of regulated cell death such as apoptosis and necroptosis. In correspondence, necrostatin-1, an inhibitor of this kinase, reduces StII cytotoxicity. However, the mechanism of cell death activated by StII does not involve caspases activation, typical molecular features of apoptosis and pyroptosis. Our results suggest that, beyond pore-formation and cell lysis, StII-induced cytotoxicity could involve other regulated intracellular mechanisms connected to RIP1-MEK1/2 –ERK1/2- pathways. This opens new perspectives and challenges the general point of view that these toxins induce a completely unregulated mechanism of necrotic cell death. This study contributes to a better understanding of the molecular mechanisms involved in toxin-cell interaction and the implications for cell functioning, with connotation for the exploitations of these toxins in clinical settings.

Sticholysin II（StII）是具有生物医学意义的成孔毒素，属于肌动孔蛋白家族。溶血球菌溶血素目前正在作为针对肿瘤细胞的疫苗平台的活性免疫调节成分，并作为向细胞溶质的核酸传递系统的关键要素。这些蛋白质在质膜上形成孔，导致离子失衡和细胞裂解。但是，几乎没有了解肌动蛋白结合膜触发的细胞内机制及其对细胞死亡的后果。在这里，我们已经在与人B细胞淋巴瘤的Raji审查了STII引起的细胞毒性和细胞内反应体外。StII的细胞毒性涉及功能性肌动蛋白的细胞骨架，诱导细胞肿胀，溶解以及细胞质内含物的释放。此外，StII诱导钙主要从内质网释放钙，激活丝裂原活化的蛋白激酶ERK，并破坏线粒体膜电位。此外，StII刺激受体相互作用蛋白激酶1（RIP1）的表达，该蛋白激酶通常与不同形式的调节性细胞死亡有关，例如凋亡和坏死性坏死。相应地，该激酶的抑制剂necrostatin-1降低了StII的细胞毒性。但是，由StII激活的细胞死亡机制并不涉及胱天蛋白酶激活，凋亡和发烧的典型分子特征。我们的结果表明，除了孔形成和细胞裂解外，StII诱导的细胞毒性可能涉及与RIP1-MEK1 / 2 –ERK1 / 2-途径有关的其他受调控的细胞内机制。这开辟了新的观点，并挑战了这些毒素诱导坏死细胞死亡的完全不受调节的机制的一般观点。这项研究有助于更好地了解涉及毒素细胞相互作用的分子机制及其对细胞功能的影响，并为在临床环境中利用这些毒素提供了内涵。