The Vibrio alginolyticus T3SS effectors, Val1686 and Val1680, induce cell rounding, apoptosis and lysis of fish epithelial cells

Vibrio alginolyticus is a Gram-negative bacterium that is an opportunistic pathogen of both marine animals and people. Its pathogenesis likely involves type III secretion system (T3SS) mediated induction of rapid apoptosis, cell rounding and osmotic lysis of infected eukaryotic cells. Herein, we report that effector proteins, Val1686 and Val1680 from V. alginolyticus, were responsible for T3SS-mediated death of fish cells. Val1686 is a Fic-domain containing protein that not only contributed to cell rounding by inhibiting Rho guanosine triphosphatases (GTPases), but was requisite for the induction of apoptosis because the deletion mutant (Δval1686) was severely weakened in its ability to induce cell rounding and apoptosis in fish cells. In addition, Val1686 alone was sufficient to induce cell rounding and apoptosis as evidenced by the transfection of Val1686 into fish cells. Importantly, the Fic-domain essential for cell rounding activity was equally important to activation of apoptosis of fish cells, indicating that apoptosis is a downstream event of Val1686-dependent GTPase inhibition. V. alginolyticus infection likely activates JNK and ERK pathways with sequential activation of caspases (caspase-8/-10, -9 and -3) and subsequent apoptosis. Val1680 contributed to T3SS-dependent lysis of fish cells in V. alginolyticus, but did not induce autophagy as has been reported for its homologue (VopQ) in V. parahaemolyticus. Together, Val1686 and Val1680 work together to induce apoptosis, cell rounding and cell lysis of V. alginolyticus-infected fish cells. These findings provide new insights into the mechanism of cell death caused by T3SS of V. alginolyticus.

溶藻弧菌（Vibrio alginolyticus）是一种革兰氏阴性菌，同时为海洋动物与人类的条件致病菌。其致病机制可能涉及Ⅲ型分泌系统（type III secretion system, T3SS）介导受感染真核细胞发生快速凋亡、细胞皱缩及渗透性裂解。本文报道，溶藻弧菌的效应蛋白Val1686与Val1680是介导T3SS诱导鱼类细胞死亡的关键因子。Val1686是一种含有Fic结构域（Fic-domain）的蛋白，它不仅通过抑制Rho家族鸟苷三磷酸酶（Rho guanosine triphosphatases, GTPases）引发细胞皱缩，同时也是诱导凋亡所必需的：缺失val1686的突变株（Δval1686）在诱导鱼类细胞皱缩与凋亡的能力上显著减弱。此外，仅向鱼类细胞中转染Val1686即可诱导细胞皱缩与凋亡，该结果进一步验证了其独立功能。值得注意的是，介导细胞皱缩活性的Fic结构域同样对激活鱼类细胞凋亡至关重要，这表明凋亡是Val1686依赖型GTP酶抑制作用的下游事件。溶藻弧菌感染可能通过依次激活半胱天冬酶（caspases）家族（caspase-8/-10、-9与-3），进而活化JNK与ERK通路，最终引发细胞凋亡。Val1680参与了溶藻弧菌T3SS依赖的鱼类细胞裂解过程，但与副溶血性弧菌（Vibrio parahaemolyticus, V. parahaemolyticus）中其同源蛋白VopQ的已有报道不同，Val1680并不会诱导自噬。综上，Val1686与Val1680协同作用，共同引发溶藻弧菌感染的鱼类细胞出现凋亡、细胞皱缩及细胞裂解。本研究为解析溶藻弧菌T3SS介导的细胞死亡机制提供了全新视角。