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.

Vibrios, which include more than 120 valid species, are an abundant and diverse group of bacteria in marine and estuarine environments. Some of these bacteria have been recognized as pathogens of both marine animals and humans, and therefore, their virulence mechanisms have attracted increasing attention. The type III secretion system (T3SS) is an important virulence determinant in many gram-negative bacteria, in which this system directly translocates variable effectors into the host cytosol for the manipulation of the cellular responses. In this study, the distribution of the T3SS gene cluster was first examined in 110 Vibrio strains of 26 different species, including 98 strains isolated from coastal areas in China. Several T3SS1 genes, but not T3SS2 genes (T3SS2α and T3SS2β), were universally detected in all the strains of four species, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, and Vibrio campbellii. The effector coding regions within the T3SS1 gene clusters from the T3SS1-positive strains were further analyzed, revealing that variations in the effectors of Vibrio T3SS1 were observed among the four Vibrio species, even between different strains in V. harveyi, according to their genetic organization. Importantly, Afp17, a potential novel effector that may exert a similar function as the known effector VopS in T3SS1-induced cell death, based on cytotoxicity assay results, was found in the effector coding region of the T3SS1 in some V. harveyi and V. campbellii strains. Finally, it was revealed that differences in T3SS1-mediated cytotoxicity were dependent not only on the variations in the effectors of Vibrio T3SS1 but also on the initial adhesion ability to host cells, which is another prerequisite condition. Altogether, our results contribute to the clarification of the diversity of T3SS1 effectors and a better understanding of the differences in cytotoxicity among Vibrio species.