Abstract:[Background] Vibrio harveyi is a Gram-negative marine bacterium that has been recognized as an opportunistic pathogen for marine animals. The type III secretion system (T3SS) is a highly-conserved virulence apparatus used by Gram-negative bacterial pathogens to cause infections. [Objective] To decipher the mechanism of T3SS of V. harveyi in inducing the death of fish cells. [Methods] The T3SS disfunctional mutant was established by the in-frame deletion of vcrD in V. harveyi strain 345, and then its complemented mutant was constructed. The wild type and mutant strains were used to infect FHM cells, and then DAPI staining, in situ detection of fragmented DNA (TUNEL assay), Caspase activity assay, and lactate dehydrogenase (LDH) release assay were carried out to reveal the features of cell death. [Results] Dying cells exhibited the features such as cell rounding, nuclear condensation, and DNA fragmentation of apoptotic cells. Caspase-3 was activated by the T3SS, which confirmed that the infection with V. harveyi rapidly induced T3SS-dependent apoptosis in fish cells. Furthermore, the infection with V. harveyi strain 345 led to release of cellular contents including LDH from infected fish cells. Importantly, the inhibition of apoptosis did not prevent cells from releasing cellular contents or rounding. [Conclusion] V. harveyi uses T3SS to induce the activation of apoptosis pathways and the rounding of fish cells, ultimately leading to cell death.
