Abstract
The type VI secretion system (T6SS) is a major virulence factor in Vibrio parahaemolyticus, but its pathogenic mechanisms are poorly understood or still not fully understood. This study investigates how two critical T6SS1 structural components, VipA1 and Hcp1, contribute to bacterial virulence and host inflammatory responses. Comparative proteomics revealed 149 secreted proteins dependent on T6SS1, including 28 core proteins requiring both VipA1 and Hcp1 for secretion. These proteins were functionally linked to metabolic pathways such as folate-mediated one-carbon metabolism and lysine degradation, as well as structural processes like flagellar assembly. Phenotypic analysis revealed that the ΔvipA1-hcp1 double mutant showed markedly attenuated virulence: 52.7% reduction in antibacterial activity compared to the wild-type strain. Biofilm formation increased 2.1-fold at 30°C and 2.8-fold at 37°C in ΔvipA1-hcp1, while swimming and swarming motility decreased by 30.9% and 35.5%. In vivo, ΔvipA1-hcp1 caused only 50% mortality in mice, compared to 91.7% for the wild-type strain, and exhibited 3- to 15-fold lower bacterial loads in the blood, liver, and spleen. Histopathological analysis confirmed that the ΔvipA1-hcp1 failed to induce tissue damage, unlike the wild-type strain. At the host interface, deletion of vipA1 and hcp1 led to significantly elevated inflammatory cytokine (IL-1β, IL-8, and IL-6) mRNA levels. Mechanistically, T6SS1 inhibited NF-κB activation by stabilizing IκBα and reducing p65 nuclear translocation (40.0% in wild-type-infected cells vs 85.8% in double mutant-infected cells). These findings establish VipA1 and Hcp1 as critical regulators of T6SS1-mediated coordinating effector secretion, virulence, immune evasion, and lethality, providing novel mechanistic insights into V. parahaemolyticus pathogenesis.