Type VI Secretion Promotes Microbial Antagonism and Contributes to Pathogenesis by AHPND-causing Vibrio parahaemolyticus

The Type VI Secretion System (T6SS) is a contractile injection apparatus which delivers effector toxins in a contact-dependent manner, and has drawn considerable attention as a determinant of pathogenicity and environmental fitness in diverse bacterial taxa. Past reports have noted that many strains of Vibrio parahaemolyticus which cause Acute Hepatopancreatic Necrosis Disease (AHPND) in shrimp harbor two functional T6SSs (T6SS1 and T6SS2), leading to speculation that these systems may contribute to virulence. Here, we evaluate the involvement of Type VI Secretion (T6S) as a pathogenicity factor during shrimp infection by two geographically distinct isolates of AHPND-causing V. parahaemolyticus (VpAHPND). Combining in vitro contact-killing assays with comparative genomic analysis, we demonstrate that these strains -- 13-306/D4 (Mexico) and 12-297/B (Vietnam) -- leverage overlapping yet distinct effector arsenals to antagonize bacterial and fungal prey under warm, high-salinity conditions. Moreover, our in silico analyses reveal a predicted T6SS effector/immunity (E/I) module encoded in proximity to the pirAB-Tn903 composite transposon on pVA1-type plasmids. We demonstrate that acquisition of a plasmid expressing this module allows the non-AHPND isolate V. parahaemolyticus BB22OP to antagonize its parental strain in a T6SS1-dependent fashion. In turn, prey equipped with a plasmid expressing only the immunity gene gain protection against BB22OP attackers armed with the full E/I module. Employing an immersion bioassay, we show that both D4 and 297B require T6S functionality for full virulence against whiteleg shrimp (Litopenaeus vannamei) postlarvae under warm, marine-like conditions, and provide preliminary evidence that T6SS-dependent interactions with host-associated bacteria may contribute to virulence. Our findings offer empirical evidence supporting the involvement of T6S in AHPND pathogenesis under conditions relevant to commercial aquaculture, inviting further studies to decipher the role/s of T6S as a virulence determinant.