vscN encodes a type III secretion system ATPase in Vibrio splendidus AJ01 that contributes to pathogenicity and Hop secretion

Abstract The type III secretion system (T3SS) is a protein secretory pathway that is commonly used to inject virulence factors into the host cells through the injectosome, which is dependent on ATPase catalyzing the unfolding of proteins. In this study, a gene designated vscN in Vibrio splendidus AJ01, which was predicted to encode the T3SS ATPase based on its sequence similarity to other T3SS ATPases, was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant VscN possessed ATPase activity, catalyzing the release of ADP and Pi from ATP with an ATPase activity of 5.86 U/mg in a time- and dose-dependent manner. To determine whether VscN contributes to the pathogenesis of V. splendidus, we first constructed a mutant, ∆vscN, by creating an in-frame deletion in the vscN gene. ∆vscN showed no difference in growth compared with the wild-type strain, but in an immersion infection test, the survival rate of Apostichopus japonicus increased from 10% to 50% after challenge with the wild-type strain and the ∆vscN strain at a level of 5 × 107 CFU/mL, respectively. After coincubation of coelomocyte with the wild-type strain or ∆vscN strain for 3 h, it was found that, in contrast to the wild-type strain, the ∆vscN strain negligibly accumulated at the cell surface of coelomocyte. In our previous study, Hop was found to be a T3SS effector, thus, fluorescence in situ hybridization and real-time reverse transcription PCR were performed to further show that that the expression of the hop gene and the secretion of Hop were attenuated in the ∆vscN mutant strain. The results not only indicated that VscN is a T3SS ATPase that is involved in the virulence of AJ01 but also confirmed that Hop is a T3SS effector whose expression and secretion depend on VscN.