Conserved domain structure and phylogenetic tree analysis of VPA1365 in V. parahaemolyticus. (A) Genetic location and organization of the T3SS2 in V. parahaemolyticus RIMD 2210633. (B) Three blue boxes represent the conserved TPR domain predicted by the INTERPRO. (C) The VPA1365 structure prediction was accomplished using the SWISS-MODEL (the blue and orange regions indicate the amino terminal and carboxy terminal, respectively). (D) The phylogenetic tree inferred from 1,000 replicates was constructed by the neighbor-joining method using MEGA-X.

Analysis of the intestinal pathological section and bacterial colonization quantity in infant rabbits. (A) The amount of WT and Δvpa1365 colonized in the small intestine. **P ≤ 0.01. (B) Observations on the small intestine were challenged with PBS, WT, and Δvpa1365, respectively. The yellow arrow, red arrow, and brown arrow represented exfoliated intestinal villous epithelial cells, damaged lamina propria, and vascular congestion, respectively. Three infected tissues from each group were analyzed, and a representative image was presented for each case. Scale bars: 50 µm.

The effects of vpa1365 on the expression and secretion of T3SS2. (A) Relative transcript levels of the T3SS2-associated genes in WT, ∆vpa1365, ∆vpa1365-vpa1365, ∆vpa1365-vtrA, and ∆vpa1365-vtrB. Results are represented as mean ± SD (n = 3, biologically independent experiments). *P ≤ 0.05 and **P ≤ 0.01. ns, no significance. (B) The T3SS2 translocon protein VopD2 secretion and RNAP of WT, ∆vpa1365, and complemented strains in LB medium with or without 0.04% bile salts. (C) The analysis of binding of VPA1365 to the promoter of the regulator vtrA. (D) The T3SS2 translocon protein VopD2 secretion and RNAP of WT, ∆vpa1365, and complemented strains in LB medium at 28°C, 40°C, pH = 8, 0.1 M NaCl, and 0.5 M NaCl, respectively.

The effects of vpa1365 on cytotoxicity and cell adhesion ability. (A) Cytotoxicity and (B) adhesion rate of HeLa cell monolayers infected with WT, ∆vpa1365, ∆vpa1365-vpa1365, and ∆vpa1365-pMMB207 at 2 h, respectively. Results are presented as mean ± SD (n = 3). Three separate experiments were performed in biological triplicates each. *P ≤ 0.05 and **P ≤ 0.01.

The effects of vpa1365 on biofilm formation and hemolytic activity. (A) The normalized biofilm formation (total amount of biofilm/growth) of WT, Δvpa1365, ∆vpa1365-vpa1365, and ∆vpa1365-pMMB207 cultured at 37°C for 31 h was stained with 0.1% crystal violet, respectively. (B) The hemolytic circle diameter of WT, Δvpa1365, ∆vpa1365-vpa1365, and ∆vpa1365-pMMB207 growing on the Wagatsuma blood agar base. (C) Relative transcript levels of virulence-associated genes in WT, ∆vpa1365, and ∆vpa1365-vpa1365. (A–C) Results are presented as mean ± SD (n = 3). Three separate experiments were performed in biological triplicates each. *P ≤ 0.05 and **P ≤ 0.01. ns, no significance. (D–F) The analysis of binding of VPA1365 to the promoter of biofilm regulator scrG, type IV pilus pilA, and mshA.

Identification of the VPA1365-binding motifs. (A) EMSA analysis of VPA1365 binding to the scrG promoter with different truncations. Six truncated fragments for the scrG promoter (left panel) were analyzed for their interaction with VPA1365 (right panel). (B) Nucleotide sequences (271 bp) of the scrG promoter (PscrG). The VPA1365-binding site is boxed in black, while −35 box and −10 box sequences are underlined. Start codon (ATG) and +1 of transcription are shown in red and with an arrow, respectively. (C) EMSA analysis of VPA1365 binding to the truncated ∆scrG promoter (∆PscrG) with the deletion of the VPA1365-binding site (140–166 bp). The fragment of ∆PscrG (left panel) was analyzed for its binding with VPA1365 (right panel). (D) The predicted consensus VPA1365-binding motif was generated from MEME-Suite analysis of the selected promoters.

The survival rate of zebrafish challenged with WT, Δvpa1365, and ∆vscN2, respectively. The number of dead zebrafish was recorded, and the percent survival rate in every group was calculated. The comparison of survival distribution between WT and mutant strains was performed using the log-rank (Mantel-Cox) test in GraphPad Prism version 7.0. **P ≤ 0.01.

The putative model of VPA1365 regulation in V. parahaemolyticus. VPA1365 indirectly contributes to the function of T3SS2 by binding to the promoter of vtrA and additionally positively regulates hemolytic activity, biofilm formation, cytotoxicity, and adhesion ability. The black solid arrows indicate direct binding or regulation, and the black dashed arrows indicate indirect or unclear regulation.