Regulatory mechanism for host-cell contact-dependent T3SS gene expression in Vibrio parahaemolyticus

Abstract

Many pathogenic bacteria regulate gene expression in response to the surrounding environment to establish infection. One such mechanism is the regulation of gene expression in response to contact with host cells. Here, we show that Vibrio parahaemolyticus, a causative agent of foodborne gastroenteritis, has a host-cell contact-dependent regulatory mechanism for virulence gene expression. Type III secretion system 2 (T3SS2), an essential virulence determinant for acute gastroenteritis encoded by V. parahaemolyticus pathogenicity island (Vp-PAI), recognizes host-cell contact by sensing high intracellular K⁺ levels and switching its secretory substrates. The switching of secretory substrates is regulated by proteins called gatekeepers. Mutants deficient in the gatekeeper genes lose the ability to switch secretory substrates and lock their secretory state into a host-cell contact-dependent manner. Transcriptomic analysis of these mutants revealed the upregulation of Vp-PAI genes, which was dependent on T3SS2 secretory activity, suggesting the presence of a negative regulator secreted by T3SS2. Comparative proteomic analyses identified a previously unrecognized T3SS2 secretory substrate, VPA1369 (VtrN), that negatively regulates Vp-PAI gene transcription. Secretion of VtrN was promoted under conditions that mimic host-cell contact. vtrN gene deletion specifically upregulated Vp-PAI gene expression, independent of T3SS2 secretory activity, indicating its role as a repressor. VtrN interacts with VtrB, a key transcription factor for Vp-PAI genes, suppressing its transcriptional activity. This mechanism illustrates how V. parahaemolyticus enhances virulence gene expression upon host-cell contact through the T3SS2 recognition system, highlighting an adaptive strategy for establishing infection.IMPORTANCEThe type III secretion system (T3SS) is a crucial virulence factor tightly regulated for optimal host manipulation and virulence. This study revealed that the expression of T3SS2, a key virulence factor in Vibrio parahaemolyticus that causes acute gastroenteritis, is strictly regulated by host-cell contact. VtrN, a negative regulator exported from the bacterium through T3SS2, plays a key role in this host-cell contact-dependent gene transcriptional process. VtrN binds directly to the master regulator of Vp-PAI, the region encoding T3SS2, and represses its transcriptional activity. Upon host-cell contact, VtrN export is promoted, leading to the derepression of Vp-PAI gene expression. Thus, V. parahaemolyticus can effectively upregulate the expression of virulence factors when interacting with the host cells. Understanding these regulatory mechanisms could lead to innovative infection control strategies, opening new avenues for research and discovery.

Keywords: Vibrio parahaemolyticus; gatekeeper; gene regulation; type III secretion system.

Fig 1

Deletions of the gatekeeper gene of T3SS2 lead to the upregulation of the Vp-PAI gene. (A) Gene transcription data from RNA-seq of V. parahaemolyticus RIMD2210633 (WT) and gatekeeper mutants (WT∆vgpA and WT∆vgpB); genes that showed more than 4-fold alteration with statistically significant differences (P ≤ 0.05) are shown. (B, C) Relative gene expression of V. parahaemolyticus WT and POR-2 (WT∆tdhAS∆vcrD1)∆vcrD2 with gatekeeper gene deletion was determined via qRT‒PCR using recA expression as an endogenous control and the expression of target genes by the parental strain as a baseline. The bars represent the average of three independent experiments. The error bars indicate 95% CI. ***, P ≤ 0.0001; **, P ≤ 0.001; *, P ≤ 0.05.

Fig 2

Identification of an unrecognized T3SS2-secreted protein that facilitates T3SS2-mediated infection and effector translocation. (A) Cytotoxic effect of V. parahaemolyticus POR-2 (WT∆tdhAS∆vcrD1) and the derivative strains on Caco-2 cells, with total cell lysis used as a positive control and uninfected cells used as a negative control. The bars represent the average of three independent experiments. The error bars indicate the standard deviations (SDs). ***, P ≤ 0.0001; **, P ≤ 0.001; *, P ≤ 0.05. (B) Growth curves of V. parahaemolyticus POR-2 and its derivative strains cultured in LB broth. The plots represent the average of three independent experiments. The error bars indicate the standard error of the mean (SEM). (C) Intracellular cAMP levels from VopT translocation in Caco-2 cells infected with VopT-CyaA-expressing isogenic V. parahaemolyticus POR-2, POR-2ΔvcrD2 and POR-2Δvpa1369 for 1.5 h. The bars represent the average of three independent experiments. The error bars indicate the SEM. (D) Production and secretion of VPA1369 with a triple FLAG tag from V. parahaemolyticus POR-2 and derivative strains cultured in LB broth with 0.04% crude bile. DnaK was used as a control for sample preparation. The figure was representative of three independent experiments. (E) Production and secretion of VPA1369 with a triple FLAG tag from V. parahaemolyticus POR-2 and POR-2ΔvcrD2 cultured in LB broth with 0.1 M NaCl or 0.1 M KCl. DnaK was used as a control for sample preparation. The figure was representative of three independent experiments. (F) Intracellular cAMP levels resulting from the translocation of VPA1369 in Caco-2 cells infected with VPA1369-CyaA-expressing isogenic V. parahaemolyticus POR-2 and POR-2ΔvcrD2 for 1.5 or 3 h. The bars represent the average of three independent experiments. The error bars indicate the SEM.

Fig 3

VPA1369 negatively regulates Vp-PAI-encoded gene expression. (A) Gene transcription levels were determined by RNA-seq of V. parahaemolyticus RIMD2210633 and the vpa1369 mutant; genes that showed more than a 4-fold change in expression with a statistically significant difference (P ≤ 0.05) are shown. (B) Relative gene expression levels in the V. parahaemolyticus POR-2 (WT∆tdhAS∆vcrD1) with vpa1369 deletion and complemented strain were determined via qRT-PCR, with recA expression as endogenous control and the expression of target genes by the parental strain as a baseline. The bars represent the average of three independent experiments. The error bars indicate 95% CI. ***, P ≤ 0.0001; **, P ≤ 0.001; *, P ≤ 0.05. (C) T3SS2-related protein production and secretion from V. parahaemolyticus POR-2 and its derivative strains cultured in LB broth. DnaK was used as a control for sample preparation. The figure was representative of three independent experiments. (D) Relative gene expression in the V. parahaemolyticus POR-2ΔvcrD2 and derivative strains was determined via qRT-PCR, with recA expression as endogenous control and the expression of target genes by the parental strain as a baseline. The bars represent the average of three independent experiments. The error bars indicate 95% CI. ***, P ≤ 0.0001; **, P ≤ 0.001; *, P ≤ 0.05.

Fig 4

VtrN interacts with VtrB and suppresses VtrB-mediated transcriptional activity of Vp-PAI expression. (A) Diagram of the T3SS2 transcriptional regulatory system. (B) β-galactosidase activity resulting from the coexpression of VtrB (pSA-vtrB) and VtrN (pBAD30-vtrN) with a β-galactosidase reporter gene under the vpa1343 promoter (pHRP309-pro-vpa1343) in E. coli MC4100 in LB broth supplemented with 0.02% arabinose. The bars represent the average of three independent experiments. The error bars indicate the SDs. (C) β-galactosidase activity of the β-galactosidase reporter gene under the vpa1343 promoter (pHRP309-pro-vpa1343) in the V. parahaemolyticus POR-2 (WT∆tdhAS∆vcrD1) and derivative strains. The bars represent the average of three independent experiments. The error bars indicate the SDs. (D) β-galactosidase activity of the β-galactosidase reporter gene under the vpa1343 promoter (pHRP309-pro-vpa1343) in vtrN mutant and complement strains cultured in LB broth. The bars represent the average of three independent experiments. The error bars indicate the SDs. (E) Protein-protein interactions between VtrN and other T3SS2 transcriptional regulators, Y2HGold/pGBKT7-53 and pGADT7-T were used as positive controls. Co-transformants were cultured on a double dropout medium lacking leucine and tryptophan (SD/‒Trp/‒Leu; DDO; top panel), DDO supplemented with 40 µg/mL X-α-Gal and 200 ng/mL aureobasidin A (DDO/X/A; middle panel) and quadruple dropout media (SD/‒Ade/‒His/‒Leu/‒Trp; QDO) supplemented with 40 µg/mL X-α-Gal and 200 ng/mL aureobasidin A (QDO/X/A; bottom panel). Y2HGold/pGBKT7-Lam and pGADT7-T were used as negative controls. (F) Diagram of the VtrB domain and truncations of VtrB tagged with a GST at the N-terminus. (G) Coomassie brilliant blue staining and immunoblotting of VtrN with a hexahistidine tag pulled down from the bacterial lysate using GST-truncated VtrB. Yellow arrows indicated pull down VtrN.

Fig 5

Schematic describing the role of VtrN in Vp-PAI regulation. VtrN directly binds VtrB to block the transcription activity during the free-living state. Upon host-cell contact, bacteria sense intracellular K⁺, and VtrN is translocated into the host cytosol, activating VtrB transcriptional activity of Vp-PAI.