The Transcriptional Regulator HlyU Positively Regulates Expression of exsA, Leading to Type III Secretion System 1 Activation in Vibrio parahaemolyticus

We initially generated a recombinant V. parahaemolyticus strain containing a promoterless bioluminescent reporter cassette (luxCDABE) transcriptionally fused to the exsA promoter region from V. parahaemolyticus. The pexsA-luxCDABE fusion was integrated into the tdhS locus of the V. parahaemolyticus RIMD2210633 genome (see Materials and Methods) (Fig. 1A). We chose the tdhS locus, as TdhS is not required for T3SS-1 activity (14). This strain (referred to as the Vp-lux strain) quantitatively reports the activity of the exsA promoter in real time by emitting bioluminescence, thereby allowing for sensitive in situ measurement of exsA promoter activation (Fig. 1B). The strain responded to magnesium and EGTA (T3SS-1-inducing conditions) by increasing exsA promoter activity as expected (Fig. 1B). A T3SS-1 protein secretion assay indicated that the Vp-lux strain secreted effectors similarly to wild-type V. parahaemolyticus (Fig. 1C).

Consequently, we employed a conjugative plasmid encoding a hyperactive transposase and its associated transposon (26) to mutagenize the Vp-lux strain. An optimized triparental mating approach resulted in the isolation of 4,302 stable transposants at an average transposition frequency of 1 × 10⁻⁶. Using a high-throughput 96-well plate formatted assay, the transposants were screened for exsA promoter activity via bioluminescence. The Vp-lux strain served as a reference for defined normal growth and light emission. The screen identified a spectrum of mutants that exhibited altered bioluminescence levels compared to those of the parent Vp-lux strain (Fig. 2). Prior to investigating specific mutants, they were divided into 4 groups (or quadrants) to facilitate downstream analyses. This was achieved by establishing categories based on an optical density at 600 nm (OD₆₀₀) of 0.4 and a counts per second (cps) value of 1,000 and produced four quadrants (separate groups), including low-bioluminescence and normal growth, normal bioluminescence and normal growth, low bioluminescence and low growth, and high bioluminescence and any growth (Fig. 2).

Mutants that fell into the low-luminescence and normal-growth quadrant were further subjected to statistical narrowing. Using a binning procedure, mutants were subdivided into three categories by luminescence emission (measured in counts per second; low bioluminescence, 0 to 100 cps; low-moderate bioluminescence, 101 to 300 cps; and moderate bioluminescence, 301 to 1,000 cps). The means and standard deviations for each group were calculated. The means and standard deviations were calculated as 63.30 ± 10.68, 155.1 ± 22.32, and 542.7 ± 126.6, respectively. Six mutants whose cps reading was less than 1 standard deviation below the mean were then selected for further characterization in the low-bioluminescence group. Within the low-moderate and moderate bioluminescence groups, 3 and 4 mutants were selected for characterization, respectively. Lastly, 5 of the most bioluminescent mutants (Fig. 2, gray box) were also selected for characterization. Details of the selected mutants and statistical data are listed in the supplemental material (see Table S1 in the supplemental material).

Using a genomic DNA marker retrieval protocol, we successfully identified chromosomal locations of transposition within mutants from the low-moderate and moderate light-emitting groups. From the low-moderate luminescence group linked to deficient exsA promoter activity, we mapped transposon insertions exclusively within Vp0529 (Fig. 3A). Notably, for Vp0529, we identified three isolates with the exact same transposon insertion site. These likely represent a single clone that generationally expanded during the mutagenesis procedure or, alternatively, this chromosomal site is favored for transposon insertion. For the moderate bioluminescence group, we mapped two transposon insertion mutants, Vpa0179 (phoX) and Vp1473 (merR operon) (Fig. S1). Within the high-luminescence group linked to increased exsA promoter activity, 3 independent transposon insertions were mapped near or within Vp1133 (hns) (Fig. 3B) and one insertion was mapped within Vp1633 (RTX toxin) (Fig. S1).

Our initial efforts focused on Vp0529 and Vp1133, since multiple independent mutant insertions were isolated from the lux reporter screen for these genes (Fig. 3) and therefore strongly implied a role in exsA regulation. In silico analysis of Vp0529 using NCBI BLAST indicated sequence similarity to HlyU, a DNA binding protein of many Vibrio species that acts as a positive regulator of virulence genes (27–31). Vp1133 encodes a histone-like nucleoid-structuring protein (H-NS) and has previously been implicated in exsA repression (32). To investigate this further, an assay that evaluates T3SS-1 protein secretion was performed. Critically, a marked decrease in many T3SS-1-dependent proteins was observed for the Vp0529 Tn5 insertion mutant compared to the Vp-lux parent strain (Fig. 4A). In contrast, the Vp1133 mutant displayed secretion of many T3SS-1-dependent proteins, comparable to the parent strain.

HlyU has previously been implicated as an H-NS derepressor, leading to rtxA1 toxin expression in V. vulnificus (33). To investigate if HlyU acts as a derepressor of H-NS in V. parahaemolyticus or, alternatively, as a bona fide transcriptional activator, we deleted hlyU in the context of the hns::Tn5 mutant (strain Vp-EL) and performed protein secretion and luciferase reporter assays. Vp-EL behaved similarly to the Δhns::Tn5 mutant, exhibiting T3SS-1 effectors and translocators along with enhanced luciferase activity (driven by the exsA promoter) (Fig. 4A and B). Therefore, in the absence of H-NS, HlyU is not required for T3SS-1 protein secretion or for exsA promoter activity. To confirm the role of HlyU for exsA promoter activity in cells expressing H-NS, we subjected the hlyU::Tn5 mutant to genetic complementation. Enhanced restoration of luminescence was observed for the hlyU complemented strain that was significantly above the level of the Vp-lux parent strain (Fig. 4C).

To validate the phenotypes measured in the hlyU::Tn5 mutant and to rule out any effects due to transposon insertion, an hlyU null mutant derived from wild-type V. parahaemolyticus was generated (strain hlyU1) and tested for protein secretion. In complete agreement with the hlyU::Tn5 mutant, T3SS-1 protein secretion for hlyU1 was deficient compared to that of wild-type V. parahaemolyticus and appeared similar to that of a ΔvscN1 T3SS-1-deficient strain (Fig. 5A). Genetic complementation of strain hlyU1 with the wild-type hlyU coding region in trans fully restored T3SS-1 protein secretion.

To investigate the role of HlyU in the context of infection-like conditions, we conducted in vitro HeLa infections with V. parahaemolyticus strains followed by a lactate dehydrogenase (LDH) release cytotoxicity assay. As expected, wild-type V. parahaemolyticus resulted in rapid HeLa cytotoxicity in a vscN1 (T3SS-1 ATPase)-dependent manner (Fig. 5B), confirming previous reports (34, 35). hlyU1 was significantly deficient in producing HeLa cytotoxicity (Fig. 5B), comparing closely to the low cytotoxicity levels observed for the ΔvscN1 strain. Genetic complementation of hlyU1 restored HeLa cytotoxicity to wild-type V. parahaemolyticus levels, indicating that HlyU contributed to V. parahaemolyticus cytotoxic activity during in vitro infection.

Taken together, the protein secretion, promoter activation, and cytotoxicity data all indicated that HlyU was required for exsA promoter activation that supported expression of T3SS-1-related genes, leading to HeLa cell cytotoxicity during infection.

HlyU is a member of the SmtB/ArsR family of regulator proteins that bind to DNA using a winged helix-turn-helix (wHTH) domain structure (27). Based on the hlyU1 deficiency for T3SS-1-associated phenotypes, we set out to investigate whether HlyU binds to V. parahaemolyticus DNA. We hypothesized that DNA near the exsA promoter (located within our lux reporter constructs) should contain sequences that direct HlyU binding to DNA. To test this hypothesis, we overexpressed and purified a HlyU-His fusion protein (see Materials and Methods). Native HlyU is 98 amino acids in length (NCBI accession no. NP_796908.1) and has a predicted molecular mass of 11.2 kDa. Overexpressed HlyU-His appeared as an approximately 11-kDa protein by SDS-PAGE (Fig. S2). Electrophoresis mobility shift assays (EMSA) next were performed using an exsA promoter DNA fragment mixed with increasing amounts of purified HlyU protein. The exsA promoter DNA fragment demonstrated multiple shifts, with concentrated DNA species appearing with larger amounts of HlyU-His (Fig. 6A). For these larger HlyU-His amounts, Sypro red and SYBR green staining revealed protein-DNA complexes which appeared as tight bands coinciding with reduced mobility through the acrylamide gel matrix (Fig. S3). In contrast, no mobility shift was apparent for increasing amounts of bovine serum albumin (BSA), indicating that the exsA promoter region did not support nonspecific protein binding (Fig. 6A). Based on densitometric analyses of HlyU-bound exsA promoter DNA (Table S2), an apparent binding affinity ranging from 5 to 6.3 μM (95% confidence interval) was determined (Fig. S4). A nonspecific DNA fragment next was mixed with purified HlyU-His. While some minor shifts were observed at low HlyU levels (as seen by smearing), no major shifts or concentrated DNA species were apparent with increasing amounts of HlyU-His (Fig. 6B). These results suggest that small amounts of HlyU-His interacted with DNA weakly, and larger HlyU amounts specifically bound the exsA promoter region DNA, leading to homogenous protein-DNA complexes (see Discussion).

We set out to identify the DNA region bound by HlyU within the exsA promoter region. Fluorescently end-labeled exsA promoter DNA was mixed with purified HlyU-His and then treated with DNase I. The reaction mixtures were purified and then subjected to a DNA footprinting assay. As shown in Fig. 6C, a 56-bp region repeatedly and specifically produced low fluorescence signals indicative of an HlyU-protected region (undigested by DNase I). Sequence examination of this DNA region resulted in the identification of a perfect 7-base inverted repeat sequence, 5′-ATATTAG-3′, separated by an A/T tract of 14 bases. Furthermore, a 10-base direct-repeat palindromic sequence centered within the A/T tract is present (TAAATTAAAT) (Fig. 6C). Bioinformatic analyses indicated that this region of DNA is highly conserved in all current V. parahaemolyticus sequences in public databases and is located upstream of exsA in every case (data not shown).

Vibrio parahaemolyticus RIMD2210633 was grown in Luria broth (LB; L3522; Sigma) or LBS (10 g tryptone, 5 g yeast extract, 20 g NaCl, pH 8.0). All mutant derivatives described in this study were derived from V. parahaemolyticus RIMD2210633 (Table 1). All Escherichia coli strains were cultured in LB. The following antibiotics (Sigma) were used in growth medium as required: chloramphenicol (5 μg/ml and 30 μg/ml for V. parahaemolyticus and E. coli, respectively), erythromycin (10 μg/ml and 75 μg/ml for V. parahaemolyticus and E. coli, respectively), kanamycin (50 μg/ml), and ampicillin (100 μg/ml). V. parahaemolyticus was routinely grown at 30°C or 37°C aerobically with shaking at 200 rpm for 16 to 18 h. Agar (A5306; Sigma) was added to medium at 1.5% (wt/vol) for solid medium preparations. All plasmids used in this study are listed in Table 1.

Primers NT393 and NT390 (synthesized by Integrated DNA Technologies [IDT]) were used in a PCR (with Phusion DNA polymerase; M0535S; New England BioLabs) with V. parahaemolyticus genomic DNA as the template. This approach amplified a contiguous DNA fragment encompassing upstream flanking sequence of tdhS to a partial coding sequence of the gene. Similarly, primers NT391 and NT392 were used to amplify a distal tdhS coding sequence and downstream flanking DNA. These DNA fragments were digested with PacI, ligated together with T4 DNA ligase, and then used as a DNA template in a PCR with primers NT393 and NT392. The resulting DNA fragment was blunt-end cloned into the Eco53kI site of pRE112 to create pΔtdhS. Primers NT337 and NT339 next were used with V. parahaemolyticus genomic DNA in a PCR to amplify the V. parahaemolyticus exsA promoter region. The resulting DNA product was subjected to restriction digestion with EcoRI and BamHI and then cloned upstream of the promoter-less luxCDABE gene cassette in pJW15. Finally, PacI digestion was used to excise the exsA-luxCDABE DNA fragment from pJW15, which was then cloned into the PacI site of pΔtdhS to generate ptdhS::exsAlux. ptdhS::exsAlux then served as a chromosomal integration suicide construct via delivery into V. parahaemolyticus using triparental conjugal mating. Chromosomal integrants were selected with medium containing chloramphenicol and then streak purified. The integrants were then subjected to allelic exchange by SacB-mediated sucrose selection. Stable integrants within the tdhS allele were screened by PCR and then confirmed by Sanger sequencing. All oligonucleotides used in this study are listed in Table 2.

A DNA fragment with the hlyU allele deleted was derived from V. parahaemolyticus genomic DNA by ligation PCR (primers are described in Table 2), cloned into pRE112, and then introduced into wild-type V. parahaemolyticus. Chloramphenicol selection for allelic exchange and then sucrose selection generated an hlyU null strain denoted the hlyU1 strain. An hlyU1 complementation construct was built using vibrio shuttle vector pVSV105 as a plasmid backbone (45). The hlyU DNA coding region was amplified by PCR using primers NT398 and NT399 and V. parahaemolyticus genomic DNA as the template. The hlyU DNA fragment was directionally cloned into pVSV105 as a KpnI and XhoI fragment. The resulting plasmid was transformed into E. coli DH5αλpir and then conjugated into the appropriate V. parahaemolyticus strains.

A mutagenesis procedure was followed, with minor modifications (26). Briefly, a conjugal mating on LBS agar allowed for the delivery of the plasposon pEVS170 into the Vp-lux strain. The mating mixture was plated onto selective LBS agar medium (pH 8.0) containing 10 μg/ml erythromycin and incubated at 22°C for 36 h. Transposants were then individually picked and grown on M9 minimal medium overnight. Finally, the transposants were grown overnight in LB (pH 8.0) supplemented with erythromycin and then frozen in 20% (vol/vol) glycerol in 96-well plate format.

Overnight cultures of the mini-Tn5 mutant library in 96-well plates were grown at 37°C in 5.0% CO₂. A volume of 200 μl of LB medium supplemented with 5 mM EGTA and 15 mM MgSO₄ was accurately pipetted into each well of a sterile 96-well clear-bottom, white-walled plate (number 3632; Corning). A sterilized 96-metal-pin well replicator (V&P Scientific) was used to sample the overnight 96-well plate cultures, and then the replicator was used to inoculate each well of the 96-well plate supplemented with LB medium. These plates were incubated with shaking at 30°C and 250 rpm for 3.5 h. Luminometry (counts per second, read at 1 s per well) and OD₆₀₀ endpoint readings were taken using a Victor X5 multilabel plate reader (PerkinElmer).

To narrow mutants down to a reasonable number for genetic characterization, we undertook a statistical binning approach. All mutants that fell below 1,000 cps and above an OD₆₀₀ of 0.4 were binned according to their counts per second into three categories: low glowers (less than 100 cps), low-moderate glowers (100 to 200 cps), and moderate glowers (200 to 1,000 cps). Statistical means and standard deviations were calculated for each group, and mutants that fell 1 standard deviation below the mean for each group were selected for characterization. These data are summarized in Table S1 in the supplemental material.

Genomic DNA from selected V. parahaemolyticus transposants was isolated and restriction enzyme digested to completion using HhaI, followed by a heat inactivation of HhaI. The digested DNA (final concentration of approximately 40 ng/μl) was then treated with T4 DNA ligase overnight. Finally, the ligated DNA was transformed into E. coli DH5αλpir using a standard procedure, and transformants containing self-replicating plasposons were selected on erythromycin LB agar medium. The plasposons were retrieved from the E. coli hosts using a standard miniprep procedure and were subjected to Sanger sequencing using the M13 forward sequencing primer. The sequencing data were compared to the RIMD2210633 reference V. parahaemolyticus genome to identify the genetic locus were transposition had occurred.

T3SS-1 protein secretion assays were performed as previously described (35). Culture conditions that support T3SS-1 expression were a starting OD₆₀₀ of 0.025 in LB supplemented with 15 mM MgSO₄ and 5 mM EGTA and a 4-h incubation at 30°C (250 rpm).

HeLa cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco 11995) supplemented with fetal bovine serum, seeded in a sterile 24-well plate (number 3526; Costar) at a density of 10⁵ cells/ml, and incubated for 16 h at 37°C in 5.0% CO₂ prior to infection. The HeLa cells were washed twice with 1 ml of phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.46 mM KH₂PO₄) before infection with selected V. parahaemolyticus strains. V. parahaemolyticus strains were cultured overnight in LB broth at 37°C and 200 rpm. The cultures were diluted in PBS and adjusted for cell number using OD₆₀₀ measurement and then transferred to phenol red-free DMEM (Gibco 21063) (without serum), resulting in bacterial suspensions of ∼5 × 10⁵ cells/ml. One milliliter of the relevant suspension was added to the appropriate wells of a HeLa cell-seeded 24-well plate for a multiplicity of infection (MOI) of approximately 5 (verified by viable plate counts). Uninoculated DMEM was added to wells containing the uninfected HeLa cells and the maximal LDH release condition controls. The 24-well plate was incubated for 4 h at 37°C and 5.0% CO₂. A cytotoxicity kit (88954; Pierce) was used according to the manufacturer's instructions. The following formula was used to calculate percent cytotoxicity: (experimental OD₄₉₀ − uninfected OD₄₉₀)/(maximal release OD₄₉₀) × 100.

Primers NT400 and NT401 were used in a PCR with V. parahaemolyticus genomic DNA to amplify the hlyU open reading frame without its stop codon. The resulting DNA fragment was digested with NdeI and XhoI and then cloned into the corresponding restriction sites within pET21a+ (Novagen), thus creating an in-frame fusion to a hexahistidine coding sequence (C-terminal His tag). The recombinant plasmid was initially transformed into DH5α and DNA sequence verified. Finally, the plasmid was moved into E. coli BL21(λDE3) for HlyU-His protein overexpression using a T7 inducible promoter system.

Overexpressed HlyU-His was purified from the soluble fraction of bacterial lysates using nickel-nitrilotriacetic acid (Ni-NTA)–agarose (Qiagen) and column chromatography as previously described (46). Extensively washed and purified HlyU-His was eluted from columns using an elution buffer (10 mM EDTA, 150 mM NaCl, 20 mM phosphate buffer).

The electrophoresis mobility shift assay (EMSA) was performed as previously described (47), with a few minor modifications. Purified HlyU-His or BSA (B9000S; New England BioLabs) was mixed with a PCR-amplified exsA promoter DNA fragment in binding buffer (10 mM Tris [pH 7.5 at 20°C], 1 mM EDTA, 0.1 M KCl, 0.1 mM dithiothreitol, 5%, vol/vol, glycerol, 0.01 mg ml⁻¹ BSA). A PCR-amplified nleH1 gene fragment (derived from EPEC genomic DNA) served as an unrelated nonspecific DNA control. Six percent Tris-borate-EDTA (TBE)–polyacrylamide gels were prerun with 1.5 μl of 6× TBE loading dye (6 mM Tris, 0.6 mM EDTA, 30%, vol/vol, glycerol, 0.0006%, wt/vol, bromophenol blue, 0.0006%, wt/vol, xylene cyanol FF) and then loaded with the equilibrated protein-DNA samples. The gel was run for 4 h (100 V, 4°C) and then stained with SYBR green fluorescent DNA dye (Invitrogen) at a 1× concentration in TBE buffer and imaged using a VersaDoc MP5000 system (Bio-Rad). Protein staining and processing of TBE gels was performed using SYPRO Ruby Red (Bio-Rad) as previously described (48) and then imaged using a VersaDoc MP5000 system (Bio-Rad).

A 6-carboxyfluorescein (6-FAM)-end-labeled exsA promoter fragment was amplified by PCR using V. parahaemolyticus genomic DNA as the template with primers NT337 and NT402. Using the same EMSA binding conditions, the 6-FAM-labeled exsA promoter PCR product was mixed with purified HlyU-His or BSA and allowed to equilibrate for 30 min. Various amounts of DNase I (0.5 to 2 U) were added to the protein-DNA mixture, followed by immediate incubation at 37°C for 20 min. To stop DNase I activity, reaction mixtures were rapidly heated to 75°C for 10 min and then purified using a PCR purification kit (Qiagen). The samples were then subjected to capillary electrophoresis on an ABI-3730XL DNA Analyzer (Genome Québec Innovation Center). The chromatogram from this analysis was matched with a Sanger DNA sequencing reaction of the same exsA promoter DNA fragment. The HlyU-His protected region was identified by searching the chromatogram for a region with decreased 6-FAM fluorescence output. This experiment was repeated four times and included independent binding and DNase I digestion reactions.