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Infect Immun. Jul 2007; 75(7): 3282–3289.
Published online Apr 16, 2007. doi:  10.1128/IAI.00045-07
PMCID: PMC1932941

The HlyU Protein Is a Positive Regulator of rtxA1, a Gene Responsible for Cytotoxicity and Virulence in the Human Pathogen Vibrio vulnificus[down-pointing small open triangle]

Abstract

Vibrio vulnificus is an opportunistic human pathogen that preferentially infects compromised iron-overloaded patients, causing a fatal primary septicemia with very rapid progress, resulting in a high mortality rate. In this study we determined that the HlyU protein, a virulence factor in V. vulnificus CMCP6, up-regulates the expression of VV20479, a homologue of the Vibrio cholerae RTX (repeats in toxin) toxin gene that we named rtxA1. This gene is part of an operon together with two other open reading frames, VV20481 and VV20480, that encode two predicted proteins, a peptide chain release factor 1 and a hemolysin acyltransferase, respectively. A mutation in rtxA1 not only contributes to the loss of cytotoxic activity but also results in a decrease in virulence, whereas a deletion of VV20481 and VV20480 causes a slight decrease in virulence but with no effect in cytotoxicity. Activation of the expression of the rtxA1 operon by HlyU occurs at the transcription initiation level by binding of the HlyU protein to a region upstream of this operon.

Vibrio vulnificus is an opportunistic human pathogen that preferentially affects patients that have underlying hepatic diseases and other compromised conditions, such as hemochromatosis and beta-thalassemia, and heavy alcohol drinkers (6, 19, 32). This bacterium frequently causes fatal primary septicemia with very rapid progress, resulting in a mortality rate of more than 50% within a few days (6, 12, 20, 33). The common theme in most of these patients is that iron is present at higher than physiological levels. Some of the confirmed or putative virulence factors required for in vivo survival and growth of V. vulnificus include capsule (30, 38), protease (8), flagella (9, 24), pili (22), and siderophore vulnibactin (14). More recently it was reported that antibodies against the V. vulnificus HlyU protein were present in serum of convalescent patients who survived V. vulnificus septicemia and that an hlyU mutation resulted in a 53-fold increase of the 50% lethal dose (LD50) of V. vulnificus in the iron-normal mouse model (7). It was already known that the Vibrio cholerae HlyU predicted protein, containing a putative helix-turn-helix motif (34), activated the expression of the hemolysin gene hlyA as well as a hemolysin-coregulated protein gene hcp (35, 36). The HlyU homologue in V. vulnificus was reported to up-regulate the cytolysin/hemolysin gene vvhA and a gene encoding an elastolytic protease (7), while the hlyU mutant showed a significant decrease in cytotoxic activity to HeLa cells. It is worth mentioning that the purified V. vulnificus cytolysin (VvhA) exhibited cytolytic activity against Chinese hamster ovary cells (5) and could kill mice at low dosages by intravenous administration (10). However, when the vvhA gene was mutated, the VvhA cytolysin-negative strain still showed the same LD50 in both iron-normal and iron-overloaded mouse models (37). Furthermore, the cytotoxicity for Hep-2 cells in the cytolysin-negative strain was comparable to that of the wild type (4). Therefore, the molecular mechanisms involved in the loss of cytotoxicity and virulence of the hlyU mutant remain unknown.

In this work we demonstrate that HlyU is a positive regulator of VV20479, one of the three homologues of the V. cholerae RTX (repeats in toxin) toxin gene found in V. vulnificus CMCP6 and that we named rtxA1. RTX homologues were also identified on the small and large chromosomes of V. vulnificus strain YJ016 (2). In this study, we show that the decrease of expression of the rtxA1 gene contributes to the loss of cytotoxic activity and virulence in V. vulnificus. Furthermore, we demonstrate that the HlyU protein activates expression of the rtxA1 operon at the transcription initiation level by direct binding of HlyU to the rtxA1 upstream promoter region.

MATERIALS AND METHODS

Bacterial strains, plasmids, and primers.

The strains and plasmids used in this work are listed in Table Table1,1, whereas the list of primers is included in the supplemental material. The genomic sequence of V. vulnificus strain CMCP6 (GenBank accession no. AE016795 and AE016796) was used to generate the primers. Primers for real-time reverse transcription-PCR (RT-PCR) were designed by Primer Express software, version 2.0, while the rest of the primers were designed with a G+C content of ~50%. Bacteria were grown in trypticase soy broth with 1.5% sodium chloride (TSBS) (V. vulnificus) or Luria broth (Escherichia coli) supplemented with antibiotics as appropriate. For V. vulnificus, antibiotics were 2 μg/ml chloramphenicol and 500 μg/ml ampicillin, and for E. coli, they were 100 μg/ml ampicillin, 30 μg/ml chloramphenicol, and 50 μg/ml kanamycin. TCBS (thiosulfate-citrate-bile salts-sucrose) agar (Difco, Sparks, MD) was used as a selective medium for V. vulnificus. All plasmids to be conjugated into V. vulnificus were first transformed into E. coli S17-1λpir, which was then used as the donor strain. Plasmid transfers from E. coli to V. vulnificus were done as described previously (23).

TABLE 1.
Bacterial strains and plasmids used in this study

Construction of V. vulnificus mutants and complementation.

For functional analysis, hlyU, rtxA1, and VV20480-VV20481 deletion mutants in V. vulnificus were made using the pDM4 suicide plasmid (17) by allelic exchange and the method of splicing by overlapping extension, as described previously (27). To create the hlyU deletion mutant, primer HLYU-D1 plus HLYU-D2 and primer HLYU-D3 plus HLYU-D4 produced two separate PCR products that flanked the hlyU gene. These two fragments were used as templates in a secondary PCR using primers HLYU-D1 and HLYU-D4. The obtained PCR products were purified with a QIAquick spin column (QIAGEN, Valencia, CA) and cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA), creating plasmid pCRUD. The fragment was then digested with XhoI and SacI and subcloned into the suicide vector pDM4, creating pDMUD. For allelic exchange, exconjugants were selected on TCBS agar plate with chloramphenicol. Second recombination events were screened for resistance to sucrose (15%) and sensitivity to chloramphenicol. The strain containing the hlyU deletion was named MQ1.

A similar approach was used to construct the V. vulnificus rtxA1 and VV20480-VV20481 mutants. Primers RTXA1-D1, RTXA1-D2, RTXA1-D3, and RTXA1-D4 were used to create the rtxA1 mutant MQ2. Primers 204801-D1, 204801-D2, 204801-D3, and 204801-D4 were used to generate the VV20480-VV20481 deletion mutant MQ3. PCR amplifications from chromosome DNA were carried out to confirm the deletion of the genes of interest.

To complement the hlyU deletion mutant MQ1, primers HLYUC-F and HLYUC-R were used in the PCR to create a 372-bp fragment encompassing the hlyU gene and its putative ribosome-binding site. The fragment was cloned into the pCR2.1 vector, sequenced, and then subcloned by cleavage with XbaI and SacI into pMMB208 under the control of the Ptac promoter to generate pMMB-HLYU. This plasmid was conjugated into the hlyU deletion mutant MQ1. Similarly, the empty vector pMMB208 was conjugated into MQ1 as a control. To induce transcription of the cloned hlyU gene, 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside) was added to the solid and/or broth medium.

For the virulence tests we needed to prove there were no secondary mutations in our mutants, and since hlyU and rtxA1 are the last genes in their respective operons, we decided to use the method described by Osorio et al. (21) and constructed hlyU- and rtxA1-targeted disruptants of V. vulnificus. An internal fragment of each gene was amplified by using primers HLYU-INF and HLYU-INR for the hlyU gene or RTXA1-INF and RTXA1-INR for the rtxA1 gene. These fragments were each cloned into pCR2.1, sequenced, and then subcloned into the suicide vector pDM4 by XhoI and SacI, generating pDMHlyU and pDMRtxA1. Insertion of the suicide vector into the chromosome by a single crossover generated the hlyU and rtxA1 disruptants MQ1-1 and MQ2-1, respectively. A second crossover was selected using sucrose to eliminate the suicide plasmids and restore the wild-type (regenerated) hlyU and rtxA1 genes.

RNA purification.

V. vulnificus strains were grown to mid-log phase (optical density at 600 nm [OD600] of 0.5 to 0.8) in TSBS. Total RNA was purified using RNAWiz reagent (Ambion, Austin, TX), according to the manufacturer's instructions, and then treated with DNase Turbo DNA-free (Ambion, Austin, TX) at 37°C for 30 min to remove the residual genomic DNA.

RT-PCR and real-time RT-PCR.

Total RNA was extracted from V. vulnificus CMCP6 cells growing in TSBS and treated with DNase Turbo DNA-free as described above. Two micrograms of total RNA was used to generate cDNA using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions with primer P1 which was located in rtxA1. Then, 2 μl of the RT reaction mixture was used in the PCR reactions with primer P1 plus P2 as well as P1 plus P3. A control without the reverse transcriptase enzyme in the RT reaction was used in each PCR.

All real-time PCRs were carried out using an ABI Prism7000 sequence detection system (Applied Biosystems, Foster City, CA). Two micrograms of total RNA was used in the RT reaction with the random primer (Invitrogen, Carlsbad, CA) using Superscript II reverse transcriptase according to the manufacturer's instructions. The amplifications were performed in 96-well plates using a 25-μl total reaction volume containing 12.5 μl of 2× iTaq SYBR Green Supermix with ROX (Bio-Rad, Hercules, CA), the appropriate amount of forward and reverse primers (the final concentration for each primer is 200 nM), and 8 μl of diluted (1:10) cDNA from each RT reaction mixture as a template. The following condition was used for the PCR: 1 cycle for 5 min at 95°C and 40 cycles of PCR with denaturation at 95°C for 30 s, annealing at 58°C for 1 min, and extension for 30 s at 72°C. Each reaction was performed in triplicate. The RT reaction mixture without the reverse transcriptase enzyme was used as a negative control for each gene. We used the gap gene encoding glyceraldehyde 3-phosphate dehydrogenase as an internal control. Each experiment was carried out three times. The specificity of the PCR amplification of each primer pair was determined by constructing a melting curve after the PCR amplification. The real-time PCR results were analyzed by using SDS 7000 software (Applied Biosystems, Foster City, CA). The relative change in expression was calculated using the ΔΔCT method (where CT is the threshold cycle) (15).

Construction of a transcriptional lacZ fusion to the VV20481-VV20480-rtxA1 promoter.

The promoter region of the VV20481-VV20480-rtxA1 operon located upstream of VV20481 was fused to the promoterless lacZ gene from E. coli by using plasmid pTL61T. To amplify the upstream region of this operon, we used primers 20481P-R, with a PstI restriction site followed by bases corresponding to the 5′ end of VV20481, and 20481P-F, with an XhoI restriction site, to generate a fragment which extended up to 750-bp upstream of the putative start codon of VV20481. The PCR fragment was then cloned into the pCR2.1 vector, sequenced, and subcloned into pTL61T by using the XhoI and PstI sites, creating pTL1. The fusion plasmid and the empty vector were conjugated respectively into the various V. vulnificus strains.

β-Galactosidase assay.

V. vulnificus strains were grown overnight at 37°C in TSBS with ampicillin. Cell cultures were diluted at 1:100 into the same medium and further incubated at 37°C with shaking. Samples (0.1 ml) were taken at various time points, and the β-galactosidase activity was determined according to Miller's method (16). The background from the empty vector without the fused promoter was subtracted to obtain the results. Complementation of the hlyU mutation in these assays required the presence of a second plasmid, pMMB-HLYU; thus, we used ampicillin and chloramphenicol in the assays.

Virulence determination.

Strains were grown overnight in TSBS and then were inoculated into 10 ml of fresh TSBS by a 1:100 dilution and grown for an additional 4 h. One milliliter of the cell suspension was harvested by centrifugation, and after one wash the cells were serially diluted with phosphate-buffered saline. CD-1 mice (Charles River Laboratories, Wilmington, MA), 6 to 8 weeks old, were used in the virulence tests. For infection by intraperitoneal (i.p.) injection, mice were pretreated with 900 μg of ferric ammonium citrate by i.p. injection 30 min prior to the challenge. Mortality was monitored 24 h postinfection. The LD50 for each strain was calculated by the method of Reed and Muench (25).

Cytotoxicity assay.

To evaluate the cytotoxicity of V. vulnificus strains, the HeLa cell line derived from cervical cancer cells was used. HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and seeded in 24-well culture plates to a density of 1.0 × 105 cells/well. After growing overnight at 37°C in 5% CO2, the cells were washed once with serum-free medium. V. vulnificus strains were grown in TSBS to log phase (OD600 of ~0.6), harvested by centrifugation, and resuspended in serum-free Dulbecco's modified Eagle's medium to the appropriate concentration. The monolayers of HeLa cells were infected with bacteria at a multiplicity of infection of 10. After a 90-min incubation at 37°C in 5% CO2, the cytotoxic activity of live bacteria was measured by the released lactate dehydrogenase in the supernatant using a CytoTox96 Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI). This assay measures the conversion of tetrazolium salt to a formazan product which is red and therefore detectable by measurement of the A490 value using a Thermo Max plate reader (Molecular Devices, Sunnyvale, CA). Percent cytotoxicity is calculated by the following formula: [A490 of the supernatant of the infected cells − A490 of the supernatant of the uninfected cells]/[A490 of the supernatant of total lysate − A490 of the supernatant of the uninfected cells]. In some experiments with the mutants, we found that the A490 of the supernatant of the infected cells was lower than that of the uninfected cells due to unknown causes.

Overexpression and purification of the V. vulnificus HlyU protein.

The DNA fragment encoding HlyU was PCR amplified using primers HLYUN-F and HLYUN-R by PrimeSTAR DNA polymerase (Takara, MI) and cloned into a six-His tag expression plasmid, pET200 (Invitrogen, Carlsbad, CA), generating the plasmid pET-HLYU which encodes HlyU with an N-terminal fusion tag. The correct recombinant clone confirmed by sequencing was used for expression of His-tagged HlyU protein in E. coli BL21(DE3) Star. Two milliliters of overnight bacteria culture growing at 37°C in Luria broth supplemented with kanamycin was inoculated in 100 ml of the same fresh medium. When the OD600 reached 0.6, 0.5 mM IPTG was added to induce the expression of HlyU protein. After bacteria were grown for an additional 6 h at 30°C, the cells were collected and lysed in a native condition. The obtained soluble supernatant contained HlyU, and the recombinant protein was then purified from this fraction by affinity chromatography using Ni-nitrilotriacetic acid resin (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The concentration of the purified HlyU protein was determined by a bicinchoninic acid protein assay kit (Pierce, Rockford, IL).

Gel mobility shift assay.

The gel mobility shift assay was performed using a digoxigenin gel shift kit (second generation; Roche, Indianapolis, IN). Four subfragments from the VV20481-VV20480-rtxA1 promoter region, extending from 53 bp downstream to 750 bp upstream of the putative start codon of VV20481, were amplified by PCR and then 3′ end labeled with digoxigenin-11-ddUTP using terminal transferase. After the labeling efficiency was determined, each of the labeled probes (4 nM) was incubated with increasing amounts of the purified HlyU protein in the binding buffer [100 mM HEPES, pH 7.6, 5 mM EDTA, 50 mM (NH4)2SO4, 5 mM dithiothreitol, 1% (wt/vol) Tween 20, 150 mM KCl]. For competition analysis, a 4 nM concentration of labeled probe and 150 nM HlyU protein were incubated with increasing amounts of the unlabeled specific probe. The binding reactions were carried out at room temperature for 30 min, and then samples were separated by 6% DNA retardation gel (Invitrogen, Carlsbad, CA). The DNA-protein complex was transferred to positively charged nylon membrane by electroblotting, and then immunological detection and chemiluminescent signal detection were carried out according to the manufacturer's instructions (Roche, Indianapolis, IN).

RESULTS

The hlyU gene.

Kim et al. (7) detected antibodies against HlyU in pooled sera from convalescent-phase septicemic patients that had underlying liver cirrhosis and thus higher iron concentrations in blood than healthy people, concluding that HlyU is expressed in vivo. In addition, using the iron-normal mouse model, they determined that an hlyU mutant showed a decrease in virulence. However, since the iron-overloaded mouse model is more appropriate to reproduce the conditions found in the human infection caused by this bacterium (31, 39), we first constructed an hlyU deletion mutant, MQ1, and then evaluated its virulence in this model. Table Table22 shows that the LD50 increased from <10 CFU for the wild type to 2.4 × 105 CFU for the ΔhlyU mutant MQ1 (ca. a 104-fold increase) in the iron-overloaded mice challenged by i.p. injection, a higher decrease in virulence than that demonstrated by Kim et al. in the iron-normal mouse model (7). To test whether the deletion mutation was solely responsible for the phenotype, we constructed an hlyU-targeted disruptant of V. vulnificus by inserting the whole suicide plasmid pDM4 in the hlyU gene; then regeneration of the wild-type gene was obtained by a second crossover to eliminate the inserted plasmid. Table Table22 shows that the disruptant MQ1-1 has an LD50 of 8.4 × 104 while the regenerated wild-type derivative has recovered the wild-type high-virulence phenotype. Thus, from the results reported here, it is clear that at high iron concentrations similar to those found in compromised patients, HlyU is also a very important virulence factor of V. vulnificus; this finding motivates us to try to understand the mechanisms by which HlyU operates.

TABLE 2.
Virulence of V. vulnificus strains in the iron-overloaded mouse model

What does HlyU regulate?

It was reported that HlyU regulates the expression of the hemolysin/cytolysin gene vvhA in V. vulnificus (7); however, the vvhA mutant did not show a decrease in virulence in the mouse model (4, 37), indicating that HlyU must thus regulate the expression of other virulence-related gene(s). In order to identify the HlyU-regulated genes, we performed a microarray analysis of the ΔhlyU mutant MQ1 and the wild-type strain CMCP6 growing in TSBS. The global analysis of these transcription patterns will be published elsewhere. According to the analysis of the microarray data, in addition to the effect on genes such as vvhA, we found a gene cluster with a significant decrease in its expression level in MQ1. This cluster is located on chromosome II and includes three open reading frames (ORFs), VV20481, VV20480, and VV20479 (Fig. (Fig.1A).1A). VV20481 and VV20480 encode a predicted peptide chain release factor 1 and a predicted hemolysin acyltransferase, respectively. It is worth noting that VV20479 shows a high degree of similarity with the RTX toxin RtxA of V. cholerae (89%) (11). Since there are three homologues of RtxA in the V. vulnificus chromosome—VV12715, VV21514, and VV20479—here we named VV20479 rtxA1. To ascertain whether genes in this cluster are regulated by HlyU, we performed real-time RT-PCR on VV20481 and rtxA1. Table Table33 shows that rtxA1 and VV20481 were greatly down-regulated in the ΔhlyU mutant MQ1, as was the case with vvhA used as a positive control, while the other two RtxA homologues are not regulated by HlyU.

FIG. 1.
The VV20481, VV20480, and rtxA1 genes are transcribed as an operon. (A) Small arrows represent the location of primers. Primer P1 was for the RT reaction using RNA extracted from V. vulnificus CMCP6 cells. The generated cDNA was used as a template in ...
TABLE 3.
Real-time RT-PCR analysis

To ascertain whether these three ORFs present in the rtxA1 cluster are transcribed as an operon, we designed primers located in each of the ORFs and then performed RT-PCR. The results are shown in Fig. Fig.1B.1B. We used the primer P1, located in the 5′ end of the rtxA1 gene, for the reverse transcription reaction. Primer P2, which was in the VV20480 gene, and primer P3, located in VV20481, were used in the PCR (Fig. (Fig.1A).1A). By combination of primers P1 and P2, a fragment of ca. 590 bp was obtained, and this length is consistent with the transcript from the region spanning the 5′ end of VV20480 and the 5′ end of rtxA1; the combination of primers P1 and P3 can generate an amplicon of ca. 890 bp, which is the same length as the transcript spanning the 5′ end of VV20481 and the 5′ end of rtxA1 (Fig. (Fig.1B).1B). These RT-PCR results demonstrated that rtxA1, VV20480, and VV20481 are cotranscribed as an operon.

Cytotoxicity assay of mutations on genes in the rtxA1 operon.

rtxA1 is a homologue of the V. cholerae RTX toxin gene that we demonstrated above to be regulated by HlyU. Thus, we have focused on rtxA1 and the other two genes in this operon to determine if the shutoff or decrease of gene expression in the rtxA1 operon is the reason for the loss of cytotoxicity in the ΔhlyU mutant. We constructed two in-frame deletion mutants, ΔrtxA1 and ΔVV20480-VV20481, that were named MQ2 and MQ3, respectively. Cytotoxicity of various V. vulnificus mutants was determined using HeLa cell monolayers. Figure Figure22 shows that disruption of either the hlyU or the rtxA1 gene resulted in abolishment of the cytotoxicity phenotype in the mutant, while deleting the other two ORFs in the rtxA1 operon, VV20480 and VV20481, did not result in any changes in cytotoxicity compared to the wild-type strain. As before with the hlyU mutation, we also used a disruptant and regenerated wild type in lieu of complementation and observed successful restoration of activity to the mutated rtxA1 gene (Fig. (Fig.2).2). Therefore, RtxA1 cytotoxic activity could explain the reported remaining cytotoxicity in the vvhA mutant (4). It can also be seen that when the ΔhlyU mutant was complemented with the wild-type hlyU gene in plasmid pMMB208, its cytotoxic activity was restored to the wild-type level.

FIG. 2.
Cytotoxicity of mutants in hlyU and other genes regulated by HlyU. Bacteria were incubated with HeLa cells at a multiplicity of infection of 10 for 90 min at 37°C with 5% CO2; then the released lactate dehydrogenase in the supernatant ...

Virulence analysis of mutations on genes in the rtxA1 operon.

As in the case of the hlyU mutation, we used the iron-overloaded mouse model to assess the virulence of the deletion mutants MQ2 and MQ3. Table Table22 shows that, compared with the wild type, the LD50 of the MQ3 mutant was somewhat increased (<10 CFU of the wild type to 184 CFU of the mutant), while for MQ2, after 24 h the LD50 reached 2.5 × 103 CFU, which was ca. 103-fold higher than the level of the wild-type strain. In this experiment we also used an rtxA1 disruptant and the regenerated wild type to test whether the mutation of the rtxA1 gene was solely responsible for the phenotype. Table Table22 shows that the disruptant MQ2-1 has an LD50 of 4.82 × 103 while the regenerated wild-type strain has recovered the wild-type virulence phenotype. These results suggest that the RTX toxin of V. vulnificus is essential for virulence in the iron-overloaded mouse model and that the other two genes in this operon might also be required for the full virulence phenotype.

HlyU regulates the rtxA1 operon at the transcription initiation level.

We knew that HlyU regulates the expression of the rtxA1 operon, but we needed to determine at what level this protein operates. Thus, we cloned an 803-bp fragment (from 750 bp upstream to 53 bp downstream of the VV20481 putative start codon) containing the promoter region upstream of the promoterless lacZ gene in pTL61T and named this construct pTL1. This plasmid was conjugated into the V. vulnificus lacZ deletion mutant ALE-LAC and the ΔlacZ ΔhlyU mutant MQ4. Table Table44 shows the β-galactosidase activity of various strains in the log phase (OD600 of 0.4 to 0.8) from cultures growing in TSBS at 37°C. We observed that the β-galactosidase activity of pTL1 in MQ4 reached only about half of that in ALE-LAC (lacZ mutant). When the ΔhlyU mutant was complemented with the wild-type hlyU gene, β-galactosidase activity was restored to the wild-type level. These results demonstrate that HlyU regulates the expression of the rtxA1 operon at the transcriptional initiation level.

TABLE 4.
β-Galactosidase activity assay for the lacZ-VV20481-VV20480-rtxA1 promoter fusion

HlyU binds to the promoter region of the VV20481-VV20480-rtxA1 operon.

To determine whether HlyU directly or indirectly activates the expression of the VV20481-VV20480-rtxA1 operon, we performed gel mobility shift assays using the purified HlyU-His6 protein. Overexpression of the HlyU protein was achieved by cloning the whole hlyU gene into the pET200 vector, generating an N-terminal His tag fusion expression plasmid. Subsequent overexpression and purification of this protein were performed as described in Materials and Methods. The size of the HlyU protein with a short N-terminal His tag was determined to be ca. 13.5 kDa (data not shown). We then performed gel mobility shift assays using several subfragments (Fig. (Fig.3A)3A) of the 803-bp region that we described in the previous paragraph. When the purified HlyU protein was incubated with each of these four DNA probes, it was found that probe d (Fig. (Fig.3A)3A) consisting of the 246-bp fragment between base pairs 514 and 750 upstream of the putative start codon of VV20481 was the only one exhibiting a shift. Figure Figure3B3B shows the results of incubating this fragment with increasing concentrations of the HlyU protein in the presence of 500 ng of the nonspecific competitor poly(dI-dC). The shift of this fragment to a single band of slower mobility can already be observed at an HlyU concentration of 10 nM, with the shift becoming more pronounced at increasing concentrations. Therefore, the HlyU protein could directly interact with the promoter of the VV20481-VV20480-rtxA1 operon. Competition reactions adding the specific but unlabeled DNA fragment to the binding reaction before the addition of the HlyU protein underscored these results: the specific shifted band became less abundant with increasing concentrations of the competitor DNA (Fig. (Fig.3C).3C). These results demonstrate that the binding between HlyU and the target regulatory region is specific.

FIG. 3.
Gel mobility shift assay for the binding of the HlyU protein to the VV20481-VV20480-rtxA1 promoter region. (A) Location of the DNA probes (a, b, c, and d). (B) The labeled DNA probe d (4 nM) was mixed with increasing amounts of the HlyU protein. Lanes ...

DISCUSSION

HlyU belongs to a family of small regulatory proteins that includes NolR of Rhizobium meliloti, SmtB of Synechococcus sp., and ArsR of Staphylococcus aureus (34). Analysis of the modeled structure of the V. cholerae HlyU revealed that, although it exhibits structural features common to the SmtB/ArsR family of transcriptional repressors, it does not have the key metal-sensing residues like other members of this family (26). Furthermore, it was suggested that HlyU is the only member that has a positive regulatory function in this SmtB/ArsR family (26).

For quite some time it has been known that the hlyU gene must play an important role as a virulence factor in V. vulnificus (7). First, antibodies against HlyU were detected using an expression library of V. vulnificus that was screened by colony blot analysis using pooled convalescent-phase serum from patients recovering from septicemia caused by V. vulnificus (7). Second, a mutation in hlyU resulted in a 53-fold increase in the iron-normal mouse model. However, very little was known about the mechanisms by which HlyU acts as a virulence factor. In the study by Kim et al., it was also mentioned that vvhA, a gene encoding a hemolysin/cytolysin in V. vulnificus, was up-regulated by HlyU. However, the finding that a vvhA mutant still had a full cytotoxicity phenotype and was as virulent as the wild type suggested that HlyU must regulate another gene(s) involved in V. vulnificus virulence (4, 37). Thus, we endeavored to identify other genes that were regulated by HlyU and that were responsible for virulence. In this work, we present results that identify HlyU as a positive regulator of rtxA1, present in the small chromosome of strain CMCP6 and one of three RTX homologues identified in this strain. The other two homologues, VV21514 in the small and VV12715 in the large chromosome, are not regulated by HlyU. Homologues of rtx genes had also been previously identified on both chromosomes of the V. vulnificus strain YJ016 by in silico analysis showing high identity values (greater than 90%) with the CMCP6 genes (2). The V. cholerae RTX toxin is the second largest single-polypeptide toxin known, and its activity involves the covalent cross-linking of cellular actin, resulting in the depolymerization of actin stress fibers and an increase in paracellular permeability (1, 3, 28). Whether V. vulnificus RtxA1 behaves in the same way as the V. cholerae homologue has not yet been determined; however, it is clear from our results in this work that V. vulnificus rtxA1 is responsible for the cytotoxicity phenotype of this bacterium, since cytotoxic activity against HeLa cells was greatly decreased in a rtxA1 mutant. Concomitantly, a decrease in the expression of rtxA1 not only contributes to this loss of cytotoxic activity but also results in a decrease in virulence that is not as dramatic as that caused by the mutation in hlyU. This could mean that, in addition to rtxA1, there are other virulence factors regulated by HlyU that would account for the difference in LD50s between the hlyU and rtxA1 mutants. Because rtxA1 is the last of three genes in the operon, it is possible that expression of the first two genes, VV20481 and VV20480, also regulated by HlyU, could still affect virulence. Our experiments in this work indicate that, indeed, a mutation that inactivates these two genes results in a decrease in virulence although by a smaller margin than the mutation in rtxA1.

Because the most frequent cases of V. vulnificus infections fall into the group of patients suffering from iron overload due to various pathological conditions, we assessed whether hlyU is preferentially expressed under iron-rich conditions by semiquantitative RT-PCR analysis, and our results showed that in vitro expression of hlyU is actually unaffected by the iron concentration of the growth medium (data not shown). Furthermore, our virulence studies of the ΔhlyU mutant carried out in the iron-overloaded mouse model, demonstrated that the LD50 was up to ca. 104-fold higher than that of the wild-type strain CMCP6 and higher than the reported 53-fold change obtained with another hlyU mutant but using the iron-normal mouse model (7). These results clearly show that HlyU can operate as a virulence factor under both iron-normal and iron-rich conditions.

An insight into the mechanism of HlyU regulation of the V. vulnificus VV20481-VV20480-rtxA1 operon was obtained by using lacZ fusions that identified a region upstream of VV20481 as being positively regulated by the HlyU protein. By using gel mobility shift assays, we demonstrated that the HlyU protein directly binds to the promoter region upstream of the putative VV20481 start codon. Interestingly, this identified binding region is far from the predicted VV20481 ORF. The mechanism by which HlyU binds to this region and activates transcription initiation of the operon harboring rtxA1 remains to be elucidated.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported by grant AI065981 from the National Institutes of Health to J.H.C.

Notes

Editor: A. D. O'Brien

Footnotes

[down-pointing small open triangle]Published ahead of print on 16 April 2007.

Supplemental material for this article is available at http://iai.asm.org/.

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