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Research Article

Role of GacA in virulence of Vibrio vulnificus

Julie D. Gauthier, Melissa K. Jones, Patrick Thiaville, Jennifer L. Joseph, Rick A. Swain, Cory J. Krediet, Paul A. Gulig, Max Teplitski, Anita C. Wright

  • 1Food Science and Human Nutrition Department, 212 Aquatic Food Products Laboratory, University of Florida, Gainesville, FL 32611, USA
  • 2Department of Biological Sciences, Loyola University, New Orleans, LA 70118, USA
  • 3Molecular Genetics and Microbiology Department, R1-144 Academic Research Building, University of Florida, Gainesville, FL 32611, USA
  • 4Soil and Water Science Department, 330E Genetics Institute, University of Florida, Gainesville, FL 32611, USA
  • Correspondence
    Anita C. Wright
    acw{at}ufl.edu

    • Microbiology 2010; 156(12):3722–3733 · https://doi.org/10.1099/mic.0.043422-0

    View at publisher PubMed

    Abstract

    The GacS/GacA two-component signal transduction system regulates virulence, biofilm formation and symbiosis in Vibrio species. The present study investigated this regulatory pathway in Vibrio vulnificus, a human pathogen that causes life-threatening disease associated with the consumption of raw oysters and wound infections. Small non-coding RNAs (csrB1, csrB2, csrB3 and csrC) commonly regulated by the GacS/GacA pathway were decreased (P<0.0003) in a V. vulnificus CMCP6 ΔgacA : : aph mutant compared with the wild-type parent, and expression was restored by complementation of the gacA deletion mutation in trans. Of the 20 genes examined by RT-PCR, significant reductions in the transcript levels of the mutant in comparison with the wild-type strain were observed only for genes related to motility (flaA), stationary phase (rpoS) and protease (vvpE) (P=0.04, 0.01 and 0.002, respectively). Swimming motility, flagellation and opaque colony morphology indicative of capsular polysaccharide (CPS) were unchanged in the mutant, while cytotoxicity, protease activity, CPS phase variation and the ability to acquire iron were decreased compared with the wild-type (P<0.01). The role of gacA in virulence of V. vulnificus was also demonstrated by significant impairment in the ability of the mutant strain to cause either skin (P<0.0005) or systemic infections (P<0.02) in subcutaneously inoculated, non-iron-treated mice. However, the virulence of the mutant was equivalent to that of the wild-type in iron-treated mice, demonstrating that the GacA pathway in V. vulnificus regulates the virulence of this organism in an iron-dependent manner.

    Edited by: M. P. Stevens

    INTRODUCTION

    Vibrio vulnificus is a moderately halophilic, Gram-negative bacterium that inhabits coastal waters and colonizes fish and filter-feeding shellfish (DePaola et al., 1994; Motes et al., 1998; Tamplin & Capers, 1992; Wright et al., 1996). Individuals with underlying conditions such as liver disease, diabetes mellitus, cancer, haemochromatosis (iron-overload) or immune system dysfunction can incur life-threatening systemic disease from consumption of raw oysters or from wound infections (Blake et al., 1979; Jones & Oliver, 2009). The virulence of this bacterium in animal models has been attributed to multiple factors (Gulig et al., 2005; Jones & Oliver, 2009), including capsular polysaccharide (CPS) expression (Amako et al., 1984; Kreger et al., 1984; Simpson et al., 1987; Yoshida et al., 1985), iron acquisition (Litwin et al., 1996; Simpson & Oliver, 1983; Wright et al., 1986), pili (Gander & LaRocco, 1989; Paranjpye & Strom, 2005; Paranjpye et al., 2007), flagella (Kim & Rhee, 2003; Lee et al., 2004), quorum sensing (McDougald et al., 2001) and cytolytic haemolysin encoded by the rtxA1 gene (Lee et al., 2007; Kim et al., 2008; Liu et al., 2007). Other cytotoxin/haemolysin genes, including rtxA2 and rtxA3 (J. L. Joseph and P. A. Gulig, unpublished results) and vvhA (Wright & Morris, 1991), as well as a metalloprotease encoded by vvpE (Jeong et al., 2000; Shao & Hor, 2000), show no apparent role in the virulence of this bacterium in the mammalian system. CPS is perhaps the most definitive virulence factor, and phase variation of CPS is correlated with changes in colony morphology, whereby opaque (Op) strains are encapsulated and virulent, while translucent (Tr) variants show reduced encapsulation and virulence. Both colony types undergo reversible phase variation to the alternate phenotype (Wright et al., 1990, 1999, 2001). We recently demonstrated that pili, motility, phase variation and CPS also play a role in uptake and/or survival in the oyster host (Srivastava et al., 2009).

    The research presented herein targeted the GacS/GacA sensor/kinase two-component regulatory system, which is also referred to as VarS/VarA (Vibrio cholerae), BarA/SirA (Salmonella enterica), BarA/UvrY (Escherichia coli) and ExpS/ExpA (Erwinia carotovora ssp. carotovora) in other bacteria (Lapouge et al., 2008). This system is highly conserved in all γ-proteobacteria examined and promotes expression of small RNA (sRNA) products termed csrB and csrC (Suzuki et al., 2002; Weilbacher et al., 2003). These sRNAs function to sequester a regulatory protein, CsrA (Babitzke & Romeo, 2007; Liu & Romeo, 1997; Weilbacher et al., 2003), which in turn provides global post-transcriptional regulation of genes involved in virulence, quorum sensing and biofilm formation (Jones et al. 2008; Timmermans & Van Melderen, 2010). For example, V. cholerae VarA (virulence associated regulator) positively controls expression of cholera toxin and the major subunit of toxin-coregulated pilus (TCP) via input to HapR and the quorum-sensing system (Lenz et al., 2005). GacA has a role in the symbiotic association between Vibrio fischeri and its squid host, and mutants show defects in growth yield, siderophore production, motility and induction of host responses that limit subsequent bacterial colonization and expulsion of ineffective symbionts (Whistler & Ruby, 2003; Whistler et al., 2007). The S. enterica GacA orthologue, SirA, contributes to the regulation of the type III secretion system through hilA, and the flagellar operon and motility are regulated post-transcriptionally via the CsrA system (Goodier & Ahmer, 2001; Teplitski et al., 2003).

    We hypothesized that phenotypic traits that contribute to the virulence and survival of V. vulnificus are controlled by the GacS/GacA system. Therefore, a gacA deletion mutation was constructed in a clinical strain of V. vulnificus and examined for altered phenotypes and differential gene expression relative to the wild-type.

    METHODS

    Bacterial strains and growth assays.

    Bacteria were stored at −80 °C in 50 % (v/v) glycerol in Luria–Bertani broth with NaCl (LBN) prepared with 1 % tryptone, 0.5 % yeast extract and 1 % NaCl at pH 7. Isolated colonies were recovered on LBN agar (LBNA), prepared with 1.5 % agar and with or without antibiotics, as described below. Media were supplemented with 1 μg tetracycline ml−1 to maintain the plasmids for all in vitro phenotypic assays. Electrocompetent E. coli EC100D TransforMax cells (Epicentre Biotechnologies) and E. coli S17-1 λpir (Simon et al., 1983) were used for cloning and complementation. Unless otherwise stated, media components were purchased from Difco, and chemicals were purchased from Sigma.

    Construction and complementation of a V. vulnificus gacA deletion/insertion mutant.

    Genomic DNA was isolated from overnight cultures of V. vulnificus CMCP6 using the UltraClean Microbial DNA Isolation kit (Mo Bio Laboratories). Plasmids were purified using the QIAprep Spin Miniprep kit (Qiagen). Standard molecular biology procedures were used for PCR verification of constructs, plasmids and genomic regions. USER (uracil-specific excision reagent) Friendly Cloning (New England Biolabs) was used to construct a deletion mutation by directionally cloning 500 bp amplicons of flanking DNA from the V. vulnificus gacA locus (Gulig et al., 2009). An SmaI site was included in the gacA upstream and downstream primers for blunt-end insertion of a kanamycin-resistance marker between the joined flanking regions. The deletion construct was created in pGTR1129, a broad-host-range plasmid modified with USER ends (Gulig et al., 2009). The suicide vector was linearized by mechanical shearing and transformed into V. vulnificus via chitin induction (Gulig et al., 2009; Meibom et al., 2005). V. vulnificus ΔgacA : : aph mutants were selected on LBNA with 100 μg kanamycin ml−1. Homologous recombination into the V. vulnificus genome at gacA flanking regions was confirmed by sequencing [Interdisciplinary Center for Biotechnology Research (ICBR), University of Florida] PCR products of both the mutant and wild-type using primers (GenoMechanix) to genomic regions outside the cloned flanking regions.

    The gacA gene with its putative promoter region (BPROM analysis) was PCR-amplified from V. vulnificus CMCP6 with USER end primers and captured into pGTR1160, a USER-end modified broad-host-range vector with a tetracycline-resistance marker (Gulig et al., 2009). The gene was cloned co-directionally to the vector lac promoter, in E. coli EC100 TransforMax cells (Epicentre Biotechnologies) to create pGacA, which was transferred to conjugation-competent E. coli S17-1 λpir and introduced into the V. vulnificus ΔgacA : : aph mutant through conjugation, as previously described (Wright et al., 2001). Complemented (pGacA) and vector control transconjugates were selected on LBNA plates containing both kanamycin (100 μg ml−1) and tetracycline (7 μg ml−1), and were confirmed by PCR amplification using pGTR1160 vector forward and internal gacA reverse primers.

    RNA transcript analysis.

    Isolated colonies were inoculated into 5 ml LBN and incubated overnight at room temperature without shaking. Starter cultures were diluted to OD600 0.01 and incubated with aeration at 37 °C for 18–20 h (OD600 ∼1.5). Quadruplicate technical replicates of each biological sample (n=3) were incubated for 5 min in a 2× volume of RNAprotect Bacteria Reagent (Qiagen), centrifuged at 5000 g for 10 min, and stored at −70 °C for subsequent isolation of total RNA using RNeasy Mini (Qiagen) and Turbo DNA-free kits (Ambion). Total RNA concentration and quality were determined by A260/A280 values on a NanoPhotometer (Implen) and were verified by agarose gel electrophoresis. RNA (2 μg) was converted to cDNA by random priming using a SuperScript VILO cDNA Synthesis kit (Invitrogen), and cDNA was diluted 1 : 20 for real-time PCR. Controls without reverse transcriptase were used to determine the level of genomic DNA contamination. Specific primers were synthesized by GenoMechanix, using previously described sequences for csrB1, csrB2, csrB3 and csrC (Kulkarni et al., 2006). Primer 3 (Rozen & Skaletsky, 2000) was used to design primers for putative virulence and survival genes and for the internal control gene tufA. Reactions were conducted on a SmartCycler II thermocycler (Cepheid) in a 20 μl volume with 0.4 μl of primers (10 mM) and 2 μl template using the EXPRESS SYBR GreenER qPCR SuperMix Universal system (Invitrogen). Internal control and target gene primers were confirmed to be between 90 and 110 % efficient for fold-difference determination. Melt peaks based on the first derivative were used to confirm the sequence identity of amplicons.

    Reporter construction and bioassay for csrB–luxABCDE.

    The predicted promoter region for V. vulnificus CMPC6 csrB1 was amplified by PCR using forward and reverse primers (5′-TTCAAGCACCGCTTCCAGCTCTTTG-3′ and 5′-GTCGTTCCTTCGTCACCAACATCCTAT-3′, respectively). The resulting ∼450 bp product was cloned into the pCR-BluntII-TOPO vector (Invitrogen), and subsequently excised with EcoRI and subcloned into EcoRI-digested and calf intestinal alkaline phosphatase-treated pSB401 (Winson et al., 1998) to construct the pMT39 promoter-reporter with PcsrB1-luxCDABE. The orientation of the insert in pMT39 was confirmed by PCR. The pMT42 (PcsrB2-luxCDABE) and pMT40 (PcsrB3-luxCDABE) plasmids were constructed similarly using forward and reverse primers (5′-TCTTTGGTTTGCCAGCTTAGCGTGG-3′ and 5′-GAGGTGTCCATTGCCTTCCTTAGTA-3′; 5′-TGCCACATTGGAAAAAGGTCTT-3′ and 5′-GCTTACAAGGCTTGTAAGAGATCTC-3′, respectively). Plasmids were electroporated into E. coli MG1655 and its isogenic uvrY (gacA orthologue) negative derivative, RG133 (Goodier & Ahmer, 2001). Luminescence of the reporters was measured with a Victor3 multimode microtitre plate reader (PerkinElmer) in black clear-bottomed plates, as described previously (Teplitski et al., 2006).

    The V. vulnificus CMPC6 gacA gene with its own ribosome-binding site was amplified by PCR catalysed by Taq (New England Biolabs) using primers CJK57 (5′-AAGCTTGATCTGGAACAGAGCCA-3′) and CJK58 (5′-AAGCTTCATACGATAAACGCCAG-3′) with engineered HindIII sites (underlined). The resulting 758 bp PCR product was cloned into pCR2.1-TOPO (Invitrogen) and confirmed by sequencing (ICBR, University of Florida) with standard primers M13R and M13F. The product was subcloned into the HindIII site of pBAD18-Ap (Guzman et al., 1995), which offers tight regulation and high-level expression via the araBAD promoter, yielding pCJK5, which was then transformed into chemically competent E. coli DH5α. Transformants were confirmed to carry the plasmid by antibiotic selection and by PCR with MT13 (5′-ACTTTGCTATGCCATAGCATTTTTA-3′) and CJK58 primers. Plasmid pCJK5 was then electroporated using a Bio-Rad MicroPulser (Bio-Rad Laboratories) into electrocompetent cells of either wild-type E. coli MG1655 or its isogenic uvrY33 : : kan derivative E. coli RG133, containing pMT39, pMT40 or pMT42, as described above. The pBAD18-Ap vector was also transformed into MG1655 or RG133 containing the promoter constructs.

    Colony morphology and phase variation.

    Op versus Tr colony morphology was determined on LBNA after 24 h incubation at 37 °C, as previously described (Chatzidaki-Livanis et al., 2006a). To examine CPS phase variation, isolated colonies were inoculated into 5 ml LBN broth and shaken overnight at 37 °C. Replicate aliquots of cultures (n=3) were washed three times (10 000 g, 30 s) in PBS and diluted to 106 c.f.u. ml−1. Inocula (1 ml) were added to 45 ml 1 % Protease Peptone No. 3 (PP3) in PBS (pH 7.0) in a 250 ml flask. Cultures were incubated statically at 37 °C, streaked to duplicate LBNA plates after 7 days and incubated overnight at 37 °C, and phase variation was determined from the percentage of Tr colonies. The results are the composite of two independent experiments.

    Motility assay.

    Isolated colonies were grown statically overnight at room temperature in LBN, diluted 1 : 10 with fresh LBN, and shaken at 37 °C for approximately 2 h to achieve a cell density of 108 c.f.u. ml−1 (OD600 0.30) to generate exponential phase cells. Stationary phase cells were taken directly from overnight cultures grown at 37 °C. For each strain, six replicates were examined on LBNA or LBNA without yeast extract (LBNA/-Y) and on tryptic soy agar (TSA) plates that were prepared with 0.3, 0.5 or 0.7 % (w/v) agar. Plates were spotted with 3 μl of either exponential or stationary phase culture and incubated at 30 or 37 °C. The diameter of cell spreading was measured as an indicator of motility after 13, 21 and 24 h. Flagellar morphology was examined using NanoOrange stain (Invitrogen) on bacteria recovered from 3 % TSA plates (Grossart et al., 2000), as modified by Gulig et al. (2009).

    Protease assay.

    Strains taken from frozen stock were inoculated into fresh LBN (with 1 μg tetracycline ml−1 where appropriate) and grown to OD600 1–1.2. Cultures were then centrifuged at 4 °C at 6000 g for 30 min. The supernatant was filter-sterilized using a 0.2 μm pore-size filter and used as a crude enzyme extract for measurement of protease activity, as described elsewhere (Miyoshi et al., 1987). Azocasein solution (200 μl at 5 mg ml−1 in 0.02 M Tris/HCl buffer, pH 7.5) was mixed with 400 μl crude enzyme preparation and incubated at 37 °C for 1 h. To stop the reaction, 1.4 ml 5 % TCA was added, and the reaction was centrifuged at 1000 g for 5 min. Supernatant (1 ml) was mixed with an equal volume of NaOH (0.5 M), and A440 was measured.

    Cytotoxicity to INT-407 cell monolayers.

    RtxA1 activity was assessed by detachment of INT-407 cell monolayers, as previously described (Jeong et al., 2000). Briefly, INT-407 cells were seeded in a 24-well tissue culture plate for 2 days until monolayers reached 80–90 % confluency. Exponential phase V. vulnificus cultures were diluted in Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal bovine serum (FBS), and approximately 107 c.f.u. of V. vulnificus was added to each well for an m.o.i. of 10. Triplicate infections were used for each strain. Uninfected cells and wells with media alone were used as negative controls for lysis and background staining of wells, respectively. Gentamicin (100 μg ml−1) was added 1 h after infection, and the unwashed cells were incubated overnight. After overnight incubation, the wells were washed twice with PBS, and the cells remaining attached to the wells were stained with 0.05 % (w/v) crystal violet for 10 min at room temperature, washed four times with PBS, and the crystal violet was extracted with 95 % ethanol. The crystal violet/ethanol solution (150 μl) was transferred to a 96-well plate, and the A490 was measured using an ELx800UV microplate reader (Bio-Tek Instruments). The percentage monolayer detachment was calculated by subtracting the A490 of the medium-only control from the A490 for each infected and uninfected well. The ratio of attached infected cells to attached uninfected cells was calculated from the adjusted A490 for each infected well divided by the adjusted A490 of uninfected cells (mean of all three wells), and was converted to percentage detachment/destruction by the formula: 100−(ratio×100).

    Iron-limited growth.

    Bacterial inocula were prepared from overnight cultures grown at 37 °C and were diluted to OD600 0.3 in medium. Tetracycline (1 μg ml−1) was added to maintain plasmids where appropriate. Cells were washed twice in LBN and suspended in 1 ml LBN. For iron-limited growth, inocula were diluted 1 : 50 in LBN (35 ml) with 150 μM dipyridyl and incubated at 37 °C. The percentage growth yield was determined by OD600 and by plate counts on LBNA, and the percentage growth yield was calculated as growth in LBN with dipyridyl divided by growth in LBN (ratio×100).

    Mouse virulence.

    The virulence of V. vulnificus in terms of systemic and localized infection was determined using a previously described mouse model (DePaola et al., 2003). Seven- to 10-week-old female ICR mice (Harlan Sprague–Dawley) (n=5) were used for all experiments. Iron-treated mice were injected intraperitoneally with 250 μg iron dextran per gram body weight, while non-iron-treated mice did not receive exogenous iron. Bacteria in 0.1 ml PBS were obtained from exponential phase cultures and were injected subcutaneously (s.c.), using approximately 103 c.f.u. for injections in iron-treated mice and 106 c.f.u. for injections in mice that did not receive iron. Mice were euthanized by CO2 asphyxiation when rectal temperatures dropped below 33 °C or at 20 h post-infection. Bacterial infection of skin tissue (local infection) and liver tissues (systemic infection) was quantified by homogenization of tissues, dilution and plating on LBNA. Skin lesion c.f.u. and liver c.f.u. values were log10-transformed for statistical analysis.

    Statistics.

    Significant differences in the phenotypes of the wild-type versus the mutant were determined by Student's t test (two-tailed, type 2). Transcript fold differences between mutant and wild-type strains were determined using the

    Figure image not available in archive
    method (Livak & Schmittgen, 2001) and were based on the mean of three biological samples (individual RNA extracts). Significant differences between wild-type and mutant strains were calculated by Student's t test, comparing ΔCt values (target gene Ct−reference tufA Ct) of the wild-type versus the mutant, and fold difference was based on ΔΔCt (mutant mean ΔCt−wild-type mean ΔCt) and calculated as
    Figure image not available in archive
    .

    RESULTS

    The GacS/GacA pathway of V. vulnificus

    In order to understand the role of the GacA protein in gene expression in V. vulnificus, a deletion of gacA marked with a kanamycin-resistance gene cassette (ΔgacA : : aph) was constructed in CMCP6, as described in Methods. Strains and plasmid constructs are described in Table 1⇓. The function of the GacA pathway was compared in the mutant and wild-type strains by quantitative RT-PCR of the sRNAs that are predicted to bind to the CsrA protein in V. vulnificus, namely csrB1, csrB2, csrB3 and csrC (Kulkarni et al., 2006). In the stationary phase of growth (Table 2⇓), the ΔgacA : : aph mutant showed significantly reduced expression for sRNAs csrB1 and csrC (>1100-fold) and to a lesser extent for csrB2 (235-fold) and csrB3 (18-fold) compared with the wild-type (P=2×10−6, 2×10−6, 8×10−5 and 2×10−4, respectively). Defects in sRNA synthesis in the ΔgacA : : aph mutant were complemented in trans by introduction of the gacA gene on a plasmid, but the vector control did not restore expression. The fold differences in gene expression for csrB2 (1.71) and csrB3 (0.86) were not significantly different between the complemented and wild-type strains. Although the fold difference in gene expression between these strains remained significant (P≤0.003) for both csrB1 (8.92) and csrC (10.51), fold differences were also significantly different (P<0.0003) for the complemented mutant compared with the plasmid control (8.65 and 71.18, respectively). It should be noted that the fold differences observed between the complemented and wild-type strains were greatly reduced compared with the >1100-fold difference observed between the mutant and wild-type strains. No significant differences in gene expression were observed between mutant and wild-type strains when RNA was harvested during exponential phase growth (not shown).

    Table 1.

    Strains and plasmids used in this study

    Table 2.

    Comparison of transcript levels in V. vulnificus CMCP6 and in the ΔgacA : : aph mutant

    V. vulnificus PcsrB : : luxCDABE promoter fusions were also used in an S. enterica background to examine the effects of GacA on gene expression (not shown). Expression of V. vulnificus PcsrB1 : : luxCDABE was minimal in a Salmonella strain without a functional orthologous gacA gene (sirA3 : : cat); however, in the wild-type S. enterica, expression was similar to that of an E. coli PcsrB : : luxCDABE in the same strain. GacA regulation of V. vulnificus csrB1, csrB2 and csrB3 promoters was also confirmed by the introduction of the V. vulnificus gacA gene under the control of an arabinose-inducible promoter into a gacA-negative background in E. coli RG133. Introduction of the V. vulnificus GacA increased expression of the lux reporter gene fusions for V. vulnificus csrB1 and csrB2 promoters by about five- and 1000-fold, respectively (Fig. 1⇓). However, no increase was observed for csrB3. As expected, regulation of sRNAs remained at the basal level when grown under repressive conditions in the presence of glucose and was similar to that of the vector (pBAD18) control. Also, no changes in csr promoter activity were observed in GacA− E. coli without the gacA plasmid in the presence of either glucose or arabinose (data not shown).

    Figure image not available in archive
    Fig. 1.

    Expression of V. vulnificus PcsrB-luxCDABE promoters in a heterologous host. To reconstruct the GacA–csrB pathway of V. vulnificus, promoters for V. vulnificus CMCP6 (a) csrB1, (b) csrB2 and (c) csrB3 sRNAs were cloned upstream of a promoterless luxCDABE cassette. Regulation of the reporters was tested in an E. coli MG1655 uvrY33 : : Tn5 mutant (gacA orthologous mutant) in the presence of gacA from V. vulnificus expressed from an arabinose-inducible promoter on pCJK57 (see Table 1). Strains contained promoter reporters either with pCJK57 in the presence of arabinose (⧫) or with the pBAD18 vector control in the presence of arabinose (□), or without pCJK57 in the presence of arabinose (◊). The substitution of glucose for arabinose, as expected, eliminated complementation by gacA borne on pCJK57 (data not shown).

    GacA regulation of mRNA transcript levels

    In addition to the sRNA analyses described above, the effect of the V. vulnificus gacA deletion on mRNA transcripts of genes (n=20) that have been related to virulence or survival of V. vulnificus was also examined by quantitative RT-PCR. No significant differences in transcript levels were observed between GacA+ and GacA− strains for any of the genes in exponential phase (not shown). However, transcript levels of rpoS (3.4-fold, P=0.004), flaA (3.6-fold, P=0.025) and vvpE (8.5, P=0.001) were significantly reduced in V. vulnificus CMCP6 ΔgacA : : aph compared with the wild-type in stationary phase cultures (Table 2⇑). These effects on gene expression could not be complemented in trans for these genes.

    In vitro characterization of the gacA mutant

    Unlike V. fischeri, for which growth yield is reduced in the gacA mutant (Whistler & Ruby, 2003; Whistler et al., 2007), growth rates and yields of V. vulnificus ΔgacA : : aph were virtually identical to those of the wild-type (data not shown). However, the colony morphology of V. vulnificus was similar to that of V. fischeri in that gacA deletion mutants had colonies that were slightly smaller with less yellow pigment than the wild-type strains, and wild-type morphology was restored by complementation (data not shown).

    Colony morphology is also indicative of CPS expression in V. vulnificus, and Tr colonies are correlated with decreased virulence (Wright et al., 1990). Upon initial isolation, some ΔgacA : : aph isolates appeared to have mixed Op and Tr colonies, and the presence of the gacA deletion mutation was confirmed by DNA sequencing in both Op and Tr colony types. Therefore, Tr morphology was assumed to be the consequence of spontaneous phase variation that occurred during the process of constructing the deletion mutations. Although the morphology of Op ΔgacA : : aph isolates proved to be stable upon further isolation during standard subculture, the frequency of phase variation in the mutant compared with the wild-type strain was further investigated, using previously described conditions that enhance the rate of the Op to Tr phase shift (Chatzidaki-Livanis et al., 2006b). After 7 days in PP3 medium, the mutant was significantly (P<0.001) reduced for phase variation compared with the wild-type strain, as evidenced by the recovery of only 0.4±0.5 % Tr colonies from the mutant and 29.6±2.9 % Tr colonies from the parent strain (Table 3⇓). Complementation of the ΔgacA : : aph mutation in trans restored the frequency of Tr colonies to wild-type levels, while the vector control did not.

    Table 3.

    Phenotypic characterization of the gacA mutant

    RtxA1 cytotoxicity contributes to the virulence of V. vulnificus in mice, and RtxA1 activity is indicated by destruction of INT-407 monolayers (Lee et al., 2007). Significantly less cytotoxicity (P<0.01) was seen for the ΔgacA : : aph mutant than for the parent strain (Table 3⇑). The ability to acquire iron is also a virulence factor for V. vulnificus (Litwin et al., 1996), and growth of the wild-type versus the mutant was compared under in vitro conditions using dipyridyl for iron limitation. The gacA mutant was significantly deficient for growth yield under conditions of iron limitation compared with the wild-type (P<0.01). Deficiencies in both cytotoxicity and iron acquisition were complemented in the ΔgacA : : aph mutant by introduction of the gacA gene in trans. Protease activity was also significantly (P<0.01) decreased in the mutant compared with the wild-type, but the defect could not be complemented in trans (Table 3⇑).

    Motility was compared for the wild-type versus the ΔgacA : : aph mutant on either LBNA or TSA plates prepared with different viscosities and incubated at 30 and 37 °C for 13 and 21 h. The migration distance from the point of inoculation was measured after incubation, and the growth zone diameters were compared among strains. All strains were motile on the low-viscosity agars (0.3 %). However, the motility zone diameter differed significantly (P=0.001) on LBNA for the ΔgacA : : aph mutant (22.7 mm) compared with the wild-type (18.8 mm) strain after 13 h incubation at 30 °C. Complementing with gacA in trans under these conditions reduced motility back to the level of the wild-type (Table 3⇑). Similar results were observed with TSA with 3 % agar at 30 °C; however, significant differences between strains were not observed at 37 °C or after 24 h incubation (data not shown). None of the strains was motile on higher-viscosity (0.5, 0.7 and 1.0 %) agars, and no differences were observed between the wild-type and mutant for flagellar morphology, as all strains had a single polar flagellum (not shown). Interestingly, a pattern of concentric rings developed after extended 24 h incubation on 0.3 % agar due to the concentrated overgrowth on top of the agar plates. This pattern was seen in wild-type but not mutant strains (Fig. 2⇓); however, complementation did not fully restore this phenotype.

    Figure image not available in archive
    Fig. 2.

    Morphology of V. vulnificus ΔgacA : : aph versus that of the wild-type on motility agar. Representative growth of V. vulnificus CMCP6 on TSA motility agar (0.3 %) after incubation at 30 °C for 24 h. Plates are shown for (a) wild-type, (b) ΔgacA : : aph (ΔgacA), (c) the complemented mutant [ΔgacA (pgacA)] and the vector control [ΔgacA (vector)].

    Mouse virulence

    Subcutaneous inoculation was used in a mouse model to examine localized skin infection, as measured by c.f.u. per gram of skin tissue following infection, and systemic disease, as determined by c.f.u. per gram of liver. Decreased temperature indicates systemic infection, and a rectal temperature below 33 °C is a surrogate for death (DePaola et al., 2003). The deletion of gacA significantly impaired the capacity of V. vulnificus to cause both localized and systemic infections in mice, and this defect was related to iron pretreatment of the host. Lesions that were apparent following wild-type infections were not evident or were greatly reduced in non-iron-treated mice that were infected with the V. vulnificus gacA mutant (Fig. 3⇓). The c.f.u. g−1 recovered from both skin (P=0.0003) and liver (P=0.017) tissues was also significantly lower in the mutant compared with the wild-type (Table 4⇓). Higher body temperatures were observed with the mutant compared with wild-type infections, but differences were not significant. Conversely, the mutant was not affected for any measure of virulence in mice that were pre-treated with iron dextran. Complementation of the ΔgacA : : aph mutation with pGacA mostly restored the level of skin infection, but did not significantly change the levels of liver infection or temperature caused by the mutant strain.

    Figure image not available in archive
    Fig. 3.

    Lesions from s.c. inoculated, non-iron-treated mice. At 20 h post-inoculation, mice were euthanized, and the skin underlying the inoculation site revealed haemolytic lesions that were present in (a) wild-type infection but were reduced or absent in infection by (b) mutants, and were partially restored in (c) the mutant complemented with gacA in trans.

    Table 4.

    Role of GacA in virulence of V. vulnificus

    DISCUSSION

    This study is believed to be the first report of GacA function in V. vulnificus. GacA transduces signals from GacS in γ-proteobacteria to directly increase expression of sRNAs that in turn sequester the global regulatory protein CsrA (Baker et al., 2002; Wei et al., 2001). Our results are consistent with prior descriptions of GacS/GacA regulation in other bacteria, and the regulatory pathway in V. vulnificus CMCP6 was confirmed by observed decreases in relevant sRNA transcripts in a ΔgacA : : aph mutant compared with the parental wild-type strains and by complementation of the mutant with the gacA gene presented in trans. Loss of gacA in the mutant made csrB1 and csrC transcripts essentially undetectable, but had less of an effect on csrB2 and csrB3. However, regulation in a heterologous host (E. coli) was greater for csrB2 than for either csrB1 or csrB3, suggesting that additional layers of host-specific regulators are not present in E. coli. Our results were similar to the differential sRNA response reported for the V. cholerae gacA orthologue, varA, in which the mutant showed decreases in csrB1 and csrB3 that were 10-fold greater than those in csrB2 (Lenz et al., 2005).

    Phenotypic defects associated with the V. vulnificus gacA deletion mutation included decreased virulence in mice, as well as decreased expression of known virulence functions such as cytotoxicity and the ability to grow under conditions of iron limitation. Interestingly, the mutant exhibited wild-type virulence in iron dextran-treated mice, although its virulence was significantly reduced compared with the wild-type in non-iron-treated mice. V. vulnificus exhibits ferrophilic virulence in mouse models, whereby injection of exogenous iron greatly reduces the infective dose (Wright et al., 1981). Therefore, we hypothesize that the defect in iron acquisition related to the gacA mutation likely contributes to decreased virulence in non-iron-treated mice, although the availability of exogenous iron may compensate for this defect during infections in iron-treated mice. Defects in both cytotoxicity and in vitro iron response in the mutant were complemented in trans. However, the virulence of the gacA mutant was only partially restored by complementation for skin infection, and was not affected for liver infection or temperature decrease. Lack of complementation may reflect the instability of the plasmid vector during later stages of infection. Our results are consistent with observations of virulence defects for iron uptake-related mutants in non-iron-treated mice (Alice et al., 2008).

    Phase variation and protease activity were also decreased in the mutant compared with the wild-type. Both of these behaviours are stationary phase responses (Chatzidaki-Livanis et al., 2006a; Wright et al., 1981), which corresponds with prior observations that GacA regulation represses the expression or stability of gene transcripts related to exponential growth, while promoting gene expression related to stationary phase (Romeo, 1998). Also, decreased rpoS expression in the V. vulnificus ΔgacA : : aph mutant was consistent with the GacA control of stationary phase regulation reported in Pseudomonas fluorescens (Whistler et al., 1998) and E. coli (Mukhopadhyay et al., 2000). Genes for flaA and vvpE also showed significantly lower transcript levels in the mutant compared with the wild-type and only in stationary phase cultures. These results agree with prior reports of the positive control of protease by the GacS/GacA system in other bacteria (Heeb & Haas, 2001; Lapouge et al., 2008). Decreased rpoS expression in the V. vulnificus gacA mutant may also contribute to reduced protease activity, as an rpoS mutant lacks albuminase, caseinase and VvpE elastase activity (Hülsmann et al., 2003). Interestingly, the rpoS mutant also has reduced motility, which does not concur with results from the ΔgacA : : aph mutant in our study.

    Although the V. vulnificus mutant showed decreased expression of flaA, both motility and flagella expression were maintained compared with the parent strain. Motility on 3 % agar was enhanced compared with the wild-type strain at 30 °C but not at 37 °C, demonstrating the complexity and variability of the GacA regulon. The effects of this system on motility are potentially made even more complex by the fact that V. vulnificus possesses six flagellin genes. GacA-regulated motility has been described in several species, but mutants can show either decreased (Duffy & Defago, 2000; Goodier & Ahmer, 2001; Hammer et al., 2002; Kinscherf & Willis, 1999; Teplitski et al., 2003; Wei et al., 2001) or increased motility (Whistler & Ruby, 2003). Although this increased motility has been noted in the related species V. fischeri, gacA mutants were hyperflagellated, which was not the case for V. vulnificus. Additionally, V. vulnificus gacA mutants showed reduced growth on the surface of motility agar plates and failed to form concentric rings on this agar after extended incubation. This ring formation has previously been described as ‘swarming’ in V. vulnificus (Kim et al., 2007); however, this behaviour differs greatly from that of Vibrio parahaemolyticus, which swarms on higher-viscosity agar and uses lateral flagella genes that are not present in V. vulnificus for swarming (Stewart & McCarter, 2003). Therefore, we propose that descriptions of ‘swarming’ in V. vulnificus are not in keeping with the strictest use of the term. The concentric rings of V. vulnificus on low-viscosity agar are more likely related to stationary phase responses to nutrient limitation or perhaps to biofilm formation that occurs at the air–liquid interface. V. vulnificus vvpE mutants described elsewhere are also defective in ring formation (Kim et al., 2007), and since gacA mutants show reduced protease activity and vvpE transcript levels, these defects may also contribute to the differential growth on motility agar.

    Although GacA did not modulate capsule as indicated by colony morphology, CPS phase variation was reduced in the V. vulnificus ΔgacA : : aph mutant compared with the wild-type. A prior study showed that phase variation is RpoS-dependent in V. vulnificus (Hülsmann et al., 2003), and decreased rpoS expression may be a factor contributing to the decreased phase variation that was observed in the gacA mutant. Phase variation is dependent upon the strain and growth conditions and is likely to be a response to rapidly changing environmental conditions (Chatzidaki-Livanis et al., 2006b). Tr strains switch to Op morphology during mouse infections (Wright et al., 1990) and oyster colonization (Srivastava et al., 2009), presumably contributing to survival in the host. In Pseudomonas spp., the GacS/GacA system regulates phase variation to a Tr morphology that enhances host colonization (Han et al., 1997; Sánchez-Contreras et al., 2002). Phase variation to the rugose phenotype in V. cholerae enhances biofilm formation and is mediated through HapR (Yildiz et al., 2004), which is regulated by GacA input to the quorum-sensing system (Lenz et al., 2005). Furthermore, GacA-mediated switching in V. fischeri facilitates a symbiotic lifestyle (Whistler et al., 2007). Thus, phase variation appears essential to the life cycles of these bacteria, and these transitions are likely needed for infection/colonization of both mammalian and molluscan hosts by V. vulnificus.

    Discrepancies were found in correlating some of the defective phenotypes of the mutant with the corresponding gene expression or with complementation in trans. These anomalies may reflect the complexity of the GacA pathway. Plasmid instability, gene dosage or post-transcriptional modulation may also be contributing factors. A complication of the GacA-mediated response is that CsrA itself somehow activates expression of the sRNAs in the pathway to provide a feedback loop for homeostatic regulation that can be independent of GacA (Suzuki et al., 2002). Furthermore, as shown in the same report, both barA (gacA orthologue) deletion mutation and overexpression of barA result in decreased CsrA-mediated biofilm formation in E. coli. Thus, the autoregulatory aspects of CsrA may complicate the expected response for GacA mutants and complemented mutants. CsrA binding to mRNA can either destabilize (Baker et al., 2002) or stabilize transcripts through mechanisms that are not completely understood (Wei et al., 2001). Certain transcripts that decay rapidly in csrA+ E. coli strains are completely stable in a csrA− background (Liu et al., 1995), while a csrA mutation causes 10-fold increases in other mRNA targets (ycdT and ydeH) of CsrA (Jonas et al., 2008). Investigations of the GacS/GacA pathway are further confounded by the involvement of multiple global regulators (RpoS, HapR and LuxS). Furthermore, preliminary studies have shown some strain differences in the GacA response of V. vulnificus, and these differences are perhaps not unexpected, as plasticity of the GacA regulon is seen for closely related species (Lapouge et al., 2008).

    In summary, the gacA mutant differed greatly from the wild-type in sRNA expression and in multiple phenotypes required for virulence of V. vulnificus, demonstrating the important role of GacA in the biology of this species. Complementation of sRNA defects and for most of the altered phenotypes in the mutant confirmed this role. The signals that trigger the GacS/GacA system in E. coli have recently been shown to be the metabolic end-products formate and acetate, although these signals may vary with species, strain and growth conditions (Gonzalez Chavez et al., 2010). Further studies are needed to sort out signals, targets and alternative pathways within different species, and perhaps even different strains of bacteria that are dependent upon GacA for survival.

    Acknowledgments

    This work was supported by U.S. Department of Agriculture, Agriculture and Food Research Initiative (USDA AFRI) grant 00068375. We would also like to thank Daniel Goldberg and Marianne Fatica for technical assistance.

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