A K⁺ Uptake Protein, TrkA, Is Required for Serum, Protamine, and Polymyxin B Resistance in Vibrio vulnificus

Bacterial strains and plasmids.The bacterial strains and plasmids used for this study are listed in Table 1. V. vulnificus CKM-1 is clinical isolate from the blood of a septicemic patient (6). All strains were routinely grown in minimal medium (12.8 g of Na₂HPO₄ · 7H₂O/liter, 3 g of KH₂PO₄/liter, 10 g of NaCl/liter, 1 g of NH₄Cl/liter, 2 mM MgSO₄, 0.2 mM CaCl₂, 4 g of glucose/liter) or Luria-Bertani (LB) medium at 37°C, with aeration. Antibiotics were used as follows: ampicillin at 100 μg/ml, chloramphenicol at 34 μg/ml, and kanamycin at 100 μg/ml for Escherichia coli and ampicillin at 100 μg/ml, chloramphenicol at 3 μg/ml, kanamycin at 100 μg/ml, and rifampin at 50 μg/ml for V. vulnificus.

Molecular techniques.Standard techniques were used to construct recombinant plasmids (32). DNA fragments used in cloning were extracted from agarose gels by use of the Qiaex II kit (Qiagen, Mississauga, Ontario, Canada). PCR was carried out according to the manufacturer's recommendations by use of the Taq DNA polymerase kit (Amersham Pharmacia Biotech, Inc., Uppsala, Sweden). Nucleotide sequences were determined with an autosequencer (ABI Prism 373 DNA sequencer; Applied Biosystems).

Colony and Southern hybridization.Colony hybridization and Southern hybridization were performed as described by Sambrook et al. (32). DNA probes were labeled with [α-³²P]dCTP by use of a random priming kit (Megaprime DNA labeling system; Amersham Pharmacia Biotech), using either the PCR products or fragments excised from the recombinant plasmids as the templates. The nylon membrane with DNA was prehybridized with hybridization buffer (ExpressHyb hybridization solution; Clontech Laboratories, Inc.) for 30 min at 65°C, hybridized for 2 h at 65°C, washed, and visualized by autoradiography.

Construction of transposon mutant bank.The transposon insertional library of V. vulnificus strain CKM-1 was generated by the method described by Hensel et al. (15), with minor modifications. E. coli S17-1 λpir carrying a transposon plasmid (15) was delivered to rifampin-resistant V. vulnificus strain CKM-1 by conjugation. The transconjugants were selected by growth with kanamycin and rifampin and were tested for ampicillin sensitivity. The resultant mutants were further screened by the serum sensitivity assay as described below.

Serum sensitivity assay.Strains were grown in 96-well microtiter dishes containing LB broth at 37°C for 4 h. Bacteria were harvested, washed, and resuspended to 2 × 10⁴ CFU/ml with phosphate-buffered saline (PBS). The bacterial suspensions (50 μl) were mixed with 50 μl of fresh serum or heat-inactivated serum (56°C, 30 min) in 96-well microtiter dishes and incubated at 37°C for 1 h. The numbers of viable bacteria in serial 10-fold dilutions before and after incubation were counted on LB agar after overnight incubation at 37°C. Results were presented as percent survival relative to the original inocula. Human serum samples were obtained from individual donors, and sera from 10 different healthy donors (from the hospital center of the College of Medicine at National Cheng-Kung University) were combined.

Analysis of transposon insertion sites.Chromosomal DNAs from each Tn5 mutant were digested individually with BglII, EcoRI, KpnI, PstI, and SalI (there are no recognition sites for these five restriction enzymes within the transposon). The presence of the Tn5 mutant was screened with an α-³²P-labeled kanamycin gene by Southern hybridization. The kanamycin probe was generated by excision from plasmid pUC4K to generate a 1.2-kb SalI fragment which was then used as a template for the random priming kit.

Cloning of V. vulnificus trkA gene.Chromosomal DNA from strain SSM-1 was digested with EcoRI and inserted into identically digested pUC19. The ligation reaction mixtures were transformed into E. coli XL1B, and cells were selected by kanamycin resistance. Plasmid DNA was extracted, and the chromosomal DNA sequence flanking the transposon was obtained by DNA sequencing using primers P6 and P7 (15). The complete coding sequence of trkA was cloned from the genomic library of the CKM-1 strain (6) by colony hybridization with an α-³²P-labeled DNA fragment of the trkA gene. One positive clone was selected for DNA sequencing.

Construction of the trkA mutant.A 612-bp internal fragment of the trkA gene from pSJ1 was generated by PCR with primers AF3 (5′-GCTCGCATGCGTTCGCCACA-3′) and AR3 (5′-GACGTCGACCTGGTCGATGTT-3′). The PCR product was digested with SalI and SphI and inserted into identically digested pCVD442 (9). The resultant plasmid, pYC3, was transformed into E. coli S17-1 λpir and subsequently transferred into V. vulnificus CKM-1 via conjugation according to a previously described method (15). Transconjugants were selected by use of ampicillin and rifampin. The resultant strain was further confirmed by PCR and Southern blot analysis using the trkA probe.

Complementation analysis.A 1,529-bp fragment of the trkA gene was amplified from pSJ1 by PCR with the primers AF1 (5′-GATGAGCTCTACTATGCCGT-3′) and AR1 (5′-AGTCTAGAAAGCACTAGCCC-3′). The PCR product was digested with SacI and XbaI and inserted into identically digested pBC. The resultant plasmid, pYC2, was introduced into AKK-1 via electroporation by using the method described by McDougald et al. (21). Transformants were selected by use of ampicillin and chloramphenicol. The resultant strain was further verified by plasmid extraction and PCR.

RT-PCR.An SV total RNA isolation kit (Promega, Madison, Wis.) was used to extract RNA from wild-type CKM-1 that had been grown in LB broth for 5 h at 37°C. Primer HR1 (5′-CAGATTCATGCCGGTCAGC-3′), which is complementary to trkH mRNA, was annealed to purified RNA (1 μg) for first-strain cDNA synthesis with Superscript II RNase H reverse transcriptase (Invitrogen, Inc., Carlsbad, Calif.) according to the manufacturer's instructions. PCR amplifications were performed with Taq DNA polymerase (Invitrogen), using an aliquot (1/10) of the reverse transcription (RT) reaction mixture as the template and one of three pairs of primers designed to amplify DNA fragments corresponding to the trkA coding region (AF3 [5′-GCTCGCATGCGTTCGCCACA-3′] and AR1 [5′-AGTCTAGAAAGCACTAGCCC-3′]), the trkH coding region (HF1 [5′-CTTTAAACTCAGTGTGCGCG-3′] and HR1), and the trkA-trkH intergenic coding region (AF3 and HR1). Thirty cycles of amplification were carried out, with denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1.5 min. RNA that was subjected to PCR without prior RT was used as a negative control. One-quarter of each amplification reaction was electrophoresed on a 2% agarose gel and photographed under UV transillumination. The DNA sequences of these amplified products were confirmed by DNA sequencing.

Overexpression of TrkA in E. coli.A 1,388-bp fragment of the trkA gene was amplified from plasmid pSJ1 by PCR with primers AF2 (5′-AGGTTGGATCCGAGCTTGGTTTAT-3′) and AR2 (5′-GGCCTCTCGAGCTTTGTAGTTTTC-3′). PCR products were digested with BamHI and XhoI and inserted into identically digested pET21(b) (Novagen, Madison, Wis.) to generate plasmid pYC1. His₆-tagged TrkA was expressed in E. coli BL21(DE3) and purified under denaturing conditions by using a nickel affinity column as instructed by the manufacturer (Novagen).

Preparation of polyclonal antisera.One hundred micrograms of purified TrkA per milliliter of saline was mixed with 1 ml of Freund's incomplete adjuvant. This mixture was then injected subcutaneously into an Elite New Zealand White rabbit. Two booster doses were administered at 2-week intervals, and the antiserum was collected after 6 weeks. The antiserum was purified through a protein A column according to the manufacturer's instructions (Amersham Pharmacia Biotech).

Western blot analysis.Proteins separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis were electroblotted onto a nitrocellulose membrane (Amersham Pharmacia Biotech) and then incubated with a TrkA-specific rabbit polyclonal antibody as the primary antibody. The secondary antibody was a 1:4,000 dilution of goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (Sigma, St. Louis, Mo.). The proteins were visualized by using a chemiluminescence kit according to the manufacturer's protocol (ECL Western blotting detection reagent; Amersham Pharmacia Biotech).

Effect of K⁺ on the growth of V. vulnificus trkA.Strains were grown at 37°C in LB broth and harvested at the mid-log phase of growth. Bacteria were centrifuged for 5 min at 3,000 × g, washed twice with K⁺-free medium (12.8 g of Na₂HPO₄ · 7H₂O/liter, 3 g of NaH₂PO₄/liter, 10 g of NaCl/liter, 1 g of NH₄Cl/liter, 2 mM MgSO₄, 0.2 mM CaCl₂, 4 g of glucose/liter), and resuspended in K⁺-free medium to an optical density at 600 nm (OD₆₀₀) of 0.1. Bacterial suspensions were added at 1/100 the volume of the medium to K⁺-free medium containing different concentrations of KCl (0.01, 0.1, 1, 5, 10, 20, and 30 mM) and were incubated at 37°C. Samples were shaken gently, and aliquots removed at specified times were assayed for bacterial counts by plating of serial dilutions on LB plates. The doubling times (g) for the number of bacteria in K⁺ medium were calculated with the following equation: g = t/n = 0.301t/(logN_t − logN₀), where N₀ is the initial population number, N_t is the population number at time t, and n is the number of generations in time t.

Protamine and polymyxin B sensitivity assay.Strains were grown at 37°C in LB broth and harvested at the mid-log phase of growth. Bacteria were centrifuged for 5 min at 3,000 × g and resuspended in LB broth at an OD₆₀₀ of 0.33, and protamine (5 to 15 μg/ml) (Sigma) or polymyxin B (10 to 20 μg/ml) (Gibco-BRL, Gaithersburg, Md.) was added. The suspensions were incubated at 37°C, and bacterial lysis was monitored at specified times by the decrease in OD₆₀₀.

Virulence assay.BALB/c mice (8 to 10 weeks old) purchased from the animal center of the College of Medicine at National Cheng-Kung University were challenged by intraperitoneal (i.p.) or subcutaneous (s.c.) injection of the bacterial suspension. For experiments involving pretreatment of mice with iron dextran, mice were injected i.p. with 5 mg of iron dextran (Sigma) per mouse at 2 h preinfection. A group of eight mice was given 0.2 ml of a 10-fold serially diluted (in PBS) bacterial suspension per mouse, and mortality was recorded at 5 days postinfection. The 50% lethal dose (LD₅₀) for each strain was calculated by the method of Reed and Muench (30).

Enumeration of complemented strain TRK-1 in the spleen.Mice were sacrificed by cardiac exsanguination under anesthesia at 24 h postinfection. Spleens were removed aseptically and homogenized in 1 ml of PBS by use of a grinder. The numbers of viable bacteria in homogenates of spleen were monitored by plating serial dilutions on LB plates containing ampicillin and on LB plates containing ampicillin and chloramphenicol and by counting bacterial colonies after 24 h of incubation at 37°C. The percentage of the TRK-1 strain was calculated as 100 × (ampicillin- and chloramphenicol-resistant CFU/ampicillin-resistant CFU).

Nucleotide sequence accession number.The nucleotide sequences of trkA and trkH of V. vulnificus have been deposited in the EMBL database under accession no. AY293743 .

Production and classification of transposon mutants.Mutagenesis with the conjugative kanamycin resistance transposon Tn5 has been successfully utilized in V. vulnificus (42). Therefore, we constructed a bank of Tn5 mutants of a clinical strain of V. vulnificus, CKM-1, and screened them for susceptibility to serum by a serum sensitivity assay. Of the 3,000 Tn5 mutants screened, 15 SS mutants were isolated. Compared to the wild-type strain, which had an ∼48% survival rate relative to the original inocula after incubation with 50% (vol/vol) serum for 1 h, the SS mutants had a 10- to 1,000-fold lower survival rate. Of the 15 SS mutants, 3 were of particular interest. Compared to the remaining 12 SS mutants, which exhibited either a translucent colony morphology or a marked deficit in growth on a minimal medium, these 3 mutants had an opaque colony morphology and could grow on a minimal medium, suggesting that they probably were neither the unencapsulated mutants nor the auxotrophic mutants.

Southern blot analysis with the kanamycin gene as the probe revealed that of these three SS mutants, two (designated SSM-1 and SSM-2) contained a unique site of transposon insertion (data not shown). The DNA sequences immediately flanking the Tn5 integration sites in the SSM-1 and SSM-2 mutants were obtained as described in Materials and Methods. The resultant plasmids were sequenced, and through sequence analysis, 354 bp of SSM-1 and 456 bp of SSM-2 chromosomal DNAs beyond the transposon were located and tentatively named sequences A and B, respectively. Database searches by the BLAST algorithm (1) for homology of the two sequences to known gene sequences were performed, and several relevant sequences were identified. The A segment encoded a peptide with a high degree of sequence homology to TrkA, an NAD⁺ binding protein that was part of a low-affinity potassium uptake system of E. coli (33). The B segment encoded a peptide that showed sequence homology to a large group of σ⁵⁴-dependent response regulators that modulate cellular response to environmental signals. Cloning of the intact gene of the B segment and characterization of its gene product will be reported elsewhere.

Characterization of the genetic loci.For isolation of the complete coding sequence of the A segment, a genomic library of V. vulnificus CKM-1 DNA was subjected to colony hybridization, and a plasmid, pSJ1, obtained from a probe-reactive clone was selected for analysis. Two complete and two partial open reading frames (ORFs) were identified (Fig. 1A). The first complete ORF encompassed the insertion site of the Tn5 mutant strain SSM-1 and was identified as trkA. It was preceded by a putative ribosome binding site (GAGA) 7 bp upstream of the initiation codon and was predicted to encode a protein of 458 amino acids with a molecular mass of ∼50 kDa. The putative amino acid sequence of TrkA of V. vulnificus showed high identities to NAD⁺ binding proteins of many other bacteria implicated in K⁺ transport, including Vibrio alginolyticus TrkA (96%) (23), E. coli TrkA (78%) (33), and Salmonella enterica serovar Typhimurium SapG (78%) (29). The second ORF was located 9 bp downstream of trkA and was predicted to encode a protein of 481 amino acids with a molecular mass of ∼53 kDa. This amino acid sequence shared 92% sequence identity to TrkH from V. alginolyticus (25) and 33% sequence identity to the trkH-trkG gene products from E. coli (34) and thus was identified as TrkH of V. vulnificus. TrkA is a peripheral membrane protein bound to the inner side of the cytoplasmic membrane and is absolutely required for Trk activity in the K⁺ transport complex (5, 10, 11), whereas TrkH is an integral membrane protein and probably forms the K⁺-translocating subunit of the complex (34). In addition to those in V. vulnificus, the trkA and trkH genes clustered together on the chromosome have also been found in Archaeoglobus fulgidus (17) and V. alginolyticus (25).

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FIG. 1.

Organization of trkA-trkH and RT-PCR analysis. (A) Chromosome organization of trkA-trkH and the flanking region in V. vulnificus CKM-1. Open arrows represent the direction of transcription and the ORF of trkA-trkH. The locations of the primers used for RT-PCR are depicted by arrows below the trkA and trkH genes. (B) Cotranscription of trkA and trkH demonstrated by RT-PCR. RNA isolated from V. vulnificus CKM-1 was used as a template to generate cDNAs. The cDNAs were then used as templates in PCR with trkA-specific primers AF3 and AR1 (lane 1), with trkH-specific primers HF1 and HR1 (lane 4), and with trkA-trkH-specific primers AF3 and HR1 (lane 7). Plasmid pSJ1 was used as a positive control with the same primers (lane 3, AF3-AR1; lane 6, HF1-HR1; lane 9, AF3-HR1). RNA subjected to PCR without prior RT was used as the negative controls (lanes 2, 5, and 8).

Although only partially sequenced, an incomplete ORF upstream of trkA of V. vulnificus was homologous to the fmv gene which is analogously positioned upstream of trkA-trkH in V. alginolyticus (25). The other incomplete ORF lay downstream from V. vulnificus trkH and potentially encoded the C-terminal 91 amino acids of a protein that shared significant (46%) homology to hemolysin III (hlyIII) of Bacillus cereus (2). In this region, the chromosome organization of V. vulnificus differs from that of V. alginolyticus. In V. alginolyticus, the gene (orf1) is immediately downstream of trkH (25). The complete genome sequence of V. vulnificus CMCP6 has recently been submitted (http://www.ncbi.nlm.nih.gov/genomes/MICROBES/Complete.html ). A comparison of strain CKM-1 trkA and trkH with those of strain CMCP6 revealed that there are very few nucleotide differences between these two strains. The only points at which there are differences in the predicted trkA and trkH amino acid sequences are as follows: strain CKM-1 trkA has Asn instead of Asp at position 32, Lys instead of Glu at position 176, and His instead of Asn at position 391, and strain CKM-1 trkH has Phe instead of Ser at position 9 and Ile instead of Val at position 53.

Cotranscription of trkA and trkH.Since the trkA and trkH genes were located adjacent to each other, with the putative ribosome binding site of trkH overlapping with the stop codon of trkA, it is very likely that these two genes are transcriptionally linked and form an operon. Thus, RT-PCR analysis was used to assess whether trkA and trkH are transcribed as a single RNA transcript. As shown in Fig. 1B, the results of RT-PCR yielded products of the expected sizes (∼1,100, 500, and 1,700 bp) (lanes 1, 4, and 7) from the RNA of CKM-1. DNA sequencing confirmed that these amplified products were indeed from trkA and trkH. This indicates that the trkA and trkH genes are organized as a single bicistronic operon.

Isolation of V. vulnificus trkA mutant.To demonstrate that the observed serum-sensitive phenotype was reproducibly linked to the transposon insertion at the trkA locus, we reconstructed a trkA isogenic mutant, AKK-1. Inactivation of the wild-type trkA with the insertional disruption of the trkA gene in AKK-1 was checked by PCR using a pair of primers complementary to sequences located in the trkA and ampicillin resistance genes (data not shown). For confirmation of the lack of TrkA protein in the AKK-1 strain, Western blot analysis of the parental and AKK-1 strains was performed. The results of the Western blot analysis demonstrated that no anti-TrkA-reactive protein was produced by the AKK-1 mutant (Fig. 2).

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FIG. 2.

Confirmation of V. vulnificus trkA mutant by Western blotting. Proteins (40 μg) were isolated from the wild-type and trkA mutant strains, separated by electrophoresis, and transferred to a nitrocellulose membrane. Immunodetection of the TrkA product was performed with a rabbit polyclonal anti-TrkA antibody. Goat anti-rabbit immunoglobulin G conjugated to peroxidase was used to amplify the signals, and the reacting bands were visualized by using enhanced chemiluminescence reagents.

Effect of K⁺ on the growth of V. vulnificus trkA.Since TrkA showed a high degree of sequence homology to an NAD⁺ binding protein that was part of a low-affinity potassium uptake system of E. coli (33), the growth of the wild-type strain CKM-1 and the mutant strain AKK-1 in a K⁺-free medium and in medium with different levels of added potassium was investigated. The results showed that at a very low concentration of potassium (<0.01 mM), neither the wild type nor the mutant grew, and that at relatively high concentrations of potassium (30 mM and above), both strains grew and had approximately identical growth rates (data not shown). However, at intermediate potassium concentrations, the mutant showed attenuated growth compared to the wild type. For example, in medium containing 1, 5, 10, or 20 mM K⁺, the doubling times for the mutant were 39, 43, 40, and 34 min, respectively, whereas those for the wild type were 17, 21, 23, and 21 min, respectively. To confirm that the attenuated growth of the AKK-1 mutant at intermediate potassium concentrations was indeed due to inactivation of the trkA gene, we cloned the gene into plasmid pYC2, which was then introduced into the AKK-1 mutant. The results revealed that complementation of the AKK-1 mutant with pYC2 caused it to regain the ability to exhibit wild-type growth rates at intermediate potassium concentrations (data not shown).

TrkA is required for serum resistance.The AKK-1 strain was examined for sensitivity to serum, and the results revealed that AKK-1 had an ∼0.6% survival rate relative to the original inocula after incubation with 50% serum at 37°C for 1 h; this survival rate was identical to that of the Tn5 mutant SSM-1. However, the serum sensitivity of AKK-1 and SSM-1 was abolished by heat treating of the serum at 56°C for 30 min, indicating that complement is the sensitizing factor. The introduction of pYC2 into the AKK-1 mutant increased the level of serum resistance to be similar to that of the wild type (Table 2).

TrkA is required for protamine and polymyxin B resistance.Since TrkA shared striking homology to SapG of S. enterica serovar Typhimurium and since an S. enterica serovar Typhimurium sapG isogenic mutant has been reported to be highly susceptible to the antimicrobial peptide protamine and attenuated for virulence in mice (29), we examined the sensitivity of the AKK-1 mutant to protamine and polymyxin B. As revealed in Fig. 3, the AKK-1 mutant showed an increased sensitivity to polymyxin B and protamine compared with the wild-type strain for the concentration ranges of 5 to 15 μg/ml and 10 to 20 μg/ml, respectively. Again, resistance to protamine and polymyxin B was restored in AKK-1 containing pYC2 (data not shown).

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FIG. 3.

Bacterial killing kinetics of the wild-type and trkA mutant strains by polymyxin B (A) and protamine (B). Wild-type CKM-1 (closed symbols) and trkA mutant strain AKK-1 (open symbols) were incubated in LB broth containing different concentrations of polymyxin B (squares, 15 μg/ml; circles, 10 μg/ml; triangles, 5 μg/ml) or protamine (squares, 20 μg/ml; circles, 15 μg/ml; triangles, 10 μg/ml) at 37°C. Bacteria lysis was monitored by measuring the OD₆₀₀ value at the indicated times. The data correspond to a representative experiment performed in duplicate. Data are given as the means of the OD₆₀₀ values ± the standard errors of the means.

Reduced virulence is associated with TrkA.The virulence of the trkA isogenic mutant AKK-1 and the wild-type strain CKM-1 was studied by infecting BALB/c mice by either the i.p. or s.c. route. Mice were either untreated or were pretreated with iron dextran. As shown in Table 3, the LD₅₀ values for the mutant AKK-1 strain in normal mice were 4.0 × 10⁷ and 6.0 × 10⁵ at 72 h for infection via the i.p. and s.c. routes, respectively. These were 80- and 7-fold increases compared to the LD₅₀ values of 5.0 × 10⁵ and 9.0 × 10⁴ for wild-type infection in normal mice via the i.p. and s.c. routes, respectively. For iron-treated mice, the LD₅₀ values of the mutant AKK-1 were 1.2 × 10³ and 2.4 × 10³ for infection via the i.p. and s.c. routes, respectively. These values represented 150- and 300-fold increases compared to the LD₅₀ value of 8 for infection by the wild-type strain via the i.p. and s.c. routes (Table 3). Similar results were obtained when the experiment was repeated (Table 3). For the repeated experiment, the virulence of the mutant AKK-1 strain containing pYC2 was also determined, and the results showed that the LD₅₀ values of this complemented mutant were approximately 10- to 15-fold less than those of the uncomplemented mutant but approximately 25- to 19-fold higher than those of the wild-type strain. To investigate whether the complemented mutants were able to maintain their plasmids during the in vivo experiment, we recovered bacteria at 24 h postinfection from spleens of mice that were inoculated with the complemented or uncomplemented mutant, and their antibiotic phenotypes were determined. The results revealed that approximately 95% of the complemented mutants were unable to grow in medium containing chloramphenicol, indicating that the plasmid pYC2 was not maintained in these strains in the absence of antibiotic selective pressures and was rapidly lost within 24 h postinfection. This may explain why the level of virulence was partially restored in complemented mutants in animal infection experiments.