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. 2004 Feb;72(2):629-36.
doi: 10.1128/iai.72.2.629-636.2004.

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

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

Yu-Chung Chen et al. Infect Immun. .
Free PMC article

Abstract

Vibrio vulnificus, a highly virulent marine bacterium, is the causative agent of both serious wound infections and fatal septicemia in many areas of the world. To identify the genes required for resistance to human serum, we constructed a library of transposon mutants of V. vulnificus and screened them for hypersensitivity to human serum. Here we report that one of the isolated serum-susceptible mutants had a mutation in an open reading frame identified as trkA, a gene encoding an amino acid sequence showing high identity to that of TrkA of Vibrio alginolyticus, a protein required for the uptake of potassium. A trkA isogenic mutant was constructed via insertional inactivation, and it was significantly more easily killed by human serum, protamine, or polymyxin B than was the wild type. At K+ concentrations of 1 to 20 mM, this isogenic mutant showed attenuated growth compared to the wild-type strain. In addition, infection experiments demonstrated virulence attenuation when this mutant was administered intraperitoneally or subcutaneously to both normal and iron-treated mice, indicating that TrkA may modulate the transport of potassium and resistance to host innate defenses and that it is important for virulence in mice.

Figures

FIG. 1.
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).
FIG. 2.
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.
FIG. 3.
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.

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