Identification of the Vibrio vulnificus wbpP gene and evaluation of its role in virulence

Abstract

A wbpP gene encoding a putative UDP-N-acetyl-D-glucosamine C(4) epimerase was identified and cloned from Vibrio vulnificus. The functions of the wbpP gene, assessed by the construction of an isogenic mutant and by evaluating its phenotype changes, demonstrated that WbpP is essential in both the pathogenesis and the capsular polysaccharide biosynthesis of V. vulnificus.

FIG. 1.

V. vulnificus wbpP gene locus and relatedness of WbpP and other proteins involved in surface carbohydrate biosynthesis in Enterobacteriaceae. (A) Plasmid pNY0400 was used to determine the nucleotide sequence of wbpP. The open arrows represent the locations of a complete ORF (wbpP) and two less characterized ORFs (wzc and wbpO) and the directions of their transcription. (B) The dendrogram showing the amino acid sequence relatedness of V. vulnificus WbpP and gene products of putative polysaccharide biosynthesis genes was derived using the CLUSTALW alignment program (http://www.ebi.ac.uk/clustalw/) and is based on the amino acid sequences in the GenBank databases (NCBI).

FIG. 2.

Allelic-exchange procedure and construction of wbpP::nptI isogenic mutant. (A) Double homologous recombinations between the wild type (ATCC 29307) and plasmid pNY0421 led to an interruption of the wbpP gene and resulted in the construction of the wbpP mutant NY018. The dashed lines represent the bacterial chromosome; the full line, the plasmid DNA; the open box, the target wbpP gene; the shaded box, the nptI gene; and the large Xs, genetic crossing over. sacB, levansucrase gene. (B) PCR analysis of the wild type and NY018 generated by allelic exchanges. Molecular size markers (1-kb-plus DNA ladder; Invitrogen, Carlsbad, CA) and PCR products are indicated.

FIG. 3.

Effect of wbpP gene mutation on UDP-N-acetyl-d-glucosamine C₄ epimerase. Cultures of the wild type (WT) and NY018 (wbpP) were grown in LBS, and the UDP-N-acetyl-d-glucosamine C₄ epimerase activities were determined from samples removed at an optical density at 600 nm of 0.8. Complementation of the mutant with a functional wbpP (pNY0413) is also presented as indicated. Relative activities of the UDP-N-acetyl-d-glucosamine C₄ epimerase were measured as described in the text. The error bars represent the SEM.

FIG. 4.

Analyses of CPS. (A) The method described by Enos-Berlage and McCarter (7) was used to isolate the CPS from the wild type (WT), NY018 (wbpP), and complemented strain as indicated. After separation on 5% polyacrylamide gels, the CPS was visualized by silver staining as described by Kelley and Parker (14). (B) The relative amounts of CPS from each strain are presented based on the amount of the CPS of the wild type as 100%. The error bars represent the SEM.

FIG. 5.

Effect of wbpP mutation on virulence of V. vulnificus toward INT-407 cells. (A) INT-407 cells were infected with the wild-type, wbpP mutant, or complemented strain of V. vulnificus at various MOI for 3 h (left) or at an MOI of 10 for various incubation times (right). Thereafter, the cell cytotoxicity was determined by an LDH release assay. The data represent the means plus SEM from three independent experiments. *, P < 0.01; **, P < 0.05 relative to groups infected with the wild type of V. vulnificus at each MOI or each incubation time. (B) Microscopic observation of INT-407 infected with the V. vulnificus strains at an MOI of 10 for 3 h. From the left, uninfected (control) and infected with wild type (WT), NY018 (wbpP), or the complemented strain.

FIG. 6.

Adhesion of V. vulnificus wild-type, wbpP mutant, and complemented strains to INT-407 cells. (A) INT-407 cells were cultured on glass coverslips and infected at an MOI of 10. After incubation with the bacteria for 2 h, the INT-407 monolayers were rinsed to remove any nonadhering bacteria. Light micrographs show the adhesion of the wild-type (WT), NY018 (wbpP), and complemented strains to the INT-407 cells. (B) The adherent bacteria were quantified and expressed as the number of bacteria per cell in the coverslip tissue culture. The error bars represent the SEM.