Isolation and characterization of a Vibrio vulnificus mutant deficient in both extracellular metalloprotease and cytolysin

Abstract

We isolated a Vibrio vulnificus mutant that was deficient in both metalloprotease and cytolysin by allelic exchange. The virulence of this mutant in mice and its cytotoxicity for HEp-2 cells were comparable to those of the wild-type strain, indicating that neither factor was essential for these properties. The cytolysin, but not the protease, seemed to be important for causing damage in the alimentary tract of the mice.

FIG. 1

Detection of deletion in vvhA in the chromosome of V. vulnificus. (A) Restriction map of the insert in pFJ004, which was used to construct V. vulnificus mutants FJ201 and FJ202. The deletion (white bar), the probes used in colony (probe 1) and Southern hybridization (probe 2), and the PCR primers used in detecting the deletion in the mutants are indicated. S, SalI; C, ClaI; H, HpaI; B, BglII; Bm, BamHI. (B) Southern hybridization of the ClaI digests of the plasmid or chromosomal DNA fractionated by electrophoresis on a 1.2% agarose gel. Lanes: 1, pCVD442; 2, YJ016; 3, FJ008 (YJ016 integrated with pFJ006); 4, FJ201; 5, FJ202; M, molecular size standards.

FIG. 2

Protease and cytolysin activities in the culture supernatants of various V. vulnificus strains. Bacteria were grown at 37°C after a 1:100 dilution of an overnight culture in fresh medium. The culture supernatant was then collected at intervals, and the protease and cytolysin activities were determined (n = 3).

FIG. 3

Micrographs of upper intestines of mice challenged by force-feeding with the wild-type strain (B), the PD mutant (C), the CD mutant (D), and the DD mutant (E). Mice inoculated with PBS were used as the negative control (A). Note the sloughed and necrotic epithelial cells with condensed and pyknotic nuclei (arrowheads) and hypercellularity and congestion in lamina propria (B and C). Intestinal tissues from mice infected with the CD or DD mutant appear normal. Hematoxylin-eosin stain was used. Magnification, ×400.