Vibrio vulnificus biotype 2 serovar E gne but not galE is essential for lipopolysaccharide biosynthesis and virulence

Abstract

This work aimed to establish the role of gne (encoding UDP-GalNAc 4-epimerase activity) and galE (encoding UDP-Gal-4-epimerase activity) in the biosynthesis of surface polysaccharides, as well as in the virulence for eels and humans of the zoonotic serovar of Vibrio vulnificus biotype 2, serovar E. DNA sequence data revealed that gne and galE are quite homologous within this species (> or =90% homology). Mutation in gne of strain CECT4999 increased the surface hydrophobicity, produced deep alterations in the outer membrane architecture, and resulted in noticeable increases in the sensitivity to microcidal peptides (MP), to eel and human sera, and to phagocytosis/opsonophagocytosis. Furthermore, significant attenuation of virulence for eels and mice was observed. By contrast, mutation in galE did not alter the cellular surface, did not increase the sensitivity to MP, serum, or phagocytosis, and did not affect the virulence for fish and mice. The change in the attenuated-virulence phenotype produced by a mutation in gne was correlated with the loss of the O-antigen lipopolysaccharide (LPS), while the capsule was maintained. Complementation of a gne-deficient mutant restored the LPS structure together with the whole virulence phenotype. In conclusion, gne, but not galE, is essential for LPS biosynthesis and virulence in the zoonotic serovar of V. vulnificus biotype 2.

FIG. 1.

Dendrogram showing the amino acid sequence relatedness of V. vulnificus GalE and Gne with UDP-glucose 4-epimerases of other vibrios and related species using the TreeView software (45).

FIG. 2.

Growth curves in TSB-1 (A) and DMM-gal (B) of strain CECT4999 and its derivatives, the gne-deficient mutant (4999-2) and the galE-deficient mutant (4999-3), incubated at 28°C. An asterisk indicates that a value is significantly different from the value for the wild-type strain (CECT4999) (P < 0.05).

FIG. 3.

Immunostaining of LPS and capsule extracts with (A) rabbit anti-CECT4999 serum diluted 1:1,000 (lane 1, CECT4999; lane 2, 4999-1 [translucent variant]), (B) rabbit anti-CECT4999 serum diluted 1:1,000 (lane 1, 4999-3 [galE-deficient mutant]; lane 2, 4999-2 [gne-deficient mutant]; lane 3, C4999-2 [gne-complemented strain]), (C) rabbit anti-CECT4999 serum after absorption of the antibodies with 4999-2 cells (anti-O-antigen serum) diluted 1:10 (lane 1, CECT4999), and (D) rabbit anti-CECT400 serum after absorption with 4999-1 cells (anticapsule serum) diluted 1:10 (lane 1, CECT4999; lane 2, 4999-1; lane 3, 4999-3; lane 4, 4999-2).

FIG. 4.

Electron micrographs of strains CECT4999, 4999-3 (galE-deficient mutant), 4999-2 (gne-deficient mutant), 4999-1 (translucent variant), C4999-2 (gne-complemented strain), and C4999-3 (galE-complemented strain) after ruthenium red staining of ultrathin sections. The white arrows indicate the electron-dense periplasmic material. Bars = 0.1 μm.

FIG. 5.

Biofilm formation by CECT4999 and its derivatives on glass, polypropylene, and eel mucus measured by determining the absorbance at 540 nm (Multiskan Askcent; Labsystems). The strains used were strains CECT4999 (wild-type strain), 4999-1 (translucent variant), 4999-2 (gne-deficient mutant), 4999-3 (galE-deficient mutant), C4999-2 (gne-complemented strain), and C4999-3 (galE-complemented strain). An asterisk indicates that the value is significantly different from the value for the wild-type strain (P < 0.001).

FIG. 6.

Swimming behavior of strains CECT4999 (wild-type strain), 4999-2 (gne-deficient mutant), and 4999-3 (galE-deficient mutant).

FIG. 7.

Bactericidal activity of phagocytes from spleen, blood, and head kidney against nonopsonized or opsonized VSE strains. The strains used were strains CECT4999 (wild-type strain), 4999-1 (translucent variant), 4999-2 (gne-deficient mutant), 4999-3 (galE-deficient mutant), C4999-2 (gne-complemented strain), and C4999-3 (galE-complemented strain). An asterisk indicates that the value is significantly different from the value for the wild-type strain (P < 0.001).