A Vibrio vulnificus type IV pilin contributes to biofilm formation, adherence to epithelial cells, and virulence

Abstract

Vibrio vulnificus expresses a multitude of cell-associated and secreted factors that potentially contribute to pathogenicity, although the specific roles of most of these factors have been difficult to define. Previously we have shown that a mutation in pilD (originally designated vvpD), which encodes a type IV prepilin peptidase/N-methyltransferase, abolishes expression of surface pili, suggesting that they belong to the type IV class. In addition, a pilD mutant exhibits reduced adherence to HEp-2 cells, a block in secretion of several exoenzymes that follow the type II secretion pathway, and decreased virulence. In this study, we have cloned and characterized a V. vulnificus type IV pilin (PilA) that shares extensive homology to group A type IV pilins expressed by many pathogens, including Vibrio cholerae (PilA), Pseudomonas aeruginosa (PilA), and Aeromonas hydrophila (TapA). The V. vulnificus pilA gene is part of an operon and is clustered with three other pilus biogenesis genes, pilBCD. Inactivation of pilA reduces the ability of V. vulnificus to form biofilms and significantly decreases adherence to HEp-2 cells and virulence in iron dextran-treated mice. Southern blot analysis demonstrates the widespread presence of both pilA and pilD in clinical as well as environmental strains of V. vulnificus.

FIG. 1.

Genetic organization of the type IV pilus biogenesis gene cluster of V. vulnificus. (A) Relevant restriction sites used for cloning constructs and probes. (B) Comparison of the type IV pilus biogenesis pil genes of V. vulnificus with those of V. cholerae, A. hydrophila, A. salmonicida, and P. aeruginosa.

FIG. 2.

(A) Comparison of the amino acid sequence of V. vulnificus PilA with the homologous type IV pilins of V. cholerae (Vc PilA), A. hydrophila (Ah TapA), P. aeruginosa (Pa PilA), and V. cholerae (Vc MshA). Conserved residues around the cleavage site are shaded. The remainder of the sequence is less homologous. The inverted triangle indicates the consensus cleavage site for group A, type IV pilins. (B) Comparison of the amino acid sequence of V. vulnificus PilB with homologs from V. cholerae (Vc PilB), A. hydrophila (Ah TapB), and P. aeruginosa (Pa PilB). The location of the Walker box motif is shaded. Both alignments were generated by using the Pileup program of the Genetics Computer Group (Madison, Wis.).

FIG. 3.

Expression of PilA in V. vulnificus strains grown in TCG medium at 30°C. Whole-cell lysates were analyzed by Western blotting with anti-PilA antiserum. Lane 1, C7184 (wild-type); lane 2, C7184AΩ (pilA mutant); lane 3, C7184AΩ (pilA⁺); lane 4, C7184DΩ (pilA⁺); lane 5, PilA (purified).

FIG. 4.

Detection of pilA, pilB, pilC, and pilD transcripts by RT-PCR in V. vulnificus C7184 (wild type) (lanes 1 to 4) and C7184AΩ (pilA mutant) strains (lanes 5 to 8). The corresponding control RT reactions in which the addition of Superscript II RNase H⁻ reverse transcriptase was omitted are shown in lanes 9 to 16.

FIG. 5.

Transmission electron micrographs of V. vulnificus C7184 strains showing surface pili (indicated by arrows). Cells were negatively stained with 2% uranyl acetate on Butyar-coated grids. (A) C7184; (B) C7184AΩ; (C) C7184AΩ(pRP383); (D) P. aeruginosa PAK-NP; (E) P. aeruginosa PAK-NP(pRP383). Magnification, ×15,500.

FIG. 6.

Comparison of adherence of V. vulnificus strains to epithelial cells (HEp-2). (A) Adherence was determined with V. vulnificus strains labeled with [methyl-³H]thymidine. (B) One hundred epithelial cells were counted for each strain. Values represent the mean number ± standard deviation of adherent bacteria per coverslip (A) or the number of adherent bacteria per epithelial cell (B). Values denoted by different letters are significantly different when compared by a one-way ANOVA and Fisher's PLSD at P ≤ 0.05.

FIG. 7.

Southern blot analysis of clinical and environmental isolates of V. vulnificus, using a probe encompassing the pilA gene (see Materials and Methods). I, clinical isolates; S, shellfish isolates associated with illness; O, oyster isolates not associated with illness. M, strain MO6-24; P, PAC1; C, C7184.