Characterization of a Campylobacter jejuni VirK protein homolog as a novel virulence determinant

Abstract

Campylobacter jejuni is a leading cause of food-borne illness in the United States. Despite significant recent advances, its mechanisms of pathogenesis are poorly understood. A unique feature of this pathogen is that, with some exceptions, it lacks homologs of known virulence factors from other pathogens. Through a genetic screen, we have identified a C. jejuni homolog of the VirK family of virulence factors, which is essential for antimicrobial peptide resistance and mouse virulence.

FIG. 1.

Amino acid sequence comparison of C. jejuni CJJ81176_1087 with VirK protein family members from pathogenic bacteria. The proteins and organisms used in the alignment are as follows (from top to bottom): hypothetical protein APL_0453 of Actinobacillus pleuropneumoniae strain L20, LapB of Haemophilus influenzae strain Rd KW20, LapB of Pasteurella multocida strain Pm70, hypothetical protein NMA1569 of Neisseria meningitidis strain Z2491, Cjj81176_1087 of Campylobacter jejuni strain 81-176, VirK from Shigella flexneri serotype 2a strain 301, VirK of S. Typhimurium strain LT2, VirK from Yersinia pestis strain CO92, and a putative VirK homolog from Vibrio parahaemolyticus strain RIMD 2210633. The amino acid sequences of VirK proteins from different pathogenic bacteria were aligned using the AlignX program of InforMax 2003 software, and the output was processed for display using BOXSHADE, version 3.21 (http://www.ch.embnet.org/software/BOX_form.html). Identical residues are indicated by a filled background and conserved residues by a shaded background.

FIG. 2.

Ability of a C. jejuni 81-176 virK mutant strain to enter and survive within cultured cells. (A) Cultured cells were infected with C. jejuni 81-176 (wild type [WT]), the isogenic virK mutant, or the complemented virK mutant at an MOI of 10 for 2 h, followed by a 3-h incubation in the presence of gentamicin. Comparison of levels of invasion is shown as the percentage of bacteria that survived treatment with gentamicin relative to that for the WT strain, which was set at 100%. Values are means ± standard deviations for three independent determinations. The difference between the value for the virK mutant and that for the WT or the complemented mutant was statistically significant (P < 0.001). (B) Cultured cells were infected with C. jejuni 81-176 or the isogenic virK mutant at an MOI of 10 for 2 h. Cells were then stained with a protocol that allows extracellular and intracellular bacteria to be distinguished, and the number of internalized bacteria per cell was determined as described in Materials and Methods. Values are means ± standard deviations for three independent determinations. The difference between the values for the two strains was not statistically significant.

FIG. 3.

Subcellular localization of C. jejuni VirK. C. jejuni strain CB32, which encodes FLAG epitope-tagged VirK, was subjected to subcellular fractionation as described in Materials and Methods. The membrane fraction was treated as indicated, and the level of the extracted VirK (s) relative to the proportion that remained membrane associated (p) was determined by Western blot analysis with an antibody against the FLAG tag epitope.

FIG. 4.

C. jejuni virK is attenuated after intraperitoneal infection in a mouse colonization model. (A and B) Equal numbers of wild-type C. jejuni 81-176 and its virK mutant derivative were administered intraperitoneally to myd88^−/− mice. (A) The numbers of CFU of the wild type (filled diamonds) and the virK mutant (open circles) in the feces of the infected animals were determined at different times after infection, as indicated in Materials and Methods. (B) Colonization of tissues was evaluated by determining the CFU of the wild type (filled diamonds) and the virK mutant (open circles) in the intestine and liver. (C and D) To test the complementation of the virK mutant, myd88^−/− mice were inoculated intraperitoneally with equal numbers of the C. jejuni 81-176 virK mutant strain and its complemented derivative [virK (+virK)]. The numbers of CFU of the virK mutant (open circles) and its complemented derivative (filled triangles) in the feces (C) or tissues (D) were determined as described in Materials and Methods. Except for the fecal shedding in week 1, in all cases the difference between the number of CFU of the wild type and the virK mutant was statistically significant (P < 0.05).

FIG. 5.

Colonization of mice by C. jejuni virK after oral infection. Equal numbers of the complemented mutant strain and the virK mutant derivative were administered to myd88^−/− mice by stomach gavage. The numbers of CFU of the complemented mutant strain (filled triangles) and the virK mutant (open circles) in the feces (A) or tissues (B) of the infected animals were determined at different times after infection as described in Materials and Methods. In all cases, the differences between the complemented mutant strain and virK CFU did not reach statistical significance (P > 0.05).

FIG. 6.

C. jejuni virK exhibits increased susceptibility to antimicrobial peptides. Wild-type (WT) C. jejuni 81-176, the virK mutant, and the complemented mutant derivative [virK (+virK)] were treated with the indicated amounts of polymyxin B (PB) (as a surrogate for antimicrobial peptides) (A) or the antimicrobial peptide Magainin-1 (B), and the numbers of CFU that survived were determined as described in Materials and Methods. Values are standardized to those of the WT treated with PBS (taken as 100%) and represent means ± standard deviations for three independent determinations. Asterisks indicate values statistically significantly different (P < 0.001) from those for the WT or the complemented mutant.