Culture of Campylobacter jejuni with sodium deoxycholate induces virulence gene expression

Abstract

Campylobacter jejuni, a spiral-shaped gram-negative bacterium, is a leading bacterial cause of human food-borne illness. Acute disease is associated with C. jejuni invasion of the intestinal epithelium. Further, maximal host cell invasion requires the secretion of proteins termed Campylobacter invasion antigens (Cia). As bile acids are known to alter the pathogenic behavior of other gastrointestinal pathogens, we hypothesized that the virulence potential of Campylobacter may be triggered by the bile acid deoxycholate (DOC). In support of this hypothesis, culturing C. jejuni with a physiologically relevant concentration of DOC significantly altered the kinetics of cell invasion, as shown by gentamicin protection assays. In contrast to C. jejuni harvested from Mueller-Hinton (MH) agar plates, C. jejuni harvested from MH agar plates supplemented with DOC secreted the Cia proteins, as judged by metabolic labeling experiments. DOC was also found to induce the expression of the ciaB gene, as determined by beta-galactosidase reporter, real-time reverse transcription-PCR, and microarray analyses. Microarray analysis further revealed that DOC induced the expression of virulence genes (ciaB, cmeABC, dccR, and tlyA). In summary, we demonstrated that it is possible to enhance the pathogenic behavior of C. jejuni by modifying the culture conditions. These results provide a foundation for identifying genes expressed by C. jejuni in response to in vivo-like culture conditions.

FIG. 1.

Sodium DOC does not alter the adherence (A) or motility (B) of the C. jejuni F38011 strain. (A) Tenfold serial dilutions of the C. jejuni F38011 strain, cultured on MH and MHD agar plates for 18 h, were used to inoculate INT 407 cells. The data indicate the numbers of bacteria from MH agar plates (black bars) and MHD agar plates (gray bars) that bound to INT 407 cells 30 min postinoculation. The bars indicate the mean numbers of viable bacteria recovered per well of a 24-well tissue culture tray, and the error bars indicate the standard deviations. (B) C. jejuni cultures on MH and MHD agar plates with 0.4% agar displayed equivalent zones of migration.

FIG. 2.

Sodium DOC stimulates the synthesis of Campylobacter invasion antigens. (A) Proteins secreted by C. jejuni as determined by SDS-PAGE coupled with autoradiography of the supernatants. (B) Proteins synthesized by C. jejuni as judged by SDS-PAGE coupled with autoradiography of the WCLs (OD₅₄₀, 0.1). (C) Control, in which the proteins secreted by C. jejuni were probed with an antibody prepared against the C. jejuni CadF outer membrane protein. The metabolic labeling assays, preparation of supernatants and WCLs, and autoradiography were performed as described in Materials and Methods. Lane 1, C. jejuni F38011 harvested from an MH agar plate and radioactively labeled in medium supplemented with 1% FBS; lane 2, C. jejuni F38011 harvested from an MHD agar plate and radioactively labeled in medium supplemented with 1% FBS; lane 3, C. jejuni F38011 harvested from an MH agar plate and radioactively labeled in medium without 1% FBS; lane 4, C. jejuni F38011 harvested from an MHD agar plate and radioactively labeled in medium without 1% FBS; lane 5, 1:4 dilution of C. jejuni WCL; lane 6, 1:2 dilution of C. jejuni WCL; lane 7, 1:1 dilution of C. jejuni WCL. The migration of CadF as two protein species, indicated by the arrow (37-kDa species) and the arrowhead (32-kDa species), was due to the protein's heat-modifiable property.

FIG. 3.

Culturing the C. jejuni F38011 strain with sodium DOC alters the kinetics of invasion of INT 407 cells. Gentamicin protection assays were performed as described in Materials and Methods with C. jejuni F38011 cultured on MH and MHD agar plates for 18 h. The bacteria were incubated with INT 407 cells in the absence and presence of 128 μg/ml of Cm to inhibit protein synthesis. The numbers of internalized bacteria are indicated for C. jejuni F38011 cultured on an MH agar plate (○), C. jejuni F38011 cultured on an MHD agar plate (□), C. jejuni F38011 cultured on an MH agar plate and then incubated for 30 min with Cm (•), and C. jejuni F38011 cultured on an MHD agar plate and then incubated for 30 min with Cm (▪). The symbols indicate the mean numbers of viable bacteria recovered per well of a 24-well tissue culture tray, and the error bars indicate standard deviations.

FIG. 4.

Stimulation of ciaB promoter activity by sodium DOC is time (A) and dose (B) dependent. The data shown represent the ratios of the β-galactosidase activity of ciaB (black bars) and porA (gray bars) for the C. jejuni F38011 strain cultured on an MHD agar plate to the β-galactosidase activity of ciaB and porA for the C. jejuni F38011 strain cultured on an MH agar plate. The bars indicate the means of two separate experiments, and the error bars indicate the standard deviations; each experiment was comprised of triplicate samples.

FIG. 5.

Temporal expression of ciaB and porA in C. jejuni F38011 cultured in the presence of DOC. Real-time RT-PCR analysis was performed with total RNA extracted from the C. jejuni F38011 strain grown on MH and MHD agar plates for 3, 6, 9, 12, and 15 h. The changes in the ciaB (black bars) and porA (gray bars) transcript levels were measured using the comparative threshold cycle method, and glyA was used as the internal control.

FIG. 6.

Functional classification of the C. jejuni F38011 genes upregulated ≥1.5-fold in the presence of DOC. The chart shows the percentages of genes belonging to functional classes based on the total number of genes upregulated in the presence of DOC. The percentages are the values for functional categories containing more than 3% of the total number of genes upregulated. Functional categories with two or fewer genes were grouped together as “Others”.