Identification of Campylobacter jejuni genes involved in the response to acidic pH and stomach transit

Abstract

Campylobacter jejuni causes food- and waterborne gastroenteritis, and as such it must survive passage through the stomach in order to reach the gastrointestinal tract. While little is known about how C. jejuni survives transit through the stomach, its low infectious dose suggests it is well equipped to sense and respond to acid shock. In this study, the transcriptional profile of C. jejuni NCTC 11168 was obtained after the organism was exposed to in vitro and in vivo (piglet stomach) acid shock. The observed down-regulation of genes encoding ribosomal proteins likely reflects the need to reshuffle energy toward the expression of components required for survival. Acid shock also caused C. jejuni to up-regulate genes involved in stress responses. These included heat shock genes as well as genes involved in the response to oxidative and nitrosative stress. A role for the chaperone clpB in acid resistance was confirmed in vitro. Some genes showed expression patterns that were markedly different in vivo and in vitro, which likely reflects the complexity of the in vivo environment. For instance, transit through the stomach was characterized by up-regulation of genes that encode products that are involved in the use of nitrite as a terminal electron acceptor and down-regulation of genes that are involved in capsular polysaccharide expression. In conclusion, this study has enabled us to understand how C. jejuni modulates gene expression in response to acid shock in vitro and to correlate this with gene expression profiles of C. jejuni as it transits through the host stomach.

FIG. 1.

C. jejuni remains viable and helically shaped after a 20-min exposure to MH-MES medium at pH 4.5. (A) C. jejuni NCTC 11168 was grown to exponential phase in biphasic MH medium and exposed to MH-MES medium at pH 4.5. Samples were withdrawn after 0, 2, 4, 12, 16, and 20 min for viable count determination. Values from seven independent experiments are represented as the percent survival (with 100% being the number of viable cells present at time zero). Error bars denoting the standard errors of the means are present but are too small to be seen. (B) Scanning electron micrographs of C. jejuni prior to (0 min) and after (20 min) exposure to MH-MES medium at pH 4.5. Size bars, 1 μm. Cells from samples taken 2, 4, 12, and 16 min after acid shock were indistinguishable from those shown. mag, magnification.

FIG. 2.

Comparison of in vivo gene expression levels measured by microarray and real-time qRT-PCR. The log₂ ratio values of the microarray experiment were plotted against the log₂ relative quantity values obtained from real-time qRT-PCR.

FIG. 3.

Hierarchical clustering analysis of genes differentially expressed in the gastric environment. Genes differentially expressed (≥2-fold differential expression; P < 10⁻⁴) in the piglet stomach were subjected to hierarchical clustering using Genesis (Euclidian distance, average linkage). The genes of interest grouped into seven clusters, labeled A to G. Data from the in vitro shock experiments are shown in the first five columns (left to right: 2, 4, 12, 16, and 20 min of exposure), while in vivo gene expression ratios are shown in the last column (labeled S). A threshold log₂ value of 2 (equivalent to fourfold differential gene expression) was used in this figure. Red boxes represent up-regulated genes, green boxes represent down-regulated genes, and gray boxes denote missing data. Genes in boldface are further discussed in the text.

FIG. 4.

Hierarchical clustering analysis of genes differentially expressed in response to in vitro acid shock. Genes differentially expressed (≥2-fold differential expression; P < 10⁻⁴) in response to in vitro acid shock were subjected to hierarchical clustering using Genesis (Euclidian distance, average linkage). The three main clusters are designated A, B, and C. Each column represents gene expression after a given exposure time (e.g., 2 indicates 2 min after acid shock). A threshold log₂ value of 2 (equivalent to fourfold differential gene expression) was used in this figure. Red boxes denote up-regulated genes, green boxes designated down-regulated genes, and gray boxes represent missing data. Genes in boldface are discussed in the text.

FIG. 5.

ClpB confers protection against acid shock. Wild-type C. jejuni NCTC 11168 and deletion mutants of hrcA, hspR, and clpB were grown to exponential phase in biphasic MH cultures and exposed to MH-HCl medium at pH 3.0. Samples were withdrawn after 0, 2, 4, 6, 8, 12, 14, and 16 min for viable count determination. Data from a minimum of three independent experiments are shown as the percent survival (with 100% being the viable counts at time zero for each strain) ± standard errors of the means. (A) NCTC 11168 (▪, solid line) and ΔhrcA (•, dashed line); (B) NCTC 11168 (▪, solid line) and ΔhspR (•, dashed line); (C) NCTC 11168 (▪, solid line), ΔclpB (•, dashed line), and the complemented ΔclpB mutant (○, dotted line).

FIG. 6.

Increased sensitivity of a clpB deletion mutant in an SGF. C. jejuni NCTC 11168, ΔclpB, and the complemented ΔclpB mutant were grown to exponential phase in biphasic MH cultures and exposed to an SGF containing 10 mM lactic acid at pH 4.0. Samples were withdrawn after 0, 4, 8, 12, and 16 min for viable count determination. Data from a minimum of three independent experiments are shown as the percent survival (with 100% being the viable counts at time zero for each strain) ± standard errors of the means. Shown are NCTC 11168 (▪, solid line), the ΔclpB mutant (○, dashed line), and the complemented ΔclpB mutant (▴, dotted line).