Use of genome-wide expression profiling and mutagenesis to study the intestinal lifestyle of Campylobacter jejuni

Abstract

Campylobacter jejuni is the most common bacterial cause of diarrhea worldwide. To colonize the gut and cause infection, C. jejuni must successfully compete with endogenous microbes for nutrients, resist host defenses, persist in the intestine, and ultimately infect the host. These challenges require the expression of a battery of colonization and virulence determinants. In this study, the intestinal lifestyle of C. jejuni was studied using whole-genome microarray, mutagenesis, and a rabbit ileal loop model. Genes associated with a wide range of metabolic, morphological, and pathological processes were expressed in vivo. The in vivo transcriptome of C. jejuni reflected its oxygen-limited, nutrient-poor, and hyperosmotic environment. Strikingly, the expression of several C. jejuni genes was found to be highly variable between individual rabbits. In particular, differential gene expression suggested that C. jejuni extensively remodels its envelope in vivo by differentially expressing its membrane proteins and by modifying its peptidoglycan and glycosylation composition. Furthermore, mutational analysis of seven genes, hspR, hrcA, spoT, Cj0571, Cj0178, Cj0341, and fliD, revealed an important role for the stringent and heat shock response in gut colonization. Overall, this study provides new insights on the mechanisms of gut colonization, as well as possible strategies employed by Campylobacter to resist or evade the host immune responses.

FIG. 1.

Detection of C. jejuni transcriptome in vivo. (A and B) The RIL 48 h postinoculation with C. jejuni or PBS buffer, respectively. The arrows indicate intestine distended with gas and fluid accumulation. Total RNA was extracted from the intestinal contents, reverse transcribed using C. jejuni-specific 3′ primer, and labeled with the Cy5 dye. This labeled cDNA was cohybridized to the microarray with Cy3-labeled cDNA, obtained from in vitro-grown bacterial RNA.

FIG. 2.

Scatter plots showing the relationship between the log₂ value of the gene expression ratio obtained from hybridization experiments with bacterial cDNA derived from the same rabbit (A and B) or from two different rabbits (C). The solid lines represent the linear regression fit of the data.

FIG. 3.

Global view of genes with similar expression patterns between rabbits grouped by functional categories according to the Sanger Center C. jejuni genome database. Each row represents one gene, and each column represents the expression profile in one rabbit (the mean fold change in the expression ratio of the technical replicates). The column label corresponds to the rabbit numbering. An increasing red intensity denotes a gene for which expression was significantly increased in vivo compared to in vitro growth, and an increasing green intensity indicates a gene for which expression was significantly decreased in vivo compared to in vitro growth. A gray color indicates missing data. Genes with unknown functions are not represented.

FIG. 4.

Global view of genes with a variable expression pattern between rabbits. Each row represents one gene. Columns 1, 2, 3, 4, and 5 represent the expression profiles in rabbits 1, 2, 3, 4, and 5, respectively. For each rabbit, the microarray data correspond to the mean fold change in the expression ratio of the technical replicates. Red and green denote transcripts for which abundance was increased or decreased in vivo compared to in vitro growth, respectively. The red and green intensities are proportional to the increase or decrease, with maximal fold changes in transcript abundance of 3 and 0.33, respectively. A gray color denotes missing data.

FIG. 5.

Competitive colonization ability of seven mutants (hrcA, Cj0178, Cj0571, spoT, Cj0341, hspR, and fliD). The strains were pooled with the parent strain C. jejuni NCTC 11168 (constituting the input pool) and inoculated into four RIL. Forty-eight hours postinoculation, the intestinal contents were recovered and processed for chromosomal DNA extraction. The number of bacteria was estimated by quantitative real-time PCR for each mutant as described in Materials and Methods. The normalized competitive ratio corresponds to the ratio of the number of mutant cells to the total number of bacterial cells in the input pool divided by the ratio of the number of mutants to the total number of bacteria in the recovered pool. The data are the mean of eight determinations (four biological replicates with two technical replicates each), and the error bars represent the standard deviations.

FIG. 6.

In vivo (A) and in vitro (B) competition assays. The in vivo competitive index is the ratio of the mutant to the wild-type strain recovered in the ileal loop 48 h postinfection. Four loops were infected with a mixture of each mutant and the wild-type strain at a ratio of 1:1. The in vitro competitive index is the ratio of the mutant to the wild-type strain in MH broth at late log phase. The in vitro competition assay was performed in triplicate. The error bars indicate the standard deviations. *, statistical significance of P < 0.001.

FIG. 7.

Growth kinetics of C. jejuni NCTC 11168 and five mutants, ΔhrcA (A), ΔhspR (B), ΔspoT (C), ΔCj0178 (D), and ΔfliD (E). Biphasic MH cultures were incubated at 37°C under microaerophilic conditions. The growth kinetics were performed in triplicate, and the error bars represent the standard deviations.