Campylobacter jejuni type VI secretion system: roles in adaptation to deoxycholic acid, host cell adherence, invasion, and in vivo colonization

Abstract

The recently identified type VI secretion system (T6SS) of proteobacteria has been shown to promote pathogenicity, competitive advantage over competing microorganisms, and adaptation to environmental perturbation. By detailed phenotypic characterization of loss-of-function mutants, in silico, in vitro and in vivo analyses, we provide evidence that the enteric pathogen, Campylobacter jejuni, possesses a functional T6SS and that the secretion system exerts pleiotropic effects on two crucial processes--survival in a bile salt, deoxycholic acid (DCA), and host cell adherence and invasion. The expression of T6SS during initial exposure to the upper range of physiological levels of DCA (0.075%-0.2%) was detrimental to C. jejuni proliferation, whereas down-regulation or inactivation of T6SS enabled C. jejuni to resist this effect. The C. jejuni multidrug efflux transporter gene, cmeA, was significantly up-regulated during the initial exposure to DCA in the wild type C. jejuni relative to the T6SS-deficient strains, suggesting that inhibition of proliferation is the consequence of T6SS-mediated DCA influx. A sequential modulation of the efflux transporter activity and the T6SS represents, in part, an adaptive mechanism for C. jejuni to overcome this inhibitory effect, thereby ensuring its survival. C. jejuni T6SS plays important roles in host cell adhesion and invasion as T6SS inactivation resulted in a reduction of adherence to and invasion of in vitro cell lines, while over-expression of a hemolysin co-regulated protein, which encodes a secreted T6SS component, greatly enhanced these processes. When inoculated into B6.129P2-IL-10(tm1Cgn) mice, the T6SS-deficient C. jejuni strains did not effectively establish persistent colonization, indicating that T6SS contributes to colonization in vivo. Taken together, our data demonstrate the importance of bacterial T6SS in host cell adhesion, invasion, colonization and, for the first time to our knowledge, adaptation to DCA, providing new insights into the role of T6SS in C. jejuni pathogenesis.

Figure 1. Gene organization of T6SS clusters in Campylobacter spp.

Genes are plotted as arrows, starting from the beginning of the first gene to the end of the last gene, in order of their genomic positions. Gene sizes (defined as the distance between the annotated starts and ends) are represented by the relative length of the arrow. Arrow direction indicates the strand (i.e., forward arrows indicating genes on the positive strand and reverse arrows indicating genes on the negative strand). For each strain, genes are plotted using the color code indicated in C. jejuni 414.

Figure 2. Western blot analysis of Hcp in WT C. jejuni, the Δhcp mutant, and the ΔicmF mutant.

Equal numbers of WT, Δhcp and ΔicmF mutants were collected (see Material and Methods). Pellet and supernatant were separated and used in immunoblot assays with an anti-Hcp antibody. The estimated molecular weight of Hcp is 18 kD. The results are representative of three independent experiments.

Figure 3. Growth dynamics of WT C. jejuni, the Δhcp1 mutant, and the ΔicmF1 mutant in various sub-inhibitory concentrations of DCA.

Ten-fold serial dilutions of WT and the mutants were spotted on agar plates lacking or supplemented with DCA at concentrations ranging from 0% to 1%. Growth was inspected at 14, 18, 36 and 48 hours after incubation under microaerobic conditions. The results are representative of three independent experiments.

Figure 4. Growth responses of WT C. jejuni, Δhcp and ΔicmF mutants, and the complemented Δhcp1 strain, Δhcp1(hcp+), on agar free of or supplemented with 3% DCA.

(A) WT, Δhcp and ΔicmF mutants harboring cat (hcp1, hcp2, icmF1, icmF2), complemented Δhcp1 strain, Δhcp1(hcp+), and the control Δhcp1 mutant harboring kat (Δhcp1(kat)) grown on a DCA-free agar and (B) supplemented with 3% DCA. (C) WT, Δhcp and ΔicmF mutants harboring kat (hcp3, hcp4, icmF3, icmF4), Δhcp1(hcp+) and Δhcp1(kat) grown on a DCA-free agar and (D) supplemented with 3% DCA.

Figure 5. Expression levels of cmeA in WT C. jejuni, the Δhcp1 mutant, and the complemented Δhcp1 strain, Δhcp1(hcp+), grown in liquid media free of or supplemented with 0.1% DCA after 14 hours of growth.

The Y axis represents the ratio of cmeA copy number and the copy number of a house keeping gene, glyA, previously shown to not be affected by DCA . The graph represents results from two independent experiments. Error bars represent the standard error of the mean. P value: **≤0.01, ***≤0.001.

Figure 6. Temporal expression patterns of hcp, icmF, and cmeA during growth of C. jejuni in biphasic culture media free of or supplemented with 0.1% DCA.

The transcript levels of (A) hcp, (B) icmF, and (C) cmeA of WT C. jejuni grown in the presence or absence of 0.1% DCA at 12, 24 and 48 hours of growth. The Y axis represents the ratio of hcp, icmF or cmeA copy number and the copy number of a house keeping gene, glyA. The graphs represent results from at least three independent experiments. Error bars represent the standard error of the mean. P value: *≤0.05, **≤0.01, ***≤0.001.

Figure 7. Adhesion and invasion efficacy of WT C. jejuni, the Δhcp1 mutant, the ΔicmF1 mutant, and the complemented Δhcp1 strain, Δhcp1(hcp+), in human T84 colonic epithelial and RAW 267.4 macrophage cell lines.

(A) T84 adhesion efficacy, B) T84 invasion efficacy, (C) RAW 267.4 macrophage adhesion efficacy and (D) RAW 267.4 macrophage invasion efficacy. The graph represents results from three independent experiments. Error bars represent the standard error of the mean. P value: *≤0.05, **≤0.01, ***≤0.001.

Figure 8. Colonization potential of WT C. jejuni, the ΔicmF1 mutant, and the ΔicmF2 mutant in IL-10-deficient mice at 30 days post-infection.

The number of WT C. jejuni 43431 and the mutants in the cecum (A) and the feces (B) of individual mice were quantified by Q-PCR (see Material and Methods) and the values plotted. A solid line represents the mean value for each group. P value: *≤0.05, **≤0.01, ***≤0.001.