Growth dynamics of WT <i>C. jejuni</i>, the Δ<i>hcp1</i> mutant, and the Δ<i>icmF1</i> mutant in various sub-inhibitory concentrations of DCA.

<p>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.</p

Growth responses of WT <i>C. jejuni</i>, Δ<i>hcp</i> and Δ<i>icmF</i> mutants, and the complemented Δ<i>hcp1</i> strain, Δ<i>hcp1</i>(<i>hcp+</i>), on agar free of or supplemented with 3% DCA.

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

List of T6SS orthologs of <i>C. jejuni</i> ATCC 43431 and their proposed functions [3], [24].

<p>List of T6SS orthologs of <i>C. jejuni</i> ATCC 43431 and their proposed functions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042842#pone.0042842-Boyer1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042842#pone.0042842-Filloux1" target="_blank">[24]</a>.</p

Expression levels of <i>cmeA</i> in WT <i>C. jejuni</i>, the Δ<i>hcp1</i> mutant, and the complemented Δ<i>hcp1</i> strain, Δ<i>hcp1</i>(<i>hcp+</i>), grown in liquid media free of or supplemented with 0.1% DCA after 14 hours of growth.

<p>The Y axis represents the ratio of <i>cmeA</i> copy number and the copy number of a house keeping gene, <i>glyA</i>, previously shown to not be affected by DCA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042842#pone.0042842-MalikKale1" target="_blank">[55]</a>. The graph represents results from two independent experiments. Error bars represent the standard error of the mean. P value: **≤0.01, ***≤0.001.</p

Western blot analysis of Hcp in WT <i>C. jejuni</i>, the Δ<i>hcp</i> mutant, and the Δ<i>icmF</i> mutant.

<p>Equal numbers of WT, Δ<i>hcp</i> and Δ<i>icmF</i> mutants were collected (see <b>Material and Methods</b>). 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.</p

Primers used in this study.

<p>Primers used in this study.</p

Colonization potential of WT <i>C. jejuni</i>, the Δ<i>icmF1</i> mutant, and the Δ<i>icmF2</i> mutant in IL-10-deficient mice at 30 days post-infection.

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

Adhesion and invasion efficacy of WT <i>C. jejuni</i>, the Δ<i>hcp1</i> mutant, the Δ<i>icmF1</i> mutant, and the complemented Δ<i>hcp1</i> strain, Δ<i>hcp1</i>(<i>hcp+</i>), in human T84 colonic epithelial and RAW 267.4 macrophage cell lines.

<p>(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.</p

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

<p>The transcript levels of (A) <i>hcp</i>, (B) <i>icmF</i>, and (C) <i>cmeA</i> of WT <i>C. jejuni</i> 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 <i>hcp</i>, <i>icmF</i> or <i>cmeA</i> copy number and the copy number of a house keeping gene, <i>glyA</i>. 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.</p

Gene organization of T6SS clusters in <i>Campylobacter</i> spp.

<p>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 <i>C. jejuni</i> 414.</p