FIGURE 5.

The effect of pgp2 deletion on host-related phenotypes. A and B, PG isolated from the Δpgp2 mutant shows reduced human Nod1 activation and no change in human Nod2 stimulation. To assay the change in the ability of C. jejuni Δpgp2 PG to activate Nod proteins, human embryonic kidney cells (HEK293T) were co-transfected with either 0.2 or 2 μg of C. jejuni 81-176, Δpgp2, or Δpgp2c PG, vectors for a nuclear factor-κB (NF-κB) luciferase reporter, and either Nod1 (A) or Nod2 (B). Nod activation was determined by measuring the activity of a NF-κB luciferase reporter in comparison with the non-stimulated (NS) negative control. Positive controls used were tripeptide l-Ala-γ-d-Glu-meso-DAP (TriDAP) and MDP. Data represent the mean ± S.E. of four independent experiments. C, invasion and intracellular survival ability of the Δpgp2 strain in the INT407 epithelial cell line was assessed by a gentamicin (Gm) protection assay and showed no change in comparison with wild type. Gentamicin was added 3 h postinfection with the C. jejuni wild-type 81-176 and Δpgp2 strains. After 2 h, the gentamicin was washed off, and the cells were incubated with fresh MEM containing 3% FBS and a low dose of gentamicin. cfu were determined for each well by lysing the cells with water and plating the dilutions onto MH-trimethoprim-vancomycin plates. Data represent the mean ± S.E. of three independent experiments. D, pgp2 deletion has no effect on IL-8 secretion in the INT407 epithelial cell line. ELISA was used to quantify IL-8 levels secreted by uninfected INT407 epithelial cell lines and cells infected for 8 and 24 h with C. jejuni wild-type 81-176, Δpgp2, and Δpgp2c strains. Data represent the mean ± S.E. (error bars) of three independent experiments. E, the Δpgp2 mutant strain is defective for chick colonization. Each point represents the log cfu/g cecal contents of an individual chick 7 days postcolonization with 10⁴ cfu of the indicated C. jejuni strains. The geometric mean is denoted by a black bar. *, statistically significant difference using the unpaired Student's t test, with *, **, and *** indicating p < 0.05, p < 0.01, and p < 0.0001, respectively.