Figure 5.

Contribution of virulence factor in LC3-mediated C. jejuni invasion. (A) HeLa cells were infected with C. jejuni of WT, capA::cm^r, and cadF::cm^r. The number of intracellular C. jejuni was estimated by gentamicin protection assay at 1 h p. i. Data are expressed as means ± standard deviations (n = 4). (B) HeLa cells were infected with C. jejuni for 6 h, and detected autophagy-related proteins, p62/SQSTM and LC3 by Western blotting. (C) C. jejuni strains of WT, capA::cm^r, and cadF::cm^r or heat-killed bacteria were incubated in High glucose supplemented DMEM [DMEM(+)] for 24 h, and then bacterial cells were removed and the supernatant was collected. HeLa cells were cultured in bacterial removed supernatant for 6 h, and detected p62/SQSTM and LC3 by Western blotting. (D) Intracellular active Rac1 level at 6 h p. i. was measured by Western blotting. (E) Localization of LC3 and C. jejuni was observed by confocal microscope at 20 min p.i., and the rate of LC3-positive bacteria was quantified in C. jejuni of WT, capA::cm^r, and cadF::cm^r infected cells. WT: n = 64, capA::cm^r: n = 83, cadFcm^r: n = 84 of bacterial cells which pooled from 3 independent experiments. (F) C. jejuni and heat-killed bacteria was incubated in DMEM(+) for 24 h, and the supernatant was collected. HeLa cells were cultured in aforementioned supernatant for 6 h, and intracellular active Rac1 level was measured by Western blotting. (G) Proposed model in this study for C. jejuni infection signaling which mediated by Rac1 activated by CadF, and invasion route via LC3. All data are expressed as means ± standard deviations from 3 independent experiments. Differences were evaluated with Two-tailed Student’s t-test. *p < 0.05.