Campylobacter jejuni strains ATCC 700819 (NCTC11168) were obtained from American Type Culture Collection (ATCC). The bacteria were grown in Muller Hinton Broth (DIFCO) under a microaerobic atmosphere (5% O₂, 10% CO₂, 85%N₂) at 37°C for 48 h. The bacteria were then diluted into fresh MH and cultured under a microaerobic atmosphere at 37°C for an additional 36 h. Salmonella enterica serovar enteritidis (S. Enteritidis) was cultured in Luria-Bertani (LB) medium at 37°C with shaking.

The non-polarized cell lines HeLa, INT407 and polarized cell lines Caco-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM), high-glucose (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; GIBCO) and 50 μg/ml gentamycin (Sigma-Aldrich). The culture medium was changed every 4 days in Caco-2 cells. The culture medium was changed every 4 days. Polarized cell lines T-84 cells were cultured in Dulbecco's modified Eagle's medium nutrient mixture F-12 HAM (DMEM/F-12, 1:1; Sigma-Aldrich) supplemented with 10% FBS and 50 μg/ml gentamycin. The culture medium was changed every 2 days. All cells were incubated in 37°C in a humidified atmosphere containing 5% CO₂.

Ethylene Glycol Tetra-acetic Acid (EGTA) was purchase from Nacalai Tesque. Rhodamine Phalloidin was purchase from Molecular Probes. Methyl-β-cyclodextrin (MβCD), water soluble-cholesterol and FITC-dextran (4 kDa, 10 kDa) were purchase from Sigma-Aldrich. U18666A and Tumor necrosis factor-α (TNF-α) were purchase from Wako. Antibodies to the following were diluted in 3% BSA and used for Immunofluorescence staining ZO-1 (1:200; BD Bioscience), Alexa Fluor 568 (1:200, Molecular Probes).

Bacteria were harvested by centrifugation at the 3,000 rpm for 15 min in C. jejuni infection or 12000 rpm for 3 min in S. Enteritidis infection and supernatant was removed. The bacteria were washed by phosphate buffered saline (PBS; 137 mM NaCl, 8.1 mM anhydrous

Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4, pH 7.4), centrifuged and resuspended in PBS. The bacteria concentration was adjusted to an optical density of 600 nm (OD₆₀₀) of 1.0 by PBS. Before infection, the culture medium was removed and replaced with fresh DMEM medium without supplements. Cells were infected at a multiplicity of infection (MOI) of 100–200:1 in C. jejuni infection or 10–20:1 in S. Enteritidis infection. Infected cells were incubated at 37°C in 5% CO₂.

INT407 and HeLa cells were seeded at a density of 1.0 × 10⁵/well in 24-well plate and cultured for 3 days. Polarized Caco-2 and T84 cells were seeded at a density of 7.5 × 10⁴/well and 2.5 × 10⁵/well in 24-well plate and cultured for 7 days. For basolateral infection, Caco-2 cells were seeded at a density of 1.0 × 10⁵/well in inverted 0.33 cm² transwell insert containing 3.0 μm pores (Corning). After 6–9 h incubation, the cell seeded inserts were re-inverted into DMEM high glucose and cultured for 1 week. The cell culture medium was replenished every 2 days and cells were used for invasion experiments.

For invasion assay, cells were infected with C. jejuni for 6 h or S. Enteritidis for 1 h. After infection, the cells were incubated with gentamycin containing DMEM (100 or 500 μg/ml for C. jejuni and S. enteritidis, respectively) for 2 h to kill extracellular bacteria. After incubation, the cells were washed with PBS and lysed with 1% Triton-X in PBS for 5 min at 37°C. For adhesion experiments, cells were infected with bacteria at a MOI 20–30:1 for 1 h. After infection, the cells were washed five times with PBS and lysed with 0.1% Triton-X in PBS for 5 min at 37°C. The diluted cell lysates were plated on MH agar plates and incubated for 48 h under a microaerobic atmosphere. The number of intracellular bacteria was determined by counting colony forming units (CFU) and the values were normalized with respect to the protein concentration of the individual cells. Protein concentration was measured by using a BCA Protein Assay Kit (Thermo Fisher) according to the manufacturer's instructions.

To disrupt cellular TJs, differentiated Caco-2 or T-84 cells were incubated with 4 mM EGTA for 20 min or 20 ng/ml TNF-α for 48 h. After treatment, the cells were then washed one time with fresh free DMEM and used for infection.

To disrupt lipid rafts, cells were treated with various concentrations of MβCD (1, 5, 10 mM), which removes cholesterol from the plasma cell membrane, and U18666A (7.5, 15, 30 μM) to block intracellular cholesterol trafficking and biosynthesis for 1 h prior to infection. The concentration of each inhibitor used in this study was determined by the previous publication of Elmi et al. (2012) and Konkel et al. (2013) for MβCD or Chen et al. (1993) and Field et al. (2008). for U18666A. respectively. About U18666A, we used twice higher concentration in this study compare to the previous reports because of the incubation time difference. We checked host cell viability by protein content in each experiment. In cholesterol complement assays, MβCD treated cells were incubated with water-soluble cholesterol (150 μg/ml) containing DMEM for 1 h. After treatment, the cells were then washed one time with fresh free DMEM and used for infection. To evaluate the effect of these treatment on cells, intracellular cholesterol levels were measured with a cholesterol assay kit (Wako) according to the manufacturer's instructions.

Caco-2 cells were seeded on glass cover slips at a density of 3 × 10⁵/dish and were infected for 6 h with bacteria that had been previously incubated with the CFDA SE cell trace kit reagent (Molecular Probes) for 1 h in accordance with the manufacturer's instructions. After infection, the cells were washed with PBS and fixed with 4% paraformaldehyde in PBS at room temperature for 10 min. After washing three times with PBS, the cells were permeabilized with 0.2% Triton-X-PBS for 10 min and then treated with 3% BSA in PBS for 1 h before washing 3 times with PBS. The cells were incubated with primary antibody overnight at 4°C and washed 3 times with PBS before incubation with a secondary antibody conjugated with Alexa Fluor 568 at room temperature for 60 min and washing 3 times with PBS. For F-actin staining, cells were incubated with Rhodamine Phalloidin diluted in PBS (1: 100) at room temperature for 60 min and washing 3 times with PBS.

Caco-2 cells were seeded at a density of 1.0–2.0 × 10⁵/well on 0.33 cm² transwells for TER assessment. TER was measured using an EVOM epithelial Volt-Ohm meter (World Precision Instruments) in accordance with the manufacturer's instructions. For calcium switch assays, Caco-2 cells cultured 1 week were treated with EGTA as described above and were then washed with fresh medium and re-introduced into normal medium containing calcium. Cells were incubated at 37°C for indicated time and TER values was measured and invasion assay was performed.

Caco-2 cells were seeded at a at a density of 7.5 × 10⁴/well and cultured for 1 week in 24-well plates following treatment with EGTA or MβCD as described above. After treatment, cells were washed with HEPES buffer (10 mM HEPES, 145 mM NaCl, 10 mM glucose, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂) and incubated with HEPES buffer containing either 0.2 mg/ml 4 kDa or 10 kDa FITC-dextran for 2 h. After incubation, the cells were washed five times with HEPES buffer and lysed with 1% Triton-X. Cell lysates were centrifuged at the 12,000 rpm for 10 min and were collected supernatant. Fluorescence in supernatant was measured by using fluorescence microplate reader at excitation and emission wavelengths of 492 and 520 nm, respectively.

In all case, statistical analysis was performed by using student's t-test for pair date. Date from 3 independent experiments were evaluated. All tests were one-tailed.

Previous studies indicated that C. jejuni can invade host cells via lipid raft-mediated endocytosis in non-polarized epithelial cells (Wooldridge et al., 1996). Here we found that treatment of the non-polarized cell lines INT407 and HeLa (Figures 1A,B, Supplementary Figure 1) with methyl-β-cyclodextrin (MβCD), a potent lipid raft-mediated endocytosis inhibitor that removes cellular cholesterol, significantly and dose-dependently decreased both cellular cholesterol content and C. jejuni invasion. This reduction could be recovered by cholesterol supplementation (Figures 1C,D, Supplementary Figure 1). To further confirm the contribution of lipid rafts to C. jejuni invasion, we analyzed the effect of U18666A, an inhibitor of intracellular cholesterol trafficking, on C. jejuni invasion. U18666A treatment of INT407 cells significantly decreased C. jejuni invasion without affecting the cholesterol content (Figures 1E,F). Together these results indicated that cell surface cholesterol and lipid rafts are essential for C. jejuni invasion of non-polarized epithelial cells. Next, we investigated the dynamics of C. jejuni invasion in polarized epithelial cells using Caco-2 and T-84 cells. In contrast to non-polarized epithelial cells, MβCD treatment of polarized Caco-2 and T-84 cells did not affect C. jejuni invasion, despite the reduction in intracellular cholesterol content (Figures 1G,H, Supplementary Figure 1), suggesting that the dynamics of C. jejuni invasion might differ between non-polarized and polarized epithelial cells.

To investigate differences in C. jejuni invasion dynamics between non-polarized and polarized epithelial cells, we focused on tight junctions (TJs) formation in polarized epithelial cells. Cell polarization is established by TJ formation that separates the plasma membrane into apical and basolateral regions. Therefore, we performed invasion assays at different stages of cell culture to evaluate the relationship between TJ formation and C. jejuni invasion process. TJ formation of Caco-2 cells was assessed by measuring Trans Epithelial Resistance (TER) as previously described (Goyer et al., 2016) and this result showed significantly increase of TER values on day 6 post-seeding (Figure 2A), suggesting that mature TJs are present at this time point. As the TER values increased, the amount of C. jejuni invasion decreased. Interestingly, MβCD treatment significantly decreased C. jejuni invasion only in the short term (day 2 or 4 post-seeding) in cultured Caco-2 cells (Figure 2B). Similar results were seen for Caco-2 cells in a fluorescence microscopy assay using a CFDA SE cell tracer kit (Figures 2C–F). Based on these data, we hypothesized that the lateral or basolateral part of cells may contribute to C. jejuni invasion dynamics and C. jejuni susceptibility. Thus, we next performed an invasion assay using inverted-transwell inserts (Figure 2G). C. jejuni invasion was promoted in the basolateral region of cells, and MβCD treatment significantly reduced C. jejuni invasion only in the basolateral region (Figure 2H). These results suggested that basolateral region was critical for bacterial invasion in TJ formed cells, and that TJs provide the interface for the interaction between lipid rafts and invading C. jejuni.

To further assess the role of TJs during C. jejuni invasion of polarized epithelial cells, invasion assays were performed using the Ca²⁺ chelator EGTA to disrupt TJs. EGTA treatment was associated with lower TER values and altered localization of the TJ marker protein ZO-1 (Supplementary Figure 2). EGTA treatment promoted C. jejuni invasion, which was significantly decreased by MβCD treatment only in cells with disrupted TJs. (Figure 3A). Similar results were observed for T-84 cells and Caco-2 cells following U18666A treatment (Supplementary Figure 2). To determine whether MβCD-mediated suppression of bacterial invasion in the presence of disrupted TJs was specific to C. jejuni, we also evaluated invasion of another human enteric pathogen, S. Enteritidis in polarized cells with disrupted TJs. In contrast to C. jejuni infection, EGTA treatment did not promote S. Enteritidis invasion and MβCD treatment did not decrease S. Enteritidis invasion in polarized cells with disrupted TJs (Figure 3B). Furthermore, we analyzed how EGTA or MβCD treatment affected endocytosis function by measuring uptake of FITC-dextran. EGTA or MβCD treatment had different effects on FITC-dextran uptake than that for C. jejuni invasion (Figure 3C). These results suggested that C. jejuni invasion has greater dependence on intact TJs in polarized cells than endocytosis.

Earlier studies reported that Ca²⁺ reintroduction recovered TER values and increased TJ integrity in EGTA-treated cells (Farshori and Kachar, 1999; Ronaghan et al., 2016). Therefore, we performed a calcium switch assay to examine the effect of TJ integrity on C. jejuni invasion. Consistent with earlier reports, we showed that Ca²⁺ reintroduction into Caco-2 cells restored the TER value (Figure 4A) and ZO-1 localization (Figures 4B–F) in a time-dependent manner. Furthermore, lipid raft-mediated C. jejuni invasion was significantly decreased following TJ restoration (Figure 4G). These data strongly suggested that cellular lateral or basolateral cell regions that are normally obscured by TJs are crucial for lipid raft-mediated C. jejuni invasion in polarized epithelial cells.

Campylobacter jejuni invasion proceeds in two steps: adhesion and invasion. However, which step TJs affect is unclear. Here we first checked whether EGTA and MβCD affected C. jejuni adhesion in non-polarized INT407 cells and polarized Caco-2 cells and found no effect on adhesion by the respective compounds (Figures 5A,B). As mentioned above, during the C. jejuni invasion step, MβCD significantly decreased C. jejuni invasion of polarized cells only in EGTA-treated cells (Figure 3A). To further confirm the interaction between C. jejuni invasion and TJs, we used invasion-defective C. jejuni strains. A C. jejuni deletion mutant lacking the autotransporter protein CapA (Ashgar et al., 2007) had significantly lower invasion into host cells, particularly non-polarized cells (Figure 5C). In contrast to WT C. jejuni, invasion of the CapA mutant did not increase after EGTA treatment of Caco-2 cells to disrupt TJs (Figure 5D). These results suggested that TJs do not affect bacterial adhesion, but instead are involved in the invasion step. Moreover, an invasive factor such as CapA might have an important role in C. jejuni invasion dynamics mediated by TJs in polarized cells.

Several previous studies reported that active inflammation disrupts TJ formation and promotes barrier disruption in the intestine (Antoni et al., 2014; Lechuga and Ivanov, 2017) and proinflammatory cytokine, Tumor necrosis factor-α (TNF-α) play an important role in the intestinal barrier disruption (Ma et al., 2004). To investigate the influence of inflammation-mediated TJ disruption in C. jejuni invasion, we performed an invasion assay with polarized Caco-2 cells treated with TNF-α. Treatment of TNF-α significantly decreased TER value (Figure 6A) and induced ZO-1 localization change Figures 6B,C. Additionally, C. jejuni invasion was significantly increased and attenuated followed by MβCD treatment in TNF-α-treated polarized epithelial cells as measured by a gentamycin protection assay (Figure 6D). This result indicated that intestinal inflammation can induce barrier disruption and promote C. jejuni invasion in polarized epithelial cells.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.