Large numbers (P < 0.01) of microaerobic bacteria were isolated from the MLNs, liver, and spleen of C. jejuni-treated mice (3.08 ± 0.46, 2.03 ± 0.77, 3.12 ± 0.69 log₁₀ CFU/g, respectively), compared to control mice (0, 0, 0.43 ± 0.43 log₁₀ CFU/g, respectively). Bacteria were identified as Proteus, Acinetobacter, and Pseudomonas. C. jejuni were also isolated from the MLNs, liver, and spleen of all C. jejuni-treated mice (8/8) but not from uninfected controls (P < 0.001). Amp^R E. coli were isolated from the MLNs of 3/8 and the spleen of 1/8 C. jejuni-treated mice, but were not isolated from the liver of C. jejuni-treated mice, nor the MLNs, liver, or spleen of control mice.

E. coli translocation was increased (P = 0.03) in C. jejuni-treated monolayers (2.41 ± 0.23 log₁₀ CFU/ml), compared to controls (1.34 ± 0.17 log₁₀ CFU/ml). E. coli internalization was also increased (P = 0.04) in C. jejuni-infected monolayers (2.09 ± 0.15 log₁₀ CFU/ml), compared to controls (2.83 ± 0.04 log₁₀ CFU/ml). Permeability was not different (P = 0.75) between treatments (0.035 ± 0.011% versus 0.031 ± 0.009% apical FITC-dextran/cm²/h for control and C. jejuni-treated monolayers, respectively). E. coli were visualized within membrane-bound vacuoles within the cytoplasm of C. jejuni-treated monolayers (Figure 1), but were not observed in controls (not shown).

Treatment with MβCD or soluble cholesterol effectively decreased (P < 0.001) or increased (P < 0.001) the cholesterol of content of monolayers (9.68 ± 0.59, 1.78 ± 0.23, 24.18 ± 1.73 μg cholesterol/mg protein for untreated, MβCD-treated, and cholesterol-treated monolayers, respectively).

As observed previously, infection with C. jejuni significantly increased E. coli internalization (P = 0.03). In C. jejuni-treated monolayers, treatment with the lipid raft disruptor MβCD inhibited E. coli internalization (P = 0.04) and translocation (P = 0.04; Figure 2A and 2B). Internalization and translocation of E. coli were not different (P = 0.60 and P = 0.64, respectively), between C. jejuni/MβCD-treated and untreated monolayers. In C. jejuni-treated monolayers, there was increased internalization (P = 0.03) but not translocation (P = 0.08) of E. coli in monolayers that were treated with soluble cholesterol to augment plasma membrane cholesterol (Figure 2C and 2D). Internalization of E. coli in untreated and uninfected-cholesterol-treated monolayers was not different (P = 0.07). Cholesterol depletion and augmentation did not affect the invasion or translocation C. jejuni (results not shown).

Lipid rafts were isolated based on their buoyancy, by sucrose gradient fractionation (Figure 3), and were present in fractions 2 and 3 (as indicated by the presence of the lipid raft marker, caveolin-1). Fluorescent E. coli detected in fractions 2 and 3 represent lipid raft-associated E. coli, whereas the fluorescence in fractions 6–8 represents extracellular E. coli. Infection with C. jejuni caused E. coli to associate with lipid rafts. This effect was abolished by cholesterol depletion with MβCD.

Epifluorescent microscopy was used to visualize the association of E. coli with cholesterol and caveolin-1 (Figure 4). Accumulations of cholesterol were observed in C. jejuni-treated monolayers (Figure 4A), wherein E. coli were co-localized (Figure 4A, B). E coli co-localized with caveolin-1 in C. jejuni-treated monolayers (Figure 4C). Accumulation of cholesterol and co-localization of E. coli with cholesterol or caveolin-1 were not observed in control monolayers (not shown).

C. jejuni strains with various degrees of invasiveness were used to assess whether E. coli translocation induced by the bacterium corresponded with C. jejuni invasion. C. jejuni 81–176 was more invasive (P < 0.001) than C. jejuni CHR213 and C. fetus CHR105, as assessed by in vitro bacterial internalization assay (results not shown). Translocation of C. jejuni 81–176 (P = 0.003) and C. jejuni CHR213 (P = 0.02) was greater than that of C. fetus CHR105 (Figure 5A, black bars). Translocation of C. jejuni 81–176 and C. jejuni CHR213 did not differ (P = 0.45). All three Campylobacter strains, regardless of their degree of invasiveness, significantly promoted (P ≤ 0.04) E. coli translocation (Figure 5A, white bars); there was no difference (P ≥ 0.39) in the amount of E. coli translocation induced among the three Campylobacter strains. None of the Campylobacter strains caused changes in transepithelial electrical resistance (TER) during the 4 h experimental period (final TER was 97.7 ± 4.1%, 98.3 ± 6.8%, 94.0 ± 1.5%, and 85.6 ± 4.1% of the pre-treatment TER for control, C. jejuni 81–176, C. jejuni 213, and C. fetus CHR 105 – treated monolayers, respectively; P ≥ 0.18).

Since the presence of a functional flagellum is required for C. jejuni invasion, studies assessed the ability of flagella defective mutants to cause E. coli translocation. Isogenic FlaAFlaB mutants of C. jejuni 81–176 and CHR213 were less invasive than the wild-type (results not shown). Similarly, translocation of the isogenic FlaAFlaB mutants of C. jejuni 81–176 and CHR213 was less (P ≤ 0.004) than that of the wild-type (Figure 5B). Translocation of E. coli across the C. jejuni-treated monolayers did not differ (P ≥ 0.13) between monolayers treated with the FlaAFlaB mutants or the wild-type C. jejuni strains (Figure 5B). The final TER did not differ between treatments (results not shown). Internalization of E. coli was greater in monolayers treated with live C. jejuni 81–176 (P < 0.001) and conditioned cell culture media (P < 0.001), but not with paraformaldehyde-killed C. jejuni (Figure 6, P = 0.32). Similarly, E. coli translocation was greater in monolayers treated with live C. jejuni 81–176 (P = 0.005) and conditioned cell culture medium (P = 0.002), but not paraformaldehyde-killed C. jejuni (P = 0.77).

C. jejuni 81–176, a reference clinical strain 35, was used throughout this study. C. jejuni CHR213 and C. fetus CHR105 are clinical isolates that were characterized as previously described 36. FlaAFlaB mutants of C. jejuni 81–176 and CHR213 were constructed and characterized as described previously 36. Intestinal colonization of mice was reduced for the C. jejuni 81–176 FlaAFlaB mutant (results not shown), confirming previous observations 2122.

Inoculum was prepared by growing Campylobacter for 14–16 h in CYE broth (37°C, 100 rpm, microaerobic atmosphere) 37. E. coli HB101 was grown for 14–16 h in Columbia broth (37°C, 100 rpm; Difco, Detroit, MI).

E. coli were washed with NaHCO₃ buffer (0.1 M, pH 8.3), and incubated for 45 min in Alexa-488 carboxylic acid succinimidyl ester (0.5 mg/ml NaHCO₃ buffer; Molecular Probes, Eugene, OR). E. coli were then washed and suspended (≈10⁹ CFU/ml) in PBS containing 2% NaHCO₃ (mouse study) or antibiotic-free Dulbecco's modified Eagle medium (DMEM)/F-12 (T84 studies). Labelling did not affect bacterial viability (results not shown).

Mice (Balb/c; Charles River, Montreal, QC) were housed at Agriculture and Agri-Food Canada, Lethbridge (Alberta) under the guidelines established by the Canadian Council on Animal Care. Procedures were approved by institutional Animal Care and Biosafety Committee's. Five-week-old female mice were inoculated with C. jejuni 81–176 (10⁸ CFU in CYE containing 2% NaHCO₃) or sterile CYE (controls) by gavage on days one and two. To examine translocation of a model commensal intestinal bacterium, all mice were inoculated on day three with fluorescent non-invasive E. coli (10⁸ CFU) transformed with an ampicillin resistant (Amp^R) plasmid (pGEM-T Easy, Promega, Madison, WI). Four hours later, mice were euthanized and the MLNs, and sections of liver, spleen, and ileum were aseptically removed. Fecal pellets were also collected prior to euthanasia. Tissues and feces were weighed and homogenized in PBS. C. jejuni, microaerobic bacteria, and E. coli were enumerated by spreading serial dilutions of fecal or tissue homogenates onto Karmali agar containing selective supplement SR167 (Oxoid, Nepean, ON), non-selective Karmali agar, and MacConkey agar containing ampicillin (100 μg/ml; Difco), respectively, and incubating media at 37°C in a microaerobic (C. jejuni, microaerobic bacteria) or ambient (E. coli) atmosphere. Isolated bacteria were identified by PCR and comparative 16S rRNA sequence analysis.

T84 human colonic epithelial cells (passages 5 to 15; American Type Culture Collection, Manassas, VA) were grown in DMEM/F-12 plus 10% foetal bovine serum, 200 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 80 μg/ml tylosin, and incubated at 37°C and 5% CO₂. Medium was replenished every 2 to 3 days. For translocation studies, cells were seeded onto Transwell filters at 4 × 10⁵ cells/filter (5 μm pore size, 1.13 cm²; Costar, Corning Inc. Corning, NY). Transepithelial electrical resistance (TER) was monitored with an electrovoltohmeter (World Precision Instruments, Sarasota, FL), and monolayers were used at confluence (TER > 1000 Ω/cm²). For microscopy, cells were seeded into Lab-Tek chamber slides at 8 × 10⁴ cells/well (Nalgene Nunc International, Naperville, IL).

Monolayers were washed with PBS and antibiotic-free DMEM/F12 was added to the apical and basal compartments. Monolayers were inoculated apically with E. coli ± Campylobacter or to achieve a MOI of 100 CFU/enterocyte for each bacterial species. Control monolayers received an equivalent volume of CYE. Following 4 h incubation in microaerobic conditions, E. coli and Campylobacter recovered in the basal compartment medium were enumerated by spreading serial dilutions onto non-selective Karmali agar and incubating microaerobically at 37°C. These conditions are suitable for the growth of E. coli and Campylobacter. T84 cells maintain TER > 1000 Ω/cm² for > 24 in microaerobic atmosphere (data not shown).

To assess E. coli internalization, monolayers washed with PBS and incubated for 1 h with DMEM/F12 containing gentamicin (250 μg/ml; Sigma-Aldrich, Oakville, ON) 10. Monolayers were washed, lysed with 0.1% Triton X-100/PBS, and viable bacteria were enumerated. A preliminary experiment confirmed that E. coli were killed by the gentamicin treatment. Campylobacter internalization was determined as for E. coli, except monolayers were incubated for 3 h with DMEM/F12 containing gentamicin (500 μg/ml), as described previously 36.

For some experiments, apical compartments were inoculated with E. coli ± paraformaldehyde-fixed C. jejuni 81–176 or apical medium was replaced with conditioned DMEM/F12. Paraformaldehyde-fixed C. jejuni were prepared by incubating bacteria for 2 h in 2% paraformaldehyde. Cells were washed and suspended in antibiotic-free DMEM/F12 (≈10⁹ CFU/ml). To prepare conditioned medium, T84 monolayers (75 cm² flask) were washed with PBS, and antibiotic-free DMEM/F12 was added to the flask. Cells were inoculated with C. jejuni 81–176 (MOI 100) and incubated microaerobically overnight (37°C). Media was clarified by centrifugation, followed by filtration through a 0.2 μM syringe filter. Sterility was confirmed by viable counting.

Following the infection period, monolayers were washed with sterile Ringer's solution. A 3 kDa FITC-dextran probe (500 μl, 100 mM in Ringer's solution; Molecular Probes) was added to the apical compartment, and 1 ml of Ringer's solution added to the basal compartment and incubated for 3 h at 37°C, as describe previously 38. Samples were collected from the basal compartment and absorbance₄₈₅ was measured. Data were expressed as % apical dextran/cm²/h.

Disruption of lipid rafts was performed by membrane cholesterol depletion as validated previously. Monolayers received MβCD (50 mM or 10 mM; Sigma) plus lovastatin (1 μM; Sigma) 30 min prior to inoculation. Conversely, plasma membrane cholesterol was increased by incubating monolayers for 4 h with soluble βCD-complexed cholesterol (100 μM; Sigma) and washing with PBS prior to inoculation. These treatments did not affect viability of T84 cells, C. jejuni, or E. coli (results not shown). Plasma membrane cholesterol was measured using an Amplex red cholesterol assay kit (Molecular Probes). Total protein was measured using a Bradford protein assay (Bio-Rad Laboratories, Hercules, CA).

T84 apical membranes were fluorescently labelled by washing monolayers with Hank's buffered saline (HBSS) and incubating with Alexa-546 carboxylic acid succinimidyl ester (0.5 mg/ml HBSS, 1 h, 4°C, Molecular Probes). Monolayers were washed and inoculated with Alexa488-labelled E. coli ± C. jejuni, as described for the translocation assay. After incubation, slides were washed with PBS, and fixed in paraformaldehyde (2%). Optical sectioning was carried out by confocal laser microscopy (Leica Microsystems GmbH, Wetzler, Germany). Digitized images were analyzed with Imaris software (Bitplane AG, Zurich, Switzerland).

To visualize cholesterol-rich domains, T84 cells were stained with the fluorescent sterol-binding drug, filipin (0.5 mM in PBS; Sigma), for 30 min 39. To stain for caveolin-1, slides were rinsed with PBS, incubated with glycine (1% in PBS) for 15 min, and rinsed with PBS. Cells were permeabilized for 10 min with Triton X-100 (0.5% in PBS), blocked with BSA (2% in PBS), incubated with mouse anti-caveolin-1 antibodies (1/100 dilution in PBS; BD Bioscience, San Diego, CA) followed by Alexa-488 conjugated anti-mouse IgG (1/100 dilution in PBS; Molecular Probes).

Another set of experiments characterized the association of translocating E. coli with lipid rafts. Monolayers were inoculated with fluorescent E. coli ± C. jejuni as described for the translocation assay. Lipid rafts were isolated by sucrose gradient fractionation, as previously described 3940. For each of eight fractions that were collected, fluorescent E. coli were quantified by measuring absorbance₄₈₅, and the lipid raft marker, caveolin-1, was detected by western blot analysis using mouse anti-caveolin-1 antibodies (1/1000 dilution; BD Bioscience), and HRP-conjugated anti-mouse IgG antibodies (1/5000 dilution; Sigma) 39.

Experiments were conducted ≥ three times independently. Assays were conducted at least in triplicate, and mean values were used for analysis. Analyses were performed with GraphPad InStat software (GraphPad Software Inc., San Diego, CA). Data are expressed as means ± SEM. Data with ≥ three treatments were compared by one way ANOVA, followed by the protected Tukey-Kramer multiple comparison test. Data with two treatments were compared using an unpaired Student's t-test. Translocation incidences in mice were compared using the Fisher's exact test. P ≤ 0.05 was considered significant.