Cellular Microbiology: Pathogen-Host Cell Molecular Interactions

Cystic Fibrosis Transmembrane Conductance Regulator Reduces Microtubule-Dependent Campylobacter jejuni Invasion

Junko Kido, Takaaki Shimohata, Sachie Amano, Sho Hatayama, Anh Quoc Nguyen, Yuri Sato, Yuna Kanda, Aya Tentaku, Shiho Fukushima, Mutsumi Nakahashi, Takashi Uebanso, Kazuaki Mawatari, Akira Takahashi
Manuela Raffatellu, Editor
DOI: 10.1128/IAI.00311-17

ABSTRACT

Campylobacter jejuni is a foodborne pathogen that induces gastroenteritis. Invasion and adhesion are essential in the process of C. jejuni infection leading to gastroenteritis. The mucosal layer plays a key role in the system of defense against efficient invasion and adhesion by bacteria, which is modulated by several ion channels and transporters mediated by water flux in the intestine. The cystic fibrosis transmembrane conductance regulator (CFTR) plays the main role in water flux in the intestine, and it is closely associated with bacterial clearance. We previously reported that C. jejuni infection suppresses CFTR channel activity in intestinal epithelial cells; however, the mechanism and importance of this suppression are unclear. This study sought to elucidate the role of CFTR in C. jejuni infection. Using HEK293 cells that stably express wild-type and mutated CFTR, we found that CFTR attenuated C. jejuni invasion and that it was not involved in bacterial adhesion or intracellular survival but was associated with microtubule-dependent intracellular transport. Moreover, we revealed that CFTR attenuated the function of the microtubule motor protein, which caused inhibition of C. jejuni invasion, but did not affect microtubule stability. Meanwhile, the CFTR mutant G551D-CFTR, which had defects in channel activity, suppressed C. jejuni invasion, whereas the ΔF508-CFTR mutant, which had defects in maturation, did not suppress C. jejuni invasion, suggesting that CFTR suppression of C. jejuni invasion is related to CFTR maturation but not channel activity. When these findings are taken together, it may be seen that mature CFTR inhibits C. jejuni invasion by regulating microtubule-mediated pathways. We suggest that CFTR plays a critical role in cellular defenses against C. jejuni invasion and that suppression of CFTR may be an initial step in promoting cell invasion during C. jejuni infection.

INTRODUCTION

Campylobacter jejuni is a spiral Gram-negative bacterium that is commonly found in the gut microflora of birds or domestic animals used for food. C. jejuni is the most common cause of bacterial foodborne illness worldwide and causes gastrointestinal symptoms, such as diarrhea, fever, abdominal cramping, and gastroenteritis. Despite its frequency, the virulence factors that contribute to C. jejuni-induced gastroenteritis remain largely unknown. Genomic studies revealed that C. jejuni strains lack the type III secretion systems that are essential for the virulence of many other Gram-negative enteric pathogens (1). Thus, C. jejuni pathogenesis likely involves multifactorial virulence processes, including motility, adherence, invasion, and intracellular survival (2, 3). Indeed, a study that examined mutant C. jejuni strains that were defective in adhesion or invasion found that cultured cells infected with these strains secreted decreased amounts of the proinflammatory cytokine interleukin-8 (4, 5), which suggests that adhesion and invasion are the main pathogenic processes in C. jejuni infection (6–9).

In the human gut, the mucosal layer represents the first line of defense against bacterial adhesion and invasion (10). The binding of intestinal bacteria to host epithelial cells is assumed to play a fundamental role in intestinal bacterial colonization and disease progression (11). A previous study reported that mutant mice with a defective mucosal function had a high rate of colonization with C. jejuni in C. jejuni inoculation models (12). The mucosal layer consists of mucins, which are high-molecular-mass oligomeric glycoproteins. This layer is also critical for maintenance of gut homeostasis as it regulates water flux through the activity of several ion transporters and ion channels in the intestine (13). Among these ion channels, the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP-activated chloride channel, is one of the most important factors that govern water movement. CFTR is expressed in several tissues, such as tissues of the lung, pancreas, liver, intestine, sweat duct, and reproductive system (14, 15). In the intestinal tract, CFTR is associated with intestinal tract hydration and the clearance of intestinal contents, including bacteria (16).

CFTR activation can disrupt the water balance in the gut, leading to diarrhea in association with intestinal infections caused by different pathogens, such as Escherichia coli (17) and Vibrio cholerae (18), as well as other infectious bacteria (19). However, we previously reported that C. jejuni infection suppresses CFTR-mediated Cl− secretion in intestinal cells (20), which is a state opposite that associated with the clinical symptoms of C. jejuni infection, such as diarrhea. Thus, the relationship between CFTR suppression and C. jejuni infection is unclear.

In order to investigate the relationship between CFTR suppression and C. jejuni infection, we previously evaluated respiratory infections that occur in the presence of CFTR mutations that are also associated with cystic fibrosis (CF) (21). CF patients frequently experience respiratory bacterial infections caused by Pseudomonas aeruginosa or Burkholderia cenocepacia (22). CFTR dysfunction disrupts CFTR-mediated water flux, which in turn affects the function of the mucosal layer and attenuates bacterial clearance, such that bacteria accumulate in the respiratory tract. Epithelial cells in CF patients have increased cell surface expression of Toll-like receptors (Toll-like receptor 5 [TLR5] and TLR2), although the contribution of TLR2 has been questioned, and also have enhanced inflammatory responses (23). Moreover, P. aeruginosa is reported to suppress CFTR channel activity by secreting proteins that promote severe inflammation (24, 25). These reports indicated that CFTR plays a defensive role against respiratory infection, and a dysfunctional CFTR may promote conditions that increase the likelihood of bacterial infection.

In this study, we studied the role of CFTR in C. jejuni infection. Our data show that CFTR expression decreases the level of C. jejuni invasion. In particular, CFTR expression inhibited microtubule-mediated transport processes during C. jejuni invasion, suggesting that CFTR might be a factor protective against C. jejuni infection.

RESULTS

Exogenous CFTR expressed by cells is useful for analysis of the effect of CFTR on C. jejuni infection.In our previous study, CFTR channel activity in T-84 cells, which are cells that express CFTR endogenously, was suppressed by C. jejuni infection (Fig. 1A). To examine whether C. jejuni infection affects exogenous CFTR, we established wild-type (wt) CFTR-expressing HEK293 cells (HEK-wt-CFTR cells). CFTR expression and channel activity were confirmed by Western blotting (see Fig. S1A in the supplemental material) and an efflux assay (Fig. S1B), respectively. Interestingly, in contrast to the findings for T-84 cells, CFTR ion channel activity was not suppressed by C. jejuni infection in HEK-wt-CFTR cells (Fig. 1B). Together, these results suggest that C. jejuni infection did not directly affect CFTR function. Thus, we used HEK-wt-CFTR cells in this study to assess the contribution of CFTR to C. jejuni infection.

FIG 1
FIG 1

Change of intracellular C. jejuni numbers in the presence of CFTR expression. The rates of Cl− efflux from C. jejuni (Cj)-infected T-84 cells (n = 3) (A) and HEK-wt-CFTR cells (n = 3) (B) were estimated after 12 h of infection. After stimulation, 125I− was incorporated into the cells for 6 min and the levels of 125I− were assessed using a gamma counter. (C) HEK293 cells and HEK-wt-CFTR cells were infected with C. jejuni for 6 h. The number of intracellular bacteria was estimated by the gentamicin protection assay (n = 4). (D) HEK-wt-CFTR cells were transfected with the pLKO-CFTR vector or the pLKO.1-puro nontarget vector expressing control shRNA, and the level of CFTR protein expression was detected by Western blotting. (E) HEK-wt-CFTR cells transfected with the vector expressing shControl and HEK-wt-CFTR cells transfected with the vector expressing shCFTR were infected with C. jejuni for 6 h, and the numbers of intracellular bacteria were estimated by the gentamicin protection assay (n = 4). All data are means ± standard deviations (SDs) from 3 independent experiments. Statistical significance is indicated as follows: **, P < 0.01; NS, not significant.

The numbers of intracellular C. jejuni bacteria are decreased by CFTR expression.Adhesion and invasion processes are thought to be essential for C. jejuni virulence and infection (26), and the decreased levels of CFTR expression induced by C. jejuni may provide increased opportunities for bacterial adhesion or invasion. To test whether CFTR expression levels were related to C. jejuni invasion, we investigated the numbers of intracellular bacteria in CFTR-expressing cells infected with C. jejuni. Relative to HEK293 cells, HEK-wt-CFTR cells infected with C. jejuni for 6 h showed reduced levels of C. jejuni invasion (Fig. 1C). Next, HEK-wt-CFTR cells were transfected with a CFTR knockdown vector (pLKO-CFTR) expressing short hairpin RNA (shRNA) specific for CFTR (shCFTR). Cells transfected with the pLKO.1-puro vector expressing nontarget control shRNA (shControl) were used as controls, and the knockdown efficiency was confirmed by Western blotting (Fig. 1D). CFTR knockdown increased the numbers of intracellular bacteria, as assessed by a gentamicin protection assay (Fig. 1E). The results indicated that C. jejuni invasion was prevented by CFTR expression. Another C. jejuni strain, 81-176, which was isolated from a patient during an outbreak of Campylobacter enteritis (27), gave the same results as the NCTC11168 strain (Fig. S2A and B). Therefore, CFTR attenuated invasion by another C. jejuni strain.

CFTR attenuates microtubule-mediated C. jejuni invasion processes.Since the number of intracellular bacterial cells in host cells can be influenced at several steps, including the adhesion, invasion, and survival stages, we next assessed the relationship between CFTR and C. jejuni in the context of these processes. There were no differences in C. jejuni cell numbers, adhesion, and survival between control cells and CFTR-expressing cells (Fig. 2A and C). In contrast, C. jejuni invasion was decreased by CFTR expression during the early stage of infection (Fig. 2B). Thus, we focused on C. jejuni invasion pathways, which have two main parts: the endocytosis-like uptake pathway (26) and the microtubule-dependent intracellular transport pathway (28). Endocytosis-like uptake pathways include those involving lipid rafts or clathrin-mediated processes (29). Lipid raft domains are rich in cholesterol, which promotes bacterial adhesion and is involved in the production of bacterium-containing vacuoles for cellular uptake (30). Meanwhile, the scaffold protein clathrin binds to adaptor proteins to generate clathrin-coated vesicles and promote endocytosis (31). Methyl-β-cyclodextrin (mβ-cd) can affect lipid raft components, such as cholesterol, to inhibit lipid raft-mediated endocytosis (32). Chlorpromazine prevents clathrin formation at the cell membrane to decrease clathrin-mediated endocytosis activity (33). Here, treatment of cells with either mβ-cd or chlorpromazine dramatically decreased the extent of C. jejuni invasion of control cells. However, the CFTR-mediated suppression of C. jejuni invasion was sustained (Fig. 3A and B). These data indicate that the effect of CFTR on C. jejuni invasion is not related to the activity of endocytosis pathways.

FIG 2
FIG 2

CFTR affects C. jejuni invasion during the early phase of infection but does not affect bacterial adhesion or cellular survival. HEK-wt-CFTR cells were transfected with the vector expressing shControl or shCFTR. The cells were then infected with C. jejuni and bacterial adhesion (n = 6) (A), invasion (n = 6) (B), and intracellular survival (n = 4) (C) were assessed after 0, 1, and 3 h. All data are means ± SDs from 3 independent experiments. Statistical significance is indicated as follows: **, P < 0.01; NS, not significant.

FIG 3
FIG 3

CFTR affects microtubule tracts in C. jejuni invasion. HEK293 cells and HEK-wt-CFTR cells were treated with methyl-β-cyclodextrin (mβ-cd; 7.5 mM) (n = 4) (A), chlorpromazine (47 μM) (n = 4) (B), or nocodazole (33 μM) (n = 4) or colchicine (10 μM) (n = 4) (C) for 1 h and infected with C. jejuni for 3 h. In all experiments, the number of intracellular bacteria was estimated by the gentamicin protection assay. All data are means ± SDs from 3 independent experiments. Statistical significance is indicated as follows: **, P < 0.01; *, P < 0.05; NS, not significant.

Microtubules are dynamic structures that undergo the polymerization and depolymerization of their tubulin components to regulate both the integrity of the cell skeleton and cell transport (34). After entry into host cells, C. jejuni invasion proceeds along microtubules, which act as intracellular tracts (28). Here, treatment with the microtubule polymerization inhibitor nocodazole or colchicine (35, 36) suppressed C. jejuni invasion, but there was no difference in the number of intracellular bacteria between control cells and CFTR-expressing cells (Fig. 3C). These data imply that CFTR negatively affects the intracellular transport of C. jejuni via the microtubule tract, which in turn inhibits invasion.

CFTR expression affects the microtubule motor protein.Microtubule polymerization promotes bacterial uptake and vacuole motility (37). For C. jejuni, microtubules provide a means for invasion (28). To investigate the participation of CFTR in microtubule-mediated C. jejuni invasion, we focused on the microtubule tract and motor protein.

First, we investigated the effect of CFTR on the microtubule tract. C. jejuni infection did not change the amount of polymerized microtubules (Fig. S3A and B). The amount of polymerized microtubules in CFTR-expressing cells was estimated by Western blotting (Fig. 4A), but there were no differences between HEK293 and HEK-wt-CFTR cells. We confirmed the stability of the microtubules in CFTR-expressing cells by using the microtubule depolymerization inhibitor paclitaxel. Treatment with paclitaxel increased the level of microtubule polymerization in CFTR-expressing cells (Fig. 4B). Also, according to the level of microtubule polymerization, the number of intracellular bacteria in HEK293 cells was increased by paclitaxel treatment (Fig. 4C). However, in HEK-wt-CFTR cells, the number of intracellular bacteria did not increase significantly and the amount of polymerized microtubules was similar to that in HEK293 cells. These data indicate that CFTR does not affect the stability of the microtubule tract in microtubule-mediated transport.

FIG 4
FIG 4

CFTR expression attenuates the effect of the microtubule motor protein in C. jejuni invasion. (A) The polymerized tubulin content in HEK293 and HEK-wt-CFTR cells was assessed by Western blotting after treatment with paclitaxel (5 or 10 μM) or the DMSO vehicle for 1 h. (B) The changes in the levels of microtubule polymerization were quantified by measurement of the amount of polymerized α-tubulin and normalized with respect to the amount of β-actin that was used as a loading control (n = 4). (C) HEK293 and HEK-wt-CFTR cells were treated with paclitaxel (5 or 10 μM) or the DMSO vehicle for 1 h prior to C. jejuni infection. Intracellular bacterial numbers were assessed by the gentamicin protection assay (n = 4). (D) HEK-wt-CFTR cells transfected with the vector expressing shControl or shCFTR were treated with Na3VO4 (1 or 2 mM) for 1 h prior to C. jejuni infection. Intracellular bacterial numbers were assessed by the gentamicin protection assay (n = 4). All data are means ± SDs from 3 independent experiments. Statistical significance is indicated as follows: NS, not significant.

Next, we evaluated the effect of CFTR on the microtubule motor protein dynein. A previous study reported that the dynein inhibitor Na3O4 decreased the level of C. jejuni invasion via microtubule-mediated transport (38). Thus, we treated CFTR-expressing cells with the dynein inhibitor Na3VO4 to stop the delivery of dynein to the microtubule tract. The numbers of intracellular bacteria decreased according to the Na3VO4 content, and the differences in bacterial numbers between HEK-wt-CFTR cells transfected with the vector expressing shControl and HEK-wt-CFTR cells transfected with the vector expressing shCFTR were abolished by Na3VO4 treatment (Fig. 4D). The result for another strain, C. jejuni 81-176, was similar to that for strain NCTC11168 (Fig. S4).

These results suggest that CFTR suppresses the microtubule motor protein, which interferes with microtubule-mediated C. jejuni invasion.

CFTR maturation is essential for inhibition of C. jejuni invasion.The cell surface localization and appropriate folding are necessary for CFTR channel activity (14, 15). To examine how CFTR inhibits C. jejuni invasion, we established cell lines that stably expressed two different CFTR mutations. The G551D-CFTR mutant had a substitution of aspartic acid for glycine at amino acid position 551. This mutant has appropriate folding and trafficking to the cell surface membrane but does not have channel activity (39). Meanwhile, the ΔF508-CFTR mutant has a single deletion of a phenylalanine residue at position 508. This mutant does not have appropriate glycosylation, folding, or trafficking to the cell surface membrane (40). The expression of the mutant CFTR proteins was confirmed by Western blotting (Fig. S5A). Band C, indicative of the mature complex-glycosylated form of CFTR, was detected for both cells expressing wt CFTR and cells expressing the G551D-CFTR mutant. Bands A and B indicate the nonglycosylated and immature core-glycosylated forms of CFTR, respectively (41). Both bands A and B were detected for cells expressing the ΔF508-CFTR mutant. The ion channel activity, measured by efflux assay, showed that active ion channels were detected only for cells expressing wt CFTR (Fig. S5B). Cells expressing the G551D-CFTR mutant (HEK-G551D-CFTR cells) inhibited C. jejuni invasion, a finding similar to that for cells expressing wt CFTR (Fig. 5A). Cells expressing the G551D-CFTR knockdown mutant also had increases in intracellular bacterial cell numbers (Fig. 5B and C). These results indicate that the suppression of C. jejuni invasion by CFTR expression is not associated with CFTR ion channel activity. In contrast, expression of the ΔF508-CFTR mutant did not suppress C. jejuni invasion (Fig. 5A). A previous study showed that defects in folding and trafficking of the ΔF508-CFTR mutant could be rescued by treatment of cells with pharmacological folding correctors, such as VX-809, under low-temperature (27°C) conditions (42). Here we showed that treatment of cells with VX-809 at a low temperature facilitated CFTR maturation and increased the amount of band C, which indicates the presence of the mature complex-glycosylated form of CFTR (Fig. 5D). During CFTR maturation, C. jejuni invasion was suppressed by expressing the mature ΔF508-CFTR mutant in the presence of VX-809 (Fig. 5E). These results suggest that CFTR expression, especially expression of mature and cell surface-localized CFTR, is important for C. jejuni invasion.

FIG 5
FIG 5

Immature CFTR does not inhibit C. jejuni invasion. (A) HEK293, HEK-wt-CFTR, HEK-G551D-CFTR, or HEK-ΔF508-CFTR cells were infected with C. jejuni for 0, 1, and 3 h. The numbers of intracellular bacteria were estimated by the gentamicin protection assay (n = 4). (B) HEK-G551D-CFR cells were transiently transfected with the vector expressing shControl or shCFTR, and CFTR levels were detected by Western blotting. (C) HEK-G551D-CFTR cells transfected with the vector expressing shControl or shCFTR were infected with C. jejuni for 6 h. The numbers of intracellular bacteria were estimated by the gentamicin protection assay (n = 4). (D) HEK-ΔF508-CFTR cells were treated with VX-809 (40 μM) or the DMSO vehicle at 27°C for 24 h. CFTR expression was detected by Western blotting. Bands A, B, and C indicate nonglycosylated, immature core-glycosylated, and mature complex-glycosylated CFTR, respectively. (E) HEK-ΔF508 and HEK-wt-CFTR cells were treated with VX-809 or the DMSO vehicle at 27°C for 24 h and infected with C. jejuni for 3 h. The number of intracellular bacteria was estimated by the gentamicin protection assay (n = 6). All data are means ± SDs from 3 independent experiments. Statistical significance is indicated as follows: **, P < 0.01; *, P < 0.05; NS, not significant.

Endogenous CFTR attenuates C. jejuni invasion.Next, we confirmed the CFTR-mediated suppression of C. jejuni invasion in T-84 cells, in which CFTR is endogenously expressed. The vectors expressing shControl and shCFTR were transfected into T-84 cells by electroporation to assess whether endogenous CFTR also inhibits C. jejuni invasion. We checked the CFTR knockdown efficiency by Western blotting (Fig. 6A). Similar to the results of the experiment with HEK-wt-CFTR cells, the intracellular number of C. jejuni bacteria was increased by endogenous CFTR knockdown after 3 h of infection (Fig. 6B). Finally, we checked the endogenous CFTR expression level in C. jejuni-infected T-84 cells. We examined the mechanism of C. jejuni suppression of CFTR channel activity in T-84 cells (Fig. 1A) and found that C. jejuni attenuated CFTR gene expression and the protein level in a long-term (12-h) infection (Fig. 6C and D). Taken together, these results suggest that C. jejuni suppresses CFTR to effectively enter host intestinal cells.

FIG 6
FIG 6

Endogenous CFTR inhibits C. jejuni invasion of intestinal cells. (A) CFTR knockdown levels in T-84 cells transfected with the vector expressing shControl or shCFTR were detected by Western blotting (n = 6). (B) Induced T-84 cells transfected with the plasmid expressing shControl or shCFTR were infected with C. jejuni for 3 h, and the intracellular bacterial numbers were estimated by the gentamicin protection assay (n = 5). During the 12 h of C. jejuni infection, CFTR mRNA (n = 6) (C) and protein (n = 6) (D) expression levels were estimated in T-84 cells. All data are means ± SDs from 3 independent experiments. Statistical significance is indicated as follows: **, P < 0.01; *, P < 0.05.

DISCUSSION

In this study, we investigated the role of CFTR in C. jejuni infection. We found that overexpression of CFTR specifically inhibits C. jejuni invasion of cells (Fig. 2 and 3) and that this inhibition appears to depend on microtubules for the transport of the C. jejuni-containing vacuoles needed for bacterial invasion (Fig. 4 and 5). C. jejuni invasion of cells expressing an immature mutant CFTR that was defective in glycosylation and trafficking was not suppressed, whereas C. jejuni invasion of cells expressing a CFTR mutant that lacked ion channel activity was similar to that of HEK293 cells expressing wild-type CFTR (Fig. 6). Thus, we conclude that CFTR glycosylation and localization to the cell surface might affect bacterial interactions with cellular microtubules to suppress bacterial invasion.

The specific functions of intestinal epithelial cells, such as mucus production or cellular polarization, might affect C. jejuni invasion processes. Because we focused on the relationship between CFTR and C. jejuni infection, we overexpressed CFTR in HEK293 cells, a fibroblast line.

CFTR at the cell surface has several functional domains that have specific structures, including two membrane-spanning domains, two nucleotide-binding domains, and a regulatory region (14, 15). The carboxy-terminal domain of CFTR includes a PDZ domain that interacts with the cellular cytoskeleton by binding scaffolding proteins, such as NHERF1, N-WASP, EZRIN, and F-actin. The localization of CFTR on the cell surface helps regulate clathrin and N-WASP-mediated endocytosis pathways, as well as Rab11- and Rme-1-mediated recycling pathways. The ΔF508-CFTR mutant is thought to have impaired interactions with NHERF1 and attenuated stabilization at the cell surface (43). We considered that expression of wild-type CFTR would result in stable cell surface CFTR and increased interactions of CFTR with scaffolding proteins that in turn inhibit C. jejuni invasion. However, future studies should quantify cell surface CFTR, which was examined only at a qualitative level in this study.

During C. jejuni invasion, microtubule tracts were associated with CFTR-dependent inhibition of C. jejuni invasion (Fig. 4 and 5). Motor proteins, such as members of the dynein and kinesin families, slide along microtubules and are important for microtubule-mediated intracellular transport (44). Dynein and kinesin transport cargo toward the perinuclear region and from the perinuclear region to the cell surface, respectively. CFTR trafficking is maintained by microtubule-dependent transport involving dynein. Thus, CFTR expression could regulate the microtubule motor protein function, which might affect the delivery of C. jejuni-containing vacuoles along microtubule tracts from the cell surface. Further studies will also be needed to characterize the relationship between CFTR and motor protein function.

Recently, other bacteria causing intestinal infections, including enteropathogenic Escherichia coli (EPEC) and Salmonella enterica serovar Typhimurium, were reported to suppress CFTR ion channel activity (45, 46). Moreover, in the respiratory tract, P. aeruginosa was shown to invade host cells via a microtubule-dependent pathway that is regulated by CFTR (47). These findings suggest that the regulation of microtubule function by CFTR might be essential for protection against invasive bacterial infection in a variety of tissues.

MATERIALS AND METHODS

Bacterial strains and culture conditions. Campylobacter jejuni strains NCTC11168 (ATCC 700819) and 81-176 (ATCC BAA2151) were purchased from the American Type Culture Collection (ATCC). The bacteria were grown in Mueller-Hinton (MH) broth (catalog number 275730; Difco) at 37°C under microaerobic conditions (5% O2, 10% CO2, 85% N2) for 36 h. The bacteria were then collected by centrifugation of the medium at 12,000 rpm for 3 min, concentrated, and grown on selective supplemental medium containing Campylobacter charcoal differential agar (CCDA; Oxoid) for 36 h. Single colonies were picked and grown in MH broth for 48 h, after which the medium was centrifuged at 12,000 rpm for 3 min, diluted by placement into 15% glycerol (Wako), and stored at −80°C.

For the experiments, samples from frozen bacterial strains were grown in MH broth for 48 h under microaerobic conditions. After centrifugation at 12,000 rpm for 3 min, the supernatant was removed, the pellet was diluted by placement into fresh MH broth, and the mixture was cultured for 36 h. Bacteria were collected by centrifuging the medium at 3,000 rpm for 15 min, and the supernatant was removed. The bacterial cells were washed with phosphate-buffered saline [PBS; 137 mM NaCl, 8.1 mM anhydrous Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4), centrifuged, and resuspended in PBS. Finally, bacterial cell numbers were adjusted to an optical density of 600 nm (OD600) of 1.0.

Reagents and antibodies.Forskolin and VX-809 were purchased from Calbiochem and AdooQ Bioscience, respectively. Methyl-β-cyclodextrin (mβ-cd), chlorpromazine hydrochloride, paclitaxel, and nocodazole were purchased from Sigma-Aldrich. Antibodies to the following were diluted in 3% skim milk and used for Western blotting: CFTR (1:1,000; Millipore), α-tubulin (1:2,000; Wako), and β-actin (1:2,000; Santa Cruz).

Cell culture.Human embryonic kidney (HEK293) cells were cultured for 3 to 4 days in Dulbecco's modified Eagle's medium, high glucose (DMEM-HG; Sigma-Aldrich), supplemented with 10% fetal bovine serum (FBS; Thermo Fisher) and 50 μg/ml gentamicin (Sigma-Aldrich) [DMEM-HG(+)] at 37°C in 5% CO2. Cells of the human intestinal epithelial cell line T-84 were cultured for 7 days in Ham's F-12 medium (DMEM and F-12 medium, 1:1; Sigma-Aldrich) supplemented with 10% FBS (Thermo Fisher) and 50 μg/ml gentamicin (Sigma-Aldrich) at 37°C and 5% CO2. The culture medium was changed every 2 days.

Stable transfection of HEK293 cells.HEK293 cells were seeded at a density of 5 × 105 cells/35-mm-diameter dish and incubated for 24 h. The medium was changed to DMEM-HG without FBS and gentamicin [DMEM-HG(−)]. DMEM-HG(−) (200 μl) was mixed with 1 μg/well pcDNA-wt-CFTR vector and 4 μg/well Lipofectamine 2000 reagent (Invitrogen) for 15 min before incubation at 37°C for 3 h. The cells were then incubated overnight at 37°C in DMEM-HG(−) containing 20% FBS and 50 μg/ml gentamicin (total volume, 1.2 ml). The supernatant was removed, the cells were washed with 1 ml 0.02% EDTA-PBS, and then 1 ml 0.02% EDTA-PBS was added and the cells were incubated at 37°C for 5 min. The cells were collected by centrifugation at 800 rpm for 3 min and incubated with DMEM-HG(+) supplemented with 1 mg/ml G418, 10% FBS, and gentamicin for 10 days to produce cells that stably expressed CFTR. Colonies were picked with filter paper and incubated in a 24-well plate for 2 days before the filter was removed and the cells were grown to confluence. CFTR protein expression was assessed by Western blotting, and cells with high levels of CFTR expression were selected. Cells stably expressing mutated CFTR were produced using the same method.

Western blotting.Cultured cells were washed with PBS, diluted into radioimmunoprecipitation assay buffer (pH 7.4, 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100) plus a 10% protease inhibitor mixture (Nacalai Tesque), and centrifuged at 15,000 rpm for 10 min at 4°C, and the supernatant was collected. Protein levels were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher). Samples were added to 5× sample buffer (pH 6.8, 250 mM Tris-HCl, 5% SDS, 25% 2-mercaptoethanol, 50% glycerol, bromophenol blue) and separated on an SDS-polyacrylamide gel (7.5% or 10%). Proteins were transferred to Immobilon-P transfer membranes (Millipore) that were blocked with Tris-buffered saline with Tween 20 [TBS-T; pH 7.6, 20 mM Tris, 150 mM NaCl, 0.02% polyoxyethylene (20) sorbitan monolaurate] containing 3% skim milk for 1 h at room temperature and incubated overnight at 4°C with primary antibodies in TBS-T containing 3% skim milk. After the membranes were washed with TBS-T for 30 min, they were exposed to horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2,000; anti-mouse Ig-HRP; Biosource) for 2 h at room temperature. The membranes were then washed with TBS-T for 30 min and detected by enhanced chemiluminescence (ECL; GE Healthcare). Hyperfilm ECL (GE Healthcare) and imaging with a Fuji medical film processor (FPM 100) were used to detect CFTR expression. The β-actin protein was detected using medical X-ray film or a luminescent image analyzer (LASS-2000).

Efflux assay.Cells were seeded at a density of 5 × 105 cells/well in a 6-well plate and incubated for 4 days. The supernatants were removed, exchanged for HEPES buffer (10 mM HEPES, 145 mM NaCl, 10 mM glucose, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2) containing 125I− (2 μCi/ml), and incubated at 37°C for 1 h. The medium was removed, and the cells were washed twice with isotope-free HEPES buffer before the buffer was exchanged either for HEPES buffer or for HEPES buffer containing 10 μM forskolin. After 5 min, the supernatants were recovered for sample detection. Cells were incubated in 0.1 M NaOH at 37°C for 1 h, and samples were collected to detect intracellular 125I− using a gamma counter. The total amount of 125I− in the supernatant and cells was calculated as follows: percent efflux = [amount of 125I− secreted/(intracellular amount of 125I− + amount of 125I− secreted)] × 100.

Transient transfection with shRNA.HEK-wt-CFTR or HEK-G551D-CFTR cells were seeded at a density of 1 × 106 cells/well in 6-well plates. The cells were then washed and the medium was replaced with DMEM-HG(−) before transfection by use of the Lipofectamine 2000 reagent with 1 μg pLKO-CFTR vector or pLKO.1-puro nontarget vector expressing control shRNA (Invitrogen) for 3 h. DMEM-HG(+) containing 20% FBS and 50 μg/ml gentamicin was then added to the cells.

T84 cells were cultured for 7 days, and the cells were harvested with 0.05% trypsin-EDTA and diluted in HEPES buffer. The pLKO-CFTR vector or the pLKO.1-puro nontarget vector expressing control shRNA (2.5 to 5 μg) was mixed with T-84 cells in suspension cultures (4 × 105/24-well plate) for 20 min. The cells were electroporated 3 times at 50 V for 20 ms each time. After electroporation, the cells were suspended in culture medium for 48 h.

Invasion, adhesion, and degradation assay.For the invasion assay, HEK293 and HEK-wt-CFTR cells were seeded at a density of 4 × 105 cells/well in 6-well plates and incubated at 37°C for 4 days. The supernatants were removed and replaced with DMEM-HG(−) before the cells were infected with C. jejuni at a multiplicity of infection (MOI) of 50 for 6 h at 37°C in 5% CO2. After infection, the supernatant was replaced with DMEM-HG(−) containing 100 μg/ml gentamicin for 2 h. The supernatant was removed, and the cells were washed with PBS and lysed with PBS containing 1% Triton X-100. The lysates were plated on MH agar plates and incubated for 48 h under microaerobic conditions. HEK293 cells expressing the ΔF508-CFTR mutant (HEK-ΔF508-CFTR cells) were treated with VX-809 or the dimethyl sulfoxide (DMSO) vehicle at 27°C for 24 h before infection.

For adhesion assays, HEK293 cells and HEK-wt-CFTR cells were seeded at a density of 1 × 106 cells/well in plates with 60-mm wells. After infection, the supernatants were removed and the cells were washed with PBS three times before they were lysed at 37°C for 5 min in PBS containing 0.01% Triton X-100. The cell lysates were plated on MH agar plates and incubated for 48 h under microaerobic conditions.

In degradation assays, HEK293 cells and HEK-wt-CFTR cells were seeded at a density of 4 × 105 cells/well in 6-well plates. After infection, the supernatants were removed and replaced with DMEM-HG(−) containing 100 μg/ml gentamicin for 2, 4, 6, or 8 h. The cells were then lysed with PBS containing 1% Triton X-100, plated on MH agar plates, and incubated for 48 h under microaerobic conditions. All intracellular bacterial numbers were normalized to 1 × 107 cells.

Separation of soluble and insoluble tubulin.HEK293 cells and HEK-wt-CFTR cells were seeded at a density of 4 × 105 cells/well in 6-well plates. The supernatant was removed and the cells were washed with PBS before addition of microtubule buffer {0.1 M PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)], pH 7.6, 2 M glycerol, 5 mM MgCl2, 2 mM EGTA} containing 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, and 5 μg/ml leupeptin and incubation for 10 min at room temperature. Cells were collected and centrifuged for 10 min at 300 × g at room temperature. The resulting supernatant represented the soluble fraction. Meanwhile, pellets containing the insoluble fraction were washed again with microtubule buffer, treated with lysis buffer (25 mM Na2HPO4, pH 7.2, 400 mM NaCl, 0.5% SDS), and centrifuged for 10 min at 20,000 × g. Protein concentrations were measured with a BCA protein assay kit, and equal amounts of protein were separated by SDS-PAGE. Anti-α-tubulin and anti-β-actin antibodies were used for Western blotting.

RNA isolation and RT-PCR.Total RNA was extracted from T-84 cells using an RNeasy minikit (catalog number 74104; Qiagen) according to the manufacturer's instruction. Reverse transcription (RT) was performed with 1 μg of total RNA and a PrimeScript RT reagent kit (catalog number RR037A; TaKaRa). Quantitative real-time reverse transcription-PCR was performed in a LightCycler real-time PCR system (Roche Applied Science) with SYBR Premix Ex Taq DNA polymerase (catalog number RR820; TaKaRa). Primers specific for the 18S rRNA housekeeping gene (used as an internal control) and CFTR were as follows: for the 18S rRNA gene, forward primer 5′-AAACGGCTACCACATCCAAG-3′ and reverse primer 5′-GGCCTCGAAAGAGTCCTGTA-3′, and for CFTR, forward primer 5′-GCAGTTGATGTGCTTGGCTAG-3′ and reverse primer GAATCGTACTGCCGCACTTTG-3′. The fold change in the level of expression was calculated relative to the level of expression of the 18S rRNA gene.

Statistical analysis.Statistical analysis of all data was performed by using Student's t test for paired data. Data from 3 independent experiments were evaluated. All tests were one-tailed.

ACKNOWLEDGMENT

This work was supported by a grant-in-aid for scientific research from JSPS Kakenhi (grant number JP 15K00819).

FOOTNOTES

    • Received 22 July 2017.
    • Accepted 29 July 2017.
    • Accepted manuscript posted online 7 August 2017.

  • Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00311-17 .

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KEYWORDS

Campylobacter jejuni
Cystic Fibrosis Transmembrane Conductance Regulator
Microtubules
CFTR
Campylobacter jejuni
cell invasion
host response to infection
intracellular bacteria
microtubule

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