Campylobacter jejuni activates mitogen-activated protein kinases in Caco-2 cell monolayers and in vitro infected primary human colonic tissue

Amanda MacCallum1, Graham Haddock2 and Paul H. Everest1

1 Institute of Comparative Medicine, Sir Henry Wellcome Building, University of Glasgow Faculty of Veterinary Medicine, Bearsden Road, Glasgow G61 1QH, UK
2 Department of Paediatric Surgery, Yorkhill Hospital, Glasgow G3 8SJ, UK

Correspondence
Paul Everest
phe3d{at}udcf.gla.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The mitogen-activated protein kinases (MAPKs) play a central role in many host signalling pathways. These signalling proteins are known to be involved in host responses against invasive bacteria including generation of chemotactic and inflammatory cytokines. It was hypothesized that Campylobacter jejuni may activate MAPKs, as intestinal infection may induce a clinical and pathological picture of acute colonic inflammation. Infection of Caco-2 cell monolayers (human colonic epithelial cell line) and human colonic tissue with C. jejuni in vitro demonstrated increased MAPK activity for ERK 1/2 (p44/42 MAPK), JNK and p38 MAPKs. Kinase activity and phosphorylated forms were increased in infected Caco-2 cells and human colonic explants, suggesting that these pathways are important in inflammatory responses induced by C. jejuni in man.


Abbreviations: ERK, extracellular signal regulated kinase; JNK, c-jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MAPKK or MEK, MAPK kinase; MAPKKK/MEKK, MAPKK/MEK kinase; SAPK, stress-activated protein kinase; TEER, transepithelial electrical resistance; VAIN, variable-atmosphere incubator


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Campylobacter jejuni is the most common cause of bacterial enteritis in man, causing both watery and bloody diarrhoea with symptoms of acute inflammation in colonic mucosa (Black et al., 1988; Wassenaar & Blaser, 1999; Skirrow & Blaser, 2000; Coker et al., 2002). The mechanisms by which C. jejuni causes diarrhoeal disease and inflammation in the infected human host are not fully understood. Like other human enteric bacterial pathogens, C. jejuni infects humans by colonizing the mucus layer of the intestine followed by adherence and invasion of epithelial cells (Skirrow, 1986; Everest et al., 1992; Konkel et al., 1992, 2001; Ketley, 1997; Harvey et al., 1999; Kopecko et al., 2001). Bacteria transcytose through the host cell (Everest et al., 1992; Konkel et al., 1992; Harvey et al., 1999), to emerge on the basolateral surface, allowing access to the lamina propria. Recruitment of neutrophils occurs at the site of bacterial invasion (basolateral surface) via interleukin-8 (IL8) (Hickey et al., 1999, 2000) and release from infected enterocytes by activation of NF-{kappa}B (Mellits et al., 2002). This innate immune response involves secretion of cytokines and other mediators leading to an inflammatory response.

Mitogen-activated protein kinases (MAPKs) are a group of serine/threonine-specific protein kinases, which regulate the transcription of inflammatory cytokines in response to various extracellular and stress stimuli (Garrington & Johnson, 1999). Upon activation by upstream kinases, MAPKs phosphorylate downstream kinases and transcription factors, and transmit signals from the cell surface to the nucleus via distinct but related pathways. MAPK activation is achieved through kinase cascades, which include a MAPK kinase (MAPKK or MEK) and a MAPKK/MEK kinase (MAPKKK/MEKK). Several distinct MAPK subgroups have been identified, the best characterized of which are: extracellular signal regulated kinase (ERK) 1/2 (also known as p44/p42 MAPK), stress-activated protein kinase (SAPK)/c-jun N-terminal kinase (JNK) and p38 MAPK. The MAPK subgroups are activated by different extracellular stimuli and the signal transduced through kinase cascades, which only partially overlap. In general, the ERKs are largely involved in pathways leading to cell proliferation as a consequence of growth factor stimulation, whereas the JNK and p38 MAPKs are activated in response to a variety of cytokines and stress conditions, which include bacterial infection (Kyriakis & Avruch, 1996).

A variety of bacterial factors can stimulate or inhibit MAPKs. The best studied is LPS, which activates various MAPK pathways, in particular the p38 MAPKs (Cario et al., 2000). Jin et al. (2003) recently demonstrated that a C. jejuni cell-surface protein JlpA interacts with a host heat-shock protein Hsp 90, and activates NF-{kappa}B and p38 MAPK in Hep-2 epithelial cells. p38 MAPK plays an important role in regulation of proinflammatory responses to bacterial infections.

Host cell responses to bacterial components, and especially, host cell signal transduction, are considered to be important in the pathogenesis of disease caused by C. jejuni (Wooldridge et al., 1996; Wooldridge & Ketley, 1997; Hu & Kopecko, 1999; Konkel et al., 1999; Kopecko et al., 2001) and the resulting inflammation and fluid-loss from the gastro-intestinal mucosa. However, the actual host cell signalling pathways activated upon infection are not yet fully established. The aim of this study was to determine if MAPK activation could have a role in the signalling events associated with host cell responses to C. jejuni.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial isolates and growth conditions.
Bacteria were cultured in Mueller–Hinton broth and on Mueller–Hinton agar (Oxoid) and incubated at 37 °C in a variable-atmosphere incubator (VAIN; Don Whitley) under microaerophilic conditions in an atmosphere of 6 % hydrogen, 5 % carbon dioxide, 5 % oxygen and 84 % nitrogen. All isolates and mutants were subcultured every 24–48 h from a Microbank stock stored at –80 °C. Details of wild-type isolates and mutants are detailed in Table 1. Wild-type clinical isolates and mutants in putative virulence determinants (potentially involved in initiating inflammation within an infected host) were used for this study. Mutants were obtained from Dr Andrey Karlyshev and Dr Dennis Linton at the London School of Hygiene and Tropical Medicine, UK (11168 kpsM, cdtA, tlyA, peb3 and 11168/G1pldA, pglH) and Professor Julian Ketley at the University of Leicester, UK (11168 pglI, pglJ). Mutants were constructed using allelic replacement and insertion of a selectable marker (van Vliet et al., 1998).


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Table 1. Strains and mutants used in this study

 
Cell culture.
Caco-2 (human colon carcinoma) cells were maintained in Dulbecco's minimal essential medium (DMEM) with Glutamax-1 (Gibco) and supplemented with 10 % fetal calf serum (FCS; Gibco) without antibiotics. Cells were grown routinely in a 75 cm2 flask (Costar) at 37 °C in a 5 % CO2 humidified incubator. Confluent stock cultures were trypsinized and new stock cultures were seeded at ~4x104 cells cm–2. Confluent monolayers were fully differentiated, forming a polarized monolayer with defined apical and basolateral surfaces separated by tight junctions. Formation of polarized monolayers was determined using transepithelial electrical resistance (TEER) measurements with fully polarized and differentiated monolayers having a TEER of >200 {Omega} cm2. Selected monolayers were observed under scanning electron microscopy for the formation of microvilli, and dome formation as determined by light microscopy.

Caco-2 polarized adherence and invasion assay.
Adherence and invasion assays were performed with Caco-2 cells. These assays have been described elsewhere (Everest et al., 1992). Briefly, for the invasion assay, Caco-2 cells were seeded at 1x105 cells per well in 12-well plates (Costar) and allowed to grow until they reached confluence. The confluent monolayers were washed and inoculated with 10 µl bacterial suspension, containing varying numbers of bacteria for different experiments (between 100 and 10 000 c.f.u.). For most experiments the m.o.i. was between 100 and 1000 bacteria per eukaryotic cell. The infected monolayers were incubated for 2, 4, 8 and 24 h, respectively (Western blotting), or 24 h only (kinase assay) at 37 °C in a 6 % CO2 humidified atmosphere to allow the bacteria to adhere to and invade the cells. To kill extracellular bacteria, 250 µg gentamicin ml–1 was added after various incubation time points and the cells reincubated for 2 h before cell lysis and viable plate counts. Uninfected cells were included in the experiment as a control for kinase assays and Western blotting.

Preparation of cell lysates.
After the incubation period, the medium was removed and monolayers were washed twice with ice-cold PBS. Cells were solubilized using ice-cold cell lysis buffer A or B (see below) and scraped into 1·5 ml microcentrifuge tubes. Cell lysates were homogenized using a needle and syringe then centrifuged at either 18 000 g for 10 min at 4 °C (Western blotting) or 25 000 g for 20 min at 4 °C (kinase assay). The soluble fraction was transferred to a new microcentrifuge tube and stored at –80 °C for later analysis.

Cell lysis buffer.
The following buffers were used to solubilize the cells: buffer A was used to lyse the cells for analysis by Western immunoblotting (20 mM Tris pH 7·5, 150 mM sodium chloride, 1 mM EDTA, 1 mM EGTA, 1 % Triton-X 100, 2·5 mM sodium pyrophosphate, 1 mM {beta}-glycerophosphate, 1 mM sodium orthovanadate, 1 µg leupeptin ml–1, 1 mM PMSF). Cells were lysed in buffer B for analysis by kinase assay (10 mM Tris, 150 mM sodium chloride, 2 mM EGTA, 2 mM DTT, 1 mM orthovanadate, 1 mM PMSF, 10 µg leupeptin ml–1, 10 µg aprotinin ml–1, pH 7·4 measured at 4 °C).

Immunoprecipitation.
Cell lysates (200 µl) were incubated with primary antibody (1 : 50 to 1 : 100) (New England Biolabs) overnight with rocking at 4 °C. Samples were pre-cleared with Protein A agarose beads (Sigma) for 1 h at 4 °C. Samples were microcentrifuged for 30 s at 4 °C and the pellets were washed twice with 500 µl 1x cell lysis buffer A, being kept on ice throughout. The pellets were resuspended with 20 µl 2x Laemmli SDS-sample buffer (Sigma), vortexed and microcentrifuged for 30 s. The primary antibody used for immunoprecipitation was the same antibody used for Western blotting experiments.

SDS-PAGE and Western blotting.
Samples were boiled for 5 min and lysate proteins were separated by SDS-PAGE on 12 % acrylamide gels. Proteins were transferred onto Immobilon-P membranes (Millipore). Non-specific binding sites on the membrane were blocked using 5 % non-fat dry milk in TBS/T (50 mM Tris, 150 mM NaCl, 0·1 % Tween 20). Membranes were incubated overnight with antibody specific to phosphorylated or total MAPK (New England Biolabs) at 1 : 1000 dilution in blocking buffer. Bands were visualized by incubating with a horseradish peroxidase-conjugated anti-rabbit IgG (DAKO) at 1 : 5000 dilution in blocking buffer, for 1 h at room temperature, and subsequently enhanced by chemiluminescence detection (ECLPlus; Amersham Biosciences) and autoradiography (Hyperfilm-MP; Amersham Biosciences). Cell lysates from Caco-2 cells infected with two wild-type strains of C. jejuni (11168 and L115) were analysed by this method three times. Total MAPK activity was determined to distinguish total cell MAPK activity from activated phosphorylated MAPKs. Antibody specific to total MAPK was used at 1 : 1000 dilution and obtained from New England Biolabs.

ERK1/2 (p44/p42 MAPK) enzyme assay.
The Biotrak (ERK1/2) p44/p42 MAPK enzyme assay system from Amersham Biosciences (RPN84) was used to measure (ERK1/2) p44/p42 MAPK activity in infected and uninfected Caco-2 cells. The assay was based on the kinase-catalysed transfer of the {gamma}-phosphate group of adenosine 5'-triphosphate to a peptide that is highly selective for ERK1/2. Cells were lysed with cell lysis buffer B (as described above). The reaction was initiated by the addition of [{gamma}-32P]ATP, and following 30 min incubation the peptide was separated from unincorporated activity using a binding paper separation step. The amount of ERK1/2 activity was determined by the amount of radioactivity incorporated by the binding paper, and this was measured by scintillation counting. The assay was performed with two wild-type strains of C. jejuni (11168 and G1), 10 mutant strains of C. jejuni (Table 1) and LPS (Salmonella typhimurium), a known MAPK activator, as a positive control. Caco-2 cells were grown until confluent in 12-well tissue culture plates and infected with wild-type or mutant strains of C. jejuni or uninfected as a control, for 24 h. Following the incubation period, the monolayers were washed with PBS and solubilized in cell lysis buffer, and the lysates analysed for kinase activity according to the assay protocol. The assay was based on measuring the degree of phosphorylation of a substrate highly selective for ERK1/2 to determine relative kinase activity in lysates from both the infected and uninfected cells.

Infection of human colonic tissue.
Primary human colonic tissue explants were obtained from healthy sections of colon removed during surgery for colonic resection. Tissue explants (2 cm2) were cut into sections and divided into three pieces/sample for infection with C. jejuni and uninfected controls. They were cultured initially in DMEM with 10 % FCS and with added antibiotics for 1 h after arrival in the laboratory (penicillin/streptomycin; Gibco). After this period, tissue explants were washed, inoculated with similar numbers of bacteria as described above (or uninfected as a control) and incubated for 12 h at 37 °C, 6 % CO2 (medium was changed to antibiotic-free DMEM, 10 % FCS). Tissue was perfused with oxygen intermittently for maintenance of viability over the course of the experiment. At the end of the time points, portions of both infected and uninfected tissue were examined histologically (by fixation in formalin, wax embedding, sections cut by microtome and sections stained using haematoxylin and eosin) and both exhibited normal colonic morphology (data not shown). Explants were homogenized in cell lysis buffer A (as described above) for analysis by Western blotting. This tissue was precious and at a premium in terms of amount used; hence only one strain was used for explant infection.

MAPK inhibitors.
Caco-2 cells were infected with C. jejuni 11168 and L115, with and without the presence of MAPK inhibitors. Cells were pre-treated for 1 h with inhibitors prior to infecting with bacteria. The inhibitors used in this experiment were PD98059 (ERK inhibitor) and SP600125 (JNK inhibitor). PD98059 was added at 50 µM and SP600125 was added at 60 µM. A p38 MAPK inhibitor (SB203580) was also used in this experiment (50 µM); however, problems with solubility were experienced, as the inhibitor precipitated in culture media. All inhibitors were obtained from Calbiochem.

Data analysis.
Statistical analysis was performed using a Student's t-test (two-tailed) and a P value <0·05 was considered to be significant. All data are expressed as the mean±standard error of the mean.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Adherence and invasion of C. jejuni strains
Bacteria were left either extracellularly and intracellularly for some experiments, or representative monolayers were lysed after gentamicin treatment to determine if bacteria were intracellular in these assays. C. jejuni L115 adhered and invaded in numbers similar to those previously described for this strain (Everest et al., 1992). C. jejuni 11168 adhered at 5x104 c.f.u. and invaded at 5x103 c.f.u. Mutants derived from this strain (11168cdtA and 11168pldA) adhered and invaded to similar levels. A hypermotile variant of 11168, 11168H, adhered and invaded in greater numbers compared with the sequenced strain (106 adhesion, 104 invasion; Karlyshev et al., 2004). Mutant strains 11168peb3 and 11168pglH had decreased adhesion and invasion compared with the parent strain, and 11168kpsM had increased adhesion (8x104 c.f.u.) and markedly decreased invasion (101) compared with 11168. Strains G1, 11168pglI and pglJ were not tested for adhesion and invasion ability (data not shown). MAPK activity did not differ markedly for monolayers where bacteria were both extracellular and intracellular or intracellular only.

ERK1/2 (p44/p42 MAPK) kinase is activated in Caco-2 cells infected with C. jejuni
To detect ERK1/2 (p44/p42) MAPK activity in C. jejuni-infected Caco-2 cells, a kinase enzyme assay was performed. Radioactive counts varied between assays because of the varying specific activity of different batches of radioactivity, but uninfected controls at each time point allowed comparison within individual experiments and experimental replicates. Kinase activity was increased in C. jejuni-infected cells compared with uninfected controls. Results are shown in Fig. 1. Values shown are the means±standard errors for eight replicates (n=8). Results were statistically analysed using a paired t-test (P<0·05 is significant). ERK1/2 activity was significantly higher in the C. jejuni-infected Caco-2 cells compared with the uninfected controls. Killed bacteria had no effect on kinase levels (data not shown).



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Fig. 1. ERK1/2 (p44/p42 MAPK) activity in Caco-2 cells infected with C. jejuni. Caco-2 cells grown in 12-well plates to confluency were infected with C. jejuni strains (wild-type and mutant) for 24 h or uninfected (also for 24 h) as a control. Results obtained were a measure of picomoles of phosphate transferred per minute. The data shown in this figure has been normalized to show fold increase in the infected cells compared with the uninfected control. Values represent the mean of eight replicates, and the error bars are the standard errors of the mean. Results were statistically analysed using a Student's t-test, where P<0·05 is significant. Statistical significance is shown as an asterisk (*). Wild-type strains: 11168 and G1; and mutant strains: 11168kpsM, 11168cdtA, 11168tlyA, G1pldA, 11168H, NCTC12189, 11168pglH, 11168pglI, 11168peb3 and 11168pglJ, and LPS (from S. typhimurium).

 
This increase was independent of the strain of C. jejuni infecting the monolayers, since kinase activity was found to be increased in cells infected with all wild-type and mutant bacteria included in the experiment (Fig. 1). This increase was more than twofold in wild-type C. jejuni G1-infected cells. Strain 11168 showed a significant increase, as did strains L115 and 81-176 (data not shown). All mutants also had a significant effect on kinase activity (paired t-test); this was most marked with strain 11168tlyA and 11168H. Infection with the mutant strains 11168 kpsM, cdtA, pglH, pglI, pglJ, peb3, 11168/G1pldA and NCTC 12189 also resulted in higher ERK1/2 activity compared with uninfected cells. LPS from S. typhimurium, a known MAPK activator, was included as a positive control, and kinase activity was also upregulated in the LPS-treated cells compared with the controls. This experiment was also performed including the earlier time points of 2, 4 and 8 h post-infection (data not shown), as well as the 24 h time point. The results consistently showed that the most marked effects in terms of p44/p42 MAPK enzyme activity occurred at 24 h post-infection. These results demonstrate that C. jejuni increases ERK1/2 kinase activity inside the human enterocyte-like cell line Caco-2.

Phosphorylated ERK1/2 (p44/p42 MAPK) is increased in Caco-2 cells infected with C. jejuni
Immunoblot analysis of lysates from C. jejuni 11168- and L115-infected Caco-2 cells (although not a direct measure of kinase activity) detected differences in the amount of phosphorylated ERK1/2 in the infected cells compared with uninfected controls. Phosphorylation of ERK1 (p44) was upregulated at the later time points of 8 and 24 h (compared with uninfected controls) with strain L115 but only at 24 h with strain 11168 (Fig. 2a) compared with uninfected control cells. ERK2 (p42) phosphorylation was greatest at 2 h in uninfected cells, with an actual downregulation of p42 activity in Caco-2 cells infected with L115 at this time point compared with uninfected controls (Fig. 2a). However, increased p42 phosphorylation was evident in L115-infected cells at 8 but not 24 h post-infection compared with uninfected Caco-2 cells. Total ERK1/2 levels (Fig. 2b) were compared with phosphorylated ERK1/2 to demonstrate that there was little change in total levels compared with phosphorylated forms.



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Fig. 2. Western immunoblot detection of phosphorylated and total MAPK. Cell lysates from Caco-2 cells infected with C. jejuni 11168, L115 and uninfected controls were immunoprecipitated and separated by 12 % SDS-PAGE. Separated proteins were transferred to nitrocellulose and probed with antibody to phospho-ERK1/2 (p44/p42 MAPK) (Thr202/Tyr204) (a), phospho-p38 MAPK (Thr180/Tyr182) (c) and phospho-SAPK/JNK (Thr183/Tyr185) (e) and detected by anti-species (mouse/rabbit) IgG HRP-linked antibody. In order to distinguish total cell MAPK activity from activated phosphorylated MAPKs, blots were also probed with antibody to total-ERK1/2 (p44/p42 MAPK) (b), total-p38 MAPK (d) and total-SAPK/JNK (f). I, infected; U, uninfected. Time points were 2, 4, 8 and 24 h [4 h time point not shown (11168), 24 h time point not shown (L115) in (e)].

 
Phosphorylation of SAPK/JNK and p38 MAPK is increased in Caco-2 cells infected with C. jejuni
Involvement of the phosphorylated forms of other MAPKs in signal transduction of C. jejuni-infected cells were assessed by Western blot with antibodies to the phosphorylated forms of p38 and SAPK/JNK. Western blotting was used to examine levels of phosphorylation of p38 MAPK and SAPK/JNK in C. jejuni 11168- and L115-infected Caco-2 cells. Phosphorylation of p38 MAPK was greater at all time points with wild-type strains 11168 and L115 (Fig. 2c). Since SAPK/JNKs respond by and large to the same agonists as p38 MAPK, phosphorylation of SAPK/JNK was also increased at all the time points measured with both strains (Fig. 2e). This experiment has shown that C. jejuni infection of Caco-2 cells induces increased levels of the phosphorylated forms of both p38 and SAPK/JNK. Total p38 and SAP/JNK levels (Fig. 2d, f) were compared with phosphorylated MAPKs to demonstrate that there was little change in total levels compared with phosphorylated forms. Killed bacteria had no effect on phosphorylation levels for all kinases studied (data not shown).

Phosphorylation of ERK1/2 (p44/p42 MAPK), SAPK/JNK and p38 MAPK is increased in primary human colonic tissue infected with C. jejuni
Primary human colonic tissue explants were also infected with C. jejuni 11168, and phosphorylation of ERK1/2, SAPK/JNK and p38 MAPK was greater in the infected tissue, compared with uninfected tissue at this time point (12 h) (Fig. 3). This experiment demonstrates that C. jejuni infection increases the phosphorylated forms of MAPKs inside primary human colonic tissue.



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Fig. 3. Western immunoblot detection of phosphorylated-MAPK in primary human colonic tissue. Lysates from primary human colonic tissue infected with C. jejuni 11168 (and uninfected control) were immunoprecipitated with primary antibodies and separated by 12 % SDS-PAGE. Separated proteins were transferred to nitrocellulose and probed with antibody to phospho-ERK1/2 (p44/p42 MAPK) (Thr202/Tyr204) (a), phospho-p38 MAPK (Thr180/Tyr182) (b) and phospho-SAPK/JNK (Thr183/Tyr185) (c) and detected by anti-species IgG HRP-linked antibody. The time point was 12 h.

 
MAPK activity in C. jejuni-infected Caco-2 cells can be decreased or inhibited by MAPK inhibitors
Fig. 4 shows phosphorylation activity for different kinases in infected cells and inhibition of this activity by specific MAPK inhibitors. This demonstrates that the phosphorylated kinases are activated by C. jejuni infection. Specific inhibitors of these kinases decreased the activity of ERK1/2 and JNK upon bacterial infection. The p38 inhibitor precipitated out of solution for these experiments so we were unable to determine specific inhibition for p38 MAPK.



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Fig. 4. Action of MAPK inhibitors on infected Caco-2 cells. Caco-2 cells were infected with C. jejuni 11168 and L115, with and without the use of MAPK inhibitors. Cells were pre-treated for 1 h with inhibitors prior to infecting with bacteria. The inhibitors used were PD98059 (ERK inhibitor) and SP600125 (JNK inhibitor). PD98059 was added at 50 µM and SP600125 was added at 60 µM. Time points following infection with C. jejuni were 2, 4, 8 and 24 h. Results for each time point show cells treated with C. jejuni (11168 or L115) alone or with C. jejuni (11168 or L115) and an inhibitor (PD98059 or SP600125). PD, PD98059; SP, SP600125.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we have provided evidence that C. jejuni infection of cultured intestinal epithelial cells and human colonic tissue results in the activation of the MAPKs ERK1/2, JNK and p38. Activation of these pathways could play an important role in the pathogenesis of Campylobacter infection.

ERK1/2 (p44/p42) kinase activity and phosphorylated forms are both increased inside infected cells at comparatively late time points. Thus, increased phosphorylation of ERK1 occurred at both 8 and 24 h in cells cultured with strain L115, but not until 24 h in those cultured with strain 11168. This activation is increased in time when compared with other enteric pathogens. Strain 11168 is known to be less motile than the original parent isolate (Gaynor et al., 2004), or hypermotile variants of this strain, and so it would take more time for the strain to adhere to host cell surfaces and invade compared with a more motile isolate. This delay in getting to the cell surface probably accounts for the delayed activation of phosphorylated ERK inside infected cells. The phosphorylation of p38 MAPK and JNK was upregulated at the earlier, as well as the later, time points with both strains 11168 and L115, perhaps because this particular pathway is involved in a more immediate response to stress. Thus, MAPK activity in infected Caco-2 cells has differential time expression for the different kinases, with ERK1/2 being activated later than both p38 and JNK. Assay conditions in terms of gaseous requirements favour the eukaryotic cells not the bacteria, again perhaps delaying MAPK activation. Caco-2 cell MAPK activation upon infection with C. jejuni correlated with demonstrated increases in the in vitro infected human primary colonic tissue. Although the time points were not the same, we have demonstrated clear increases in the phosphorylated forms of MAPKs in infected human tissue. These results show that in vivo tissue culture cell models mimic the interaction of C. jejuni with human colonic tissue and justify their use in studies of C. jejuni pathogenesis.

MAPKs can also be activated via receptor tyrosine kinases and transduction of extracellular signals via clustering of integrins (Giancotti & Ruoslahti, 1999). This may be of particular relevance to enterocyte infection by C. jejuni. C. jejuni binds to fibronectin via CadF protein (Monteville & Konkel, 2002, Monteville et al., 2003), and this protein is involved in both adhesion and invasion of C. jejuni into epithelia (Monteville & Konkel, 2002, Monteville et al., 2003). Fibronectin is bound to cell-surface integrin molecules on the basolateral cell surface and integrin clustering is associated with tyrosine phosphorylation of cellular cytoplasmic contents leading to host cell cytoskeletal rearrangements, presumably facilitating bacterial entry into the cell (Giancotti & Ruoslahti, 1999). Tyrosine kinase inhibitors also decrease C. jejuni invasion inside host cells (Wooldridge & Ketley, 1997; Wooldridge et al., 1996). Thus, we have a link between campylobacter–host cell adhesion and invasion, integrin clustering, receptor tyrosine kinase activation and phosphorylation with MAPK cellular pathway activation in C. jejuni infection of host enterocytes.

Campylobacter is most likely to activate MAPKs by bacterial adhesion and invasion or a bacterial component, engaging a cell-surface receptor and the signal being transduced to the relevant signalling pathway via upstream regulators. Indeed, this ligand–receptor interaction has been demonstrated by C. jejuni JlpA surface protein binding to Hsp 90 resulting in p38 MAPK activation (Jin et al., 2003). Bacterial LPS can also trigger MAPK activation (Cario et al., 2000). Shigella flexneri invasion and intracellular LPS can also activate JNK (Girardin et al., 2001). JNK phosphorylates c-Jun, which is a component of the transcription factor AP-1, also considered to be an important regulator of the inflammatory response (Foletta et al., 1998). Previous studies have shown MAPK activation to be associated with infection of epithelial cells by various enteropathogens (Hobbie et al., 1997; Warny et al., 2000; Meyer-ter-Vehn et al., 2000; Berin et al., 2002). A number of other bacteria or bacterial factors have been shown to modulate the activity of various MAPKs. For example, Escherichia coli LPS can activate p38 MAPK (Berin et al., 2002), Salmonella LPS can activate ERK via PI-3-kinase/phospholipase D pathways (Hobbie et al., 1997), Clostridium difficile toxin A can activate p38, ERK and c-Jun (Warny et al., 2000), Helicobacter pylori stimulates ERK 1 and 2 (Meyer-ter-Vehn et al., 2000), and Yersinia YopJ/P inhibits p38 and JNK MAPKs (Orth et al., 1999). Inhibitors of MAPK pathways are being increasingly used to determine the role of MAPK pathways in the control of inflammation due to bacterial infection. After reporting that Listeria monocytogenes stimulates ERK1/2 activity upon attachment to HeLa cells, Tang et al. (1998) have further shown that actual invasion of the cell requires the ERK2 pathway. S. typhimurium has been shown to activate ERK, JNK and p38 MAPKs in intestinal Henle-407 and HeLa cells. The authors have associated this with an increase in the production of the proinflammatory cytokine IL8 (Berin et al., 2002). Enteropathogenic E. coli (EPEC) infection has also been shown to activate ERK1/2, this time in T84 cells, and again this was associated with the inflammatory response, since IL8 expression and the degradation of I{kappa}B{alpha} (the primary inhibitor of NF-{kappa}B) were stimulated (Czerucka et al., 2001). The secretion of IL8 from the basal aspect of infected intestinal cells has been shown to be involved in the transepithelial migration of neutrophils during the inflammatory response to C. jejuni (Hickey et al., 1999, 2000). In contrast, YopJ/P from Yersinia spp. has been found to inhibit MAPK activity (Orth et al., 1999).

Our results indicate that the host enterocyte responds to C. jejuni infection by stimulation of MAPK signalling pathways. These responses may lead to host inflammatory reactions in terms of proinflammatory cytokine release and accumulation of neutrophils within the infected intestinal mucosa. Indeed such events characterize the microscopic histology of C. jejuni enterocolitis (Lambert et al., 1979; Price et al., 1979, 1984; van Spreeuwel et al., 1985). Understanding the signalling events triggered by C. jejuni infection may lead to the development of novel therapeutic strategies to limit the clinical consequences of inflammatory diarrhoea.


   ACKNOWLEDGEMENTS
 
This study was supported by the Biotechnology and Biological Sciences Research Council (BBSRC).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 21 February 2005; revised 18 April 2005; accepted 10 May 2005.



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