Campylobacter jejuni inhibits the absorptive transport functions of Caco-2 cells and disrupts cellular tight junctions

Amanda MacCallum1, Simon P. Hardy2 and Paul H. Everest1

1 Institute of Comparative Medicine, University of Glasgow Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK
2 Department of Pharmacy and Biomolecular Sciences, University of Brighton, Lewes Road, Brighton BN2 4GJ, UK

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Caco-2 cells are models of absorptive enterocytes. The net transport of fluid from apical to basolateral surfaces results in ‘domes' forming in differentiated monolayers. Here, the effect of Campylobacter jejuni on this process has been examined. C. jejuni caused no changes in short-circuit current upon infection of Caco-2 cell monolayers in Ussing chambers. Thus, no active secretory events could be demonstrated using this model. It was therefore hypothesized that C. jejuni could inhibit the absorptive function of enterocytes and that this may contribute to diarrhoeal disease. C. jejuni infection of fluid-transporting (‘doming’) Caco-2 cells resulted in a significant reduction in dome number, which correlated with a decrease in tight junction integrity in infected monolayers, when measured as transepithelial electrical resistance. Defined mutants of C. jejuni also reduced dome numbers in infected monolayers. C. jejuni also altered the distribution of the tight junction protein occludin within cell monolayers. The addition to monolayers of extracellular gentamicin prevented these changes, indicating the contribution of extracellular bacteria to this process. Thus, tight junction integrity is required for fluid transport in Caco-2 cell monolayers as leaky tight junctions cannot maintain support of transported fluid at the basolateral surface of infected cell monolayers. Inhibition of absorptive cell function, changes in epithelial resistance and rearrangement of tight junctional proteins such as occludin represent a potential diarrhoeal mechanism of C. jejuni.


Abbreviations: ISC, short-circuit current; LPF, low-power field; TEER, transepithelial electrical resistance; VAIN, variable-atmosphere incubator


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Campylobacter jejuni is the most frequent cause of food-borne diarrhoeal disease in developed countries and probably also in developing countries (Skirrow & Blaser, 2000; Coker et al., 2002). The organism causes a range of symptoms within an infected human host, from small-volume bloody diarrhoea resembling dysentery, watery diarrhoea with blood staining, to watery diarrhoea with no evidence of mucosal inflammation (Black et al., 1988; Wassenaar & Blaser, 1999; Skirrow & Blaser, 2000; Coker et al., 2002). How the organism causes diarrhoea within an infected host is as yet unknown.

In previous studies, Caco-2 cells have been used as a model of intestinal epithelium for the interaction of C. jejuni with host cells (Everest et al., 1992, Konkel et al., 1992, Harvey et al., 1999). The cells have similar properties to colonic enterocytes, in that they form brush borders with microvilli and maintain tight junctions, which dominate the transepithelial resistance values, thus exhibiting the properties of a polarized cell line (Delie & Rubas, 1997). In addition, they are used as models for absorptive epithelium as they exhibit the property of transporting fluid from their apical surface to their basolateral surface (Delie & Rubas, 1997). This process is demonstrable in the formation of ‘domes' across the monolayer when cultured on solid supports. The cells also transport fluid when grown on permeable supports but dome formation cannot be assessed when using these filter systems.

We hypothesized that C. jejuni may inhibit absorption of fluid across epithelia as a mechanism of diarrhoeal disease. This hypothesis was examined by studying the effect of infection on dome formation in Caco-2 cell monolayers. In the absence of any increase in detectable epithelial conductance (representing active secretory events), we reasoned that if C. jejuni could disrupt or inhibit dome formation, or collapse domes once formed, then this would inhibit fluid absorption, a potential mechanism of diarrhoeal disease. In this study, we examined the ability of C. jejuni to collapse fluid-transporting domes within Caco-2 cell monolayers and how the collapse of domes relates to electrical resistance of the monolayer and cellular rearrangement of the tight junction protein occludin.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacteria.
Strains used in this study were as follows: wild-type strains – 11168 (Parkhill et al., 2000), L115 (strain from a child with severe colitis; Everest et al., 1992, 1993), G1 (strain causing diarrhoea and Guillain–Barre syndrome), 81-176 (clinical isolate causing inflammatory diarrhoea in human volunteers; Black et al., 1988); mutant strains – NCTC 12189 (Newell et al., 1985; Everest et al., 1993), 11168 kpsM (capsule-minus mutant; Karlyshev et al., 2000), 11168 cdtA (cytolethal distending toxin mutant – mutation in A subunit of toxin), 11168 tlyA (mutant in what is characterized as a haemolysin in other pathogenic bacteria but may be a possible methylase or regulator in C. jejuni; Martino et al., 2001; Zhang et al., 2002), 11168/G1 pldA (mutant in contact-dependent phospholipase A), 11168 pglH, pglI, pglJ (N-linked bacterial protein glycosylation; Karlyshev et al., 2004; Larsen et al., 2004), 11168 peb3 (major antigenic protein – transport protein/colonization factor; Pei et al., 1991; Linton et al., 2002), 11168 flhF (totally aflagellate mutant), 11168 fliD (flagella mutant). We have used both wild-type clinical isolates and mutants in putative virulence determinants for this study. The rationale for studying the particular mutants that were chosen was that the genes they lack are known to code for proteins involved in colonization and adherence factors to cells or animal mucosa, or they have been postulated to have a role in the pathogenesis of diarrhoeal disease. 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/G1 pldA, pglH), Professor Julian Ketley at the University of Leicester, UK (11168 pglI, pglJ) and Professor Charles Penn, University of Birmingham, UK (11168 flhF and fliD).

Bacteria were grown in Mueller–Hinton broth and agar (Oxoid) and incubated at 37 °C in a variable-atmosphere incubator (VAIN; Don Whitley Scientific) in an atmosphere of 6 % hydrogen, 5 % carbon dioxide, 5 % oxygen and 84 % nitrogen.

Dome formation in Caco-2 cells.
Caco-2 cells were seeded at 2x104 cells cm–2 in 12-well plates and incubated until confluent. Cells were maintained in Dulbecco's minimal essential medium (DMEM) with 10 or 20 % fetal calf serum without antibiotics. The confluent monolayers were washed and then inoculated with 10 µl bacterial suspension, containing varying numbers of bacteria for different experiments (between 103 and 109 c.f.u.). Infected monolayers were incubated for up to 7 days at 37 °C in a 6 % carbon dioxide humidified atmosphere. Monolayers for the investigation of bacterial invasion were also treated with 200 µg gentamicin ml–1 for 4 h, for experiments involving bacteria invading cells. The bacteria were left on the monolayers for 24 h prior to gentamicin treatment for low (1000 c.f.u.) inoculum experiments. After 4 h, the cells were washed and replaced with DMEM without gentamicin for the remainder of the experiment. Gentamicin kills extracellular bacteria, allowing the effects of intracellular bacteria alone to be investigated. To check that bacteria were indeed intracellular, representative monolayers were lysed using 1 % Triton-X and viability was counted on Mueller–Hinton agar. Domes were counted daily by microscopy for infected and uninfected monolayers. Results were expressed as domes per low-power field (LPF) (x4 objective). At least 10 microscopic fields were counted per monolayer for each time point. Three replicates of infected and uninfected cells per experiment were performed and each experiment was repeated three times on different days.

Measurement of transepithelial electrical resistance (TEER).
Caco-2 cells were grown on 24 mm diameter semi-permeable filters of 0·4 µm pore size in Transwell units (Costar). Cells were used 10–12 days post-confluence when they were fully differentiated. Bacteria were added at 103–105 c.f.u. for different experiments. Infected monolayers were incubated for up to 7 days at 37 °C in a 6 % carbon dioxide humidified atmosphere. TEER was measured daily using the Millicell electrical resistance meter (Millicell ERS; Millipore) and monolayer resistance was determined by the calculation – monolayer resistance minus blank resistance (Transwell without Caco-2 cells)xarea of the Transwell (4·7 cm2 for 24 mm filters)=monolayer resistance ({Omega} cm2). Blank electrical resistance values were usually 30 {Omega} cm2. Cell monolayers were considered fully differentiated when showing electrical resistances of >200 {Omega} cm2.

Ussing chambers.
Confluent monolayers of cells were examined by using Ussing-type chambers as described previously (Hardy et al., 1999). Short-circuit current (ISC) was monitored whilst voltage-clamping (VCC600 amplifier; Physiologic Instruments) the Caco-2 epithelia at 0 mV with the mucosal bath as ground. Transepithelial resistance (RT) was calculated using Ohm's law from voltage pulses of 1 mV for 0·35 ms. All readings were automatically corrected for electrode offsets and solution resistance and recorded online using a Powerlab/8SP (AD Instruments). The bathing solutions contained 113 mM NaCl, 4·5 mM KCl, 25 mM NaHCO3, 1·2 mM Na2HPO4, 1·1 mM CaCl2, 1·2 mM MgCl2, 10 mM glucose, pH 7·4 when gassed at 37 °C. Reduced-chloride Ringer's solution (18 mM chloride) was the same as the standard Ringer's except the NaCl was replaced with equimolar sodium gluconate and the CaCl2 was increased to 5·7 mM (to compensate for the chelating effect of gluconate on calcium). Chloride conductance of the apical membranes was measured by using a chloride gradient with reduced chloride in the mucosal bath. Permeabilization of the basolateral membrane was achieved using nystatin at 0·36 mg ml–1 as described by Sheppard et al. (1993).

Occludin staining.
Tight junction occludin distribution was investigated by fluorescent antibody staining in infected and uninfected cells. Occludin is a tight junction protein whose functions include maintaining tight junction integrity. When occludin is disrupted, tight junction integrity is lost, electrical resistance is decreased and water may be lost from the paracellular pathway (Simonovic et al., 2000). Immunofluorescent staining was performed on both uninfected and infected monolayers. Infected monolayers were stained when TEER of the monolayer had fallen below 200 {Omega} cm2, indicating that tight junction integrity was lost. Cells were grown on glass coverslips to confluence, and when differentiated they were infected for the appropriate time, then fixed with paraformaldehyde (3 %), pH 7·4, in PBS for 15 min. Cells were then rinsed and permeabilized with 0·2 % Triton-X 100 in PBS for 15 min and blocked in 1 % BSA in PBS. Monolayers were incubated with anti-occludin antibody (Chemicon; 1 : 1000 dilution) for 1 h, followed by fluorescein-conjugated anti-rabbit IgG (1 : 1000) antibody for 1 h. Monolayers were washed and mounted with Antifade reagent (Molecular Probes). Monolayers were photographed by using a Nikon digital camera on a Leica fluorescent microscope.

Host cell viability.
Host cell viability was determined using live/dead staining (Molecular Probes) and apoptosis by fluorescent terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) reaction. DMEM pH was measured for both uninfected and infected monolayers for each time point.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
C. jejuni infection of Caco-2 cells eliminates domes within the monolayer
Caco-2 monolayers exhibited on average 10 domes per LPF at 20 days post-confluence (Fig. 1). C. jejuni infection of Caco-2 cells at high inocula caused dome numbers to collapse within 24 h (Fig. 2). C. jejuni strains 11168, G1, 81-176 (not shown) and L115 (shown) at a dose of 109 c.f.u. reduced dome number at 24 h but not at earlier time points. Using smaller inocula, C. jejuni infection of Caco-2 monolayers resulted in a time-dependent reduction in the number of domes with the maximum effect observed 6 days after infection (Fig. 3). Fig. 3 shows results for strains 11168, G1 and 81-176. Strain L115 gave identical results in these experiments (data not shown). Small numbers of bacteria inoculated on day 1 (103 c.f.u.) grew slowly under the assay conditions and reached a threshold level over several days where sufficient bacteria were able to induce this phenomenon (107 c.f.u.) (Fig. 3). Uninfected monolayers maintained dome formation over the course of the experiment although there was some variation in doming observed each day. Consistently, domes were maintained from uninfected monolayers over the course of the experiment, contrasting markedly with dome loss from infected monolayers.



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Fig. 1. Caco-2 cell monolayer showing one dome (x400 high power).

 


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Fig. 2. C. jejuni infection of Caco-2 cells with a large initial inoculum both collapses domes and decreases TEER within 24 h. (a) C. jejuni strain L115 at a dose of 109 c.f.u. causes domes on Caco-2 monolayers to be lost at 24 h but not at early time points. Strains 11168, 81-176 and G1 gave similar results but are not shown for clarity. (b) Dome collapse correlates with decreases in TEER at 24 h post-infection using a high initial inoculum for both C. jejuni L115. Again strains 11168, 81-176 and G1 gave similar results but are not shown. Error bars represent standard error of the means.

 


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Fig. 3. C. jejuni infection of Caco-2 cells collapses dome formation. Bars represent standard errors for all replicates and all experiments (three replicates repeated three times on different days). y axis on the left of the figure represents domes per LPF (x40) and y axis on the right of the figure represents c.f.u. per well as judged by surface plate counts of extracellular bacteria. Results for strains 11168, G1 and 81-176 are shown. Strain L115 gave identical results in these experiments (data not shown). Neg. refers to uninfected monolayers at all time points.

 
Bacteria must be both intracellular and extracellular for domes to be inhibited
Addition of gentamicin 24 h post-infection abolished the ability of C. jejuni to reduce dome numbers (Fig. 4), whereas dome numbers were retained in infected monolayers, where bacteria resided intracellularly. Results shown are for strains G1 and 11168 but strains L115 and 81-176 gave identical results. This effect was observed after incubation of infected monolayers for 4 days. Thus, only when bacteria are both intracellular and extracellular are domes lost from the monolayer. This suggests that bacteria residing outside cells, perhaps adherent or close to the cell surface, exert an influence on cell transport functions.



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Fig. 4. Bacteria must be both intracellular and extracellular for domes to be inhibited. Bars represent standard errors for all replicates in all experiments. Results shown are for strains G1 and 11168 but strains L115 and 81-176 gave identical results. Three replicates repeated three times on different days. Domes per LPF (x40). Uninfected, Uninfected monolayer; G1 ex, G1 extracellular and intracellular bacteria present, no gentamicin treatment; G1 in, G1 intracellular bacteria only, gentamicin treated; Uninfected+Gent, uninfected monolayer, gentamicin treated; Uninfected–Gent, uninfected monolayer, not gentamicin treated; 11168 ex, 11168 extracellular and intracellular bacteria present, no gentamicin treatment; 11168 in, 11168 intracellular bacteria only, gentamicin treated.

 
Electrical resistance of C. jejuni-infected Caco-2 cells
Normal differentiated polarized Caco-2 cells have tight junctions with a TEER of >200 {Omega} cm2. Measurement of TEER in Caco-2 cell monolayers infected with a large initial inoculum significantly reduced the electrical resistance of the monolayer after 24 h but not at earlier time points (Fig. 2b). This fall in TEER coincided precisely with the loss of domes from an infected monolayer at this time point (Fig. 2a).

To confirm the results obtained by using the Millicell electrical resistance meter, TEER measurements were also calculated from Caco-2 monolayers mounted in Ussing chambers under short-circuit conditions (Fig. 5). The TEER of the Caco-2 monolayers infected initially with lower numbers of C. jejuni (1000 c.f.u.) was again effectively abolished in monolayers infected with C. jejuni 11168 for 5 days [C. jejuni 11168, (mean) 52 {Omega} cm2; C. jejuni L115, (mean) 90 {Omega} cm2; uninfected controls, (mean) 182 {Omega} cm2, n=6]. Again, the fall in TEER coincided with loss of domes from the infected monolayers. Mutations in cytolethal distending toxin (cdtA) and the proposed haemolysin/regulator/methylase (tlyA) had little effect on TEER in infected cells, indicating no role for these particular mutants in TEER collapse.



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Fig. 5. Change in TEER of C. jejuni-infected Caco-2 cells. Percentage change in TEER in Caco-2 monolayers, measured in Ussing chambers and infected with 1000 bacteria per well as the initial inoculum. Results are expressed as mean values±standard errors of all experiments (three replicates repeated three times on different days). Results shown are for strain 11168. L115 and 81-176 gave identical results in these assays.

 
Measurement of Caco-2 cell ISC in intact monolayers and apical membranes
Any reduction in TEER may represent an increase in tissue conductance (due to net fluid secretion). To examine this we performed experiments on the effect of infection by C. jejuni strains 11168 and L115 on ISC across intact Caco-2 cell monolayers, but were unable to detect any alterations from uninfected monolayers when measured up to 24 h after infection (data not shown). Subsequently, we measured the current across the apical membranes, having permeabilized the basolateral membrane with nystatin. To detect any changes in tissue chloride conductance a concentration gradient of 122 mM serosal bathing solution versus 18 mM mucosal bathing solution was created. Again, we found no significant differences in the measured ISC in the apical membranes of infected and uninfected cells up to 24 h after infection (data not shown). In summary, we have found no evidence to suggest that the fall in TEER is due to increases in ISC as a result of secretory activity induced by C. jejuni.

TEER of infected Caco-2 cells is affected by both extracellular and intracellular bacteria but not intracellular bacteria alone
The fall in TEER of C. jejuni-infected Caco-2 monolayers after 5 days was prevented by exposure of the monolayers to gentamicin for 4 h, applied 24 h after infection of the monolayers (data not shown). This was true for strains 11168, G1, L115 and 81-176. These findings show that intracellular organisms alone were unable to alter TEER. Representative monolayers were lysed after gentamicin treatment to determine if bacteria were intracellular in these assays. Experiments yielded intracellular C. jejuni in numbers similar to those described previously for these strains (Everest et al., 1992).

The tight junction protein occludin is rearranged in infected cells
Fig. 6 demonstrates changes in the cellular distribution of occludin. These changes were observed in Caco-2 cell monolayers infected with C. jejuni at the time points when TEER was reduced but not before.



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Fig. 6. Occludin staining in uninfected and C. jejuni 11168-infected Caco-2 cells. (a) Uninfected Caco-2 cells stained with fluorescein-labelled antibody to occludin demonstrating local concentration in tight junctions of cells. (b) 11168-infected Caco-2 cells at day 6 post-infection; as domes have been lost and TEER is <200 {Omega} cm2. Other strains tested showed identical results.

 
Host cell viability
Concern over the viability of host cells infected for a longer than usual time (greater than 24 h) allowed us to investigate host cell viability and media pH changes in infected and uninfected monolayers. Caco-2 cells in an infected monolayer were viable as judged by vital staining, so the collapse of domes and tight junction disruption caused by C. jejuni is not simply a result of cell death. Likewise, there was no change in tissue culture medium pH over the course of the experiment for infected, compared to uninfected, monolayers (data not shown).

Role of putative bacterial virulence factors in dome collapse and tight junction integrity
All mutants tested, with one exception, behaved like wild-type isolates in our assays. Thus, we have been unable to find a role for a defined bacterial molecule causing the effects we describe. The exception was C. jejuni strain NCTC 12189, a genetically undefined mutant, where infection of doming monolayers showed no collapse of domes, loss of TEER or occludin changes (data not shown).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Caco-2 cells mimic intestinal absorptive epithelium by forming domes, reflecting their ability to transport fluid and electrolytes from the apical to the basolateral surface of the monolayer. We have shown that C. jejuni infection of Caco-2 cells causes domes to collapse in infected monolayers, reflecting a net loss of absorptive function. The collapse of transporting domes from infected cells coincides with rearrangement of the tight junction protein occludin, one of the proteins responsible for tight junction integrity and maintenance of monolayer electrical resistance. We propose that domes collapse because tight junctions become ‘leaky’ due to disruption of proteins integral to their function. Fluid accumulated under the dome of transporting Caco-2 cells and between the plastic of the tissue culture plate escapes via the leaky tight junctions known as the paracellular pathway. Accumulation of epithelial fluid on the basolateral side of the epithelia is usually maintained by a sufficiently high TEER. This is dependent, at least in part, upon correct functioning of occludin within tight junctions. Thus, collapse of epithelial fluid transport, maintenance of occludin and decrease in monolayer electrical resistance, as a consequence of failure of cell tight junctions, are all mediated by C. jejuni infection.

One previous study (Bras & Ketley, 1999) has shown no disruption of Caco-2 cell tight junction integrity by C. jejuni, as measured by electrical resistance at early time points (2, 4, 6, 8, 10 h post-infection compared to Salmonella sp., which disrupt tight junction integrity by a fall in electrical resistance as early as 2 h post-infection). However, in the same publication, Bras & Ketley (1999) showed that C. jejuni can decrease the electrical resistance of Caco-2 cells 24 h post-infection using an inoculum of 1–2x109 ml–1. Our data confirm these findings in that for an initial large inoculum, dome formation was lost 24 h post-infection, which was found to correlate with loss of TEER for infected but not uninfected monolayers. For the results presented in both this paper and that of Bras & Ketley (1999), bacteria are present extracellularly (interacting with the apical cell surface) inside the cells (after bacterial invasion) and have passed through or between cells (transcytosis), indicating the importance of extracellular bacteria for disruption of TEER. The time taken for C. jejuni to disrupt cellular tight junctions in vitro is longer than that observed for Salmonella typhimurium (Jepson et al., 1995) and enteropathogenic Escherichia coli (Canil et al., 1993; Berkes et al., 2003). The extended time span may reflect the conditions of a tissue culture system (cultured in 6 % carbon dioxide rather than microaerophilic conditions), which will favour eukaryotic cell survival and growth and may be suboptimal for the organism being studied. C. jejuni takes longer to replicate under the assay conditions we employed and therefore host cell–bacteria interaction is delayed, accounting for the longer time points at which the organism exerts a biological effect on epithelial cell function. The small numbers of bacteria used initially (1000 organisms), which then replicate over subsequent days, extend the time for the biological effects of the infection to occur; hence experimental conditions are monitored over 5–7 days for each experiment. However, using a larger initial inoculum reduces the time for dome collapse and fall in TEER, indicating that a ‘threshold’ number of organisms must be reached for these phenomena to occur (Bras & Ketley, 1999). The finding that C. jejuni takes longer to increase the permeability of the paracellular pathway compared with other enteropathogens should not detract from the fact that C. jejuni can alter this important aspect of cell physiology, with all the resulting implications that these events may play an important role in the pathogenesis of diarrhoea caused by these organisms.

C. jejuni strain NCTC 12189, an aflagellate, non-motile spontaneous laboratory mutant (Dolby & Newell, 1985), was the only strain found that did not collapse domes or decrease TEER over the experimental time course (data not shown). Thus, we reasoned that flagella may play a role in the process of dome collapse, perhaps via the secreted Cia effector proteins (Konkel et al., 1999a, b, 2001) previously shown to be actively inserted inside infected cells via flagella. However, the defined flagella mutants flhF and fliD were able to collapse domes in these assays as effectively as the wild-type parent (data not shown). NCTC 12189 is also exquisitely sensitive to antibiotics, suggesting additional physiological defects, so we suspect this strain may also harbour other mutations, which may be responsible for its performance in these experiments. We examined a number of other mutants defective in various bacterial structural and secreted molecules in our assay system but these acted in a manner indistinguishable from the wild-type in our experiments. It has been postulated that CdtA and TlyA are possibly involved in the pathogenesis of diarrhoeal disease due to C. jejuni, but in our assays these mutants behaved like the wild-type clinical isolates. The kpsM mutant has previously been shown to be less virulent in a ferret diarrhoeal model (Bacon et al., 2001) but behaves like the wild-type strain in our studies. The kpsM mutant is killed by serum and as it lacks a capsule it may be more efficiently killed by phagocytes. So the kpsM mutant may not be so readily able to cause diarrhoea in the ferret model because it is killed by the environment of the host intestinal tract and hence less able to colonize the epithelium. Our epithelial cell models have no such host-selective pressures and hence we would never see such an effect in our system.

The observation that extracellular bacteria seem to be important for the collapse of domes, loss of TEER and rearrangement of occludin suggests an important role for bacteria outside cells, perhaps subverting host cell signalling by an as yet unknown mechanism. It is possible that bacterial secreted proteins from organisms outside cells influence the absorptive transport processes of enterocyte-like cells. Thus, the Cia or FlaC effectors look attractive candidates for such a role in host cell subversion.

Tight junctional integrity per se has never been determined in natural human disease due to C. jejuni. However, the histopathology of intestinal biopsies of acute disease shows intense neutrophil infiltration in the infected mucosa and neutrophils in diarrhoeal faeces (Skirrow & Blaser, 2000). For these neutrophils to be present in mucosa and faeces they presumably have had to traverse intestinal epithelial tight junctions in response to bacterial invasion, which in turn must lose their tight junctional integrity in order to allow neutrophils to migrate through the epithelial monolayer. Using a macaque model of C. jejuni diarrhoeal disease, Russell et al. (1993) demonstrated colonic damage with intercellular junctions widened by electron microscopy. As previously mentioned, this may be a consequence of infiltrating neutrophils or the direct effects of the organism on the mucosa.

Our data suggest that C. jejuni inhibits absorption in infected Caco-2 cells, as we could detect no secretory activity in infected cell monolayers. If the tight junctional integrity of the intestinal epithelium is lost, electrolyte and fluid absorption are likely to be compromised. If these observations are manifested in vivo it is likely that they contribute to the clinical manifestations of diarrhoea. The in vitro experimental system employed here could provide a useful model for investigation of how C. jejuni effector molecules mediate this process.


   ACKNOWLEDGEMENTS
 
This study was financially supported by the Biotechnology and Biological Sciences Research Council (BBSRC). Grateful thanks to Brendan Wren, Andrey Karleyshev, Julian Ketley and Charles Penn for the generous gifts of mutants for this study.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 7 February 2005; revised 1 April 2005; accepted 20 April 2005.



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