School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4233, USA
Correspondence
Michael E. Konkel
konkel{at}mail.wsu.edu
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ABSTRACT |
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Abbreviations: CadF, Campylobacter adhesion to Fibronectin; Fn, fibronectin; FAK, focal adhesion kinase; ECM, extracellular matrix
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INTRODUCTION |
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Campylobacter jejuni is a Gram-negative, microaerophilic bacterium, and is recognized as the most frequent cause of gastrointestinal disease in developed countries. The ability of C. jejuni to colonize the gastrointestinal tract of humans is proposed to be essential for disease (Allos & Blaser, 1995; Wassenaar & Blaser, 1999
). Fauchère et al. (1986)
found that C. jejuni isolated from individuals with fever and diarrhoea exhibited greater binding to epithelial cells than strains isolated from individuals without fever and diarrhoea. Numerous studies have been done to identify and characterize C. jejuni adhesins (De Melo & Pechère, 1990
; Fauchère et al., 1989
; Jin et al., 2001
; Kelle et al., 1998
; Kervella et al., 1993
; Konkel et al., 1997
; McSweegan & Walker, 1986
; Moser & Schröder, 1995
; Moser et al., 1992
, 1997
; Pei & Blaser, 1993
; Pei et al., 1998
; Schröder & Moser, 1997
). Based on these studies, C. jejuni appears to synthesize a number of adhesive molecules.
Fibronectin (Fn) is a 220 kDa glycoprotein that is present at regions of cell-to-cell contact in the gastrointestinal epithelium, thereby providing a potential binding site for pathogens (Quaroni et al., 1978). Several bacterial pathogens, including Staphylococcus aureus (Kuusela, 1978
; Rydén et al., 1983
), Streptococcus pyogenes (Jaffe et al., 1996
; Myhre & Kuusela, 1983
), Salmonella enteritidis (Baloda et al., 1985
), Escherichia coli (Fröman et al., 1984
; Visai et al., 1991
), Neisseria gonorrhoeae (van Putten et al., 1998
), Mycobacterium avium (Schorey et al., 1996
) and Treponema species (Dawson & Ellen, 1990
, 1994
; Thomas et al., 1985
), bind Fn. We identified a 37 kDa outer-membrane protein in C. jejuni that mediates the binding of the organism to the extracellular matrix (ECM) component Fn (Konkel et al., 1997
). The cadF (Campylobacter adhesion to Fn) gene has thus far been found to be conserved among C. jejuni and Campylobacter coli isolates (Konkel et al., 1999
). In vivo studies have suggested that the CadF protein is required for the colonization of chickens by C. jejuni (Ziprin et al., 1999
).
Published work indicates a relationship between the pericellular Fn matrix and the cytoskeleton (Gumbiner, 1996; Miyamoto et al., 1998
; van der Flier & Sonnenberg, 2001
). In mammalian cells, the actin cytoskeleton is necessary for a variety of cellular processes including control of cell-to-cell and cell-to-substrate interactions. Actin nucleation occurs at membrane-associated sites called focal adhesions. Focal adhesions are the sites at which the bundles of actin filaments (stress fibres) are cross-linked with membrane-associated adhesion molecules (e.g. integrins) and extracellular molecules (Gumbiner, 1996
; Sarkar, 1999
). The integrin molecules bind extracellularly to matrix components and intracellularly associate with protein complexes consisting of vinculin, talin,
-actinin, paxillin, tensin, zyxin and focal adhesion kinase (FAK) (Miyamoto et al., 1998
; Tachibana et al., 1995
). The integrins are transmembrane glycoprotein receptors composed of heterodimeric
ß subunits (Danen & Yamada, 2001
; Hynes, 1992
; van der Flier & Sonnenberg, 2001
; Vuori, 1998
). The two subunits are noncovalently associated with one another (1 : 1) in the membrane. There are 18 known
subunits and 8 known ß subunits (van der Flier & Sonnenberg, 2001
). Different
and ß subunit combinations dictate the specificity of cell-to-cell and cell-to-ECM recognition. The
5ß1 integrin receptor specifically binds Fn (Hynes, 1992
; Miyamoto et al., 1998
). Integrin occupancy and clustering is associated with tyrosine phosphorylation of cellular cytoplasmic proteins including FAK and paxillin, and is a means of regulating host signal transduction events leading to actin rearrangements (Miyamoto et al., 1998
; Tachibana et al., 1995
). As stated earlier, certain bacterial pathogens are known to utilize host cell ECM and cytoskeleton components to their benefit. For example, N. gonorrhoeae appears to utilize a Fn-mediated uptake pathway involving integrin receptors (van Putten et al., 1998
). The binding of N. gonorrhoeae to Fn is proposed to trigger the uptake of the organism via integrin receptors by stimulating the host cell signalling pathways that are responsible for cytoskeletal rearrangement.
Because C. jejuni isolates possess a minimum of three different adhesive molecules including CadF, JlpA and PEB1 (Jin et al., 2001; Konkel et al., 1997
; Pei et al., 1998
), we sought to determine the step of the infectious process in which the CadF outer-membrane protein participates. We investigated the binding properties of two C. jejuni clinical isolates, F38011 and 81-176, to the INT 407 cell line (human embryonic intestinal cells) and the participation of the INT 407 cells in C. jejuni uptake. While the host cell cytoskeletal components involved in C. jejuni F38011 uptake have not been examined previously, C. jejuni 81-176 has been reported to be internalized via a novel pathway exclusively involving microtubules (Bacon et al., 2000
; Hu & Kopecko, 1999
, 2000
; Kopecko et al., 2001
; Oelschlaeger et al., 1993
). Based on the experiments performed herein implicating microfilaments in C. jejuni uptake, additional experiments were done to determine if the host cell signalling events known to be associated with cytoskeletal rearrangement occur during C. jejuni entry.
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METHODS |
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Binding of C. jejuni to immobilized ECM.
Binding of C. jejuni isolates to human plasma Fn (Sigma) was assessed as previously described (Konkel et al., 1997). Specificity of binding was determined by preincubating Fn-coated coverslips with a 1 : 50 dilution of a rabbit anti-human Fn antibody (Telios Pharmaceuticals), preincubating C. jejuni isolates with a 1 : 50 dilution of a goat anti-C. jejuni 37 kDa serum, or by the addition of 100 µg Fn ml-1. For each coverslip, the bacteria in each of three randomly chosen fields were counted.
Gel electrophoresis and immunoblot analysis.
Bacterial whole-cell extracts (an equivalent of 0·1 OD600 units) were solubilized in single strength electrophoresis sample buffer and incubated at 95 °C for 5 min. Proteins were separated in SDS-12·5 % PAGE minigels as previously described (Laemmli, 1970) and electrophoretically transferred to PVDF membranes (Immobilon P; Millipore). The membranes were washed three times in PBS and incubated for 18 h at 4 °C with a 1 : 500 dilution of the goat anti-C. jejuni 37 kDa serum in PBS pH 7·4/0·01 % Tween-20 containing 20 % foetal bovine serum (FBS). Bound antibodies were detected using peroxidase-conjugated rabbit anti-goat IgG (Sigma) at a 1 : 2000 dilution and 4-chloro-1-naphthol (Sigma) as the chromogenic substrate.
Bactericidal concentration of gentamicin.
Assays were performed to determine the concentration of gentamicin and length of incubation time required to kill each isolate used in this study. C. jejuni isolates, C. freundii and S. typhimurium were suspended in Minimal Essential Medium (MEM) (Gibco Invitrogen) supplemented with 1 % FBS (MEM-1 % FBS) or 10 % FBS (MEM-10 % FBS) at an approximate concentration of 2x106 c.f.u. ml-1. Aliquots (1 ml) of the bacterial suspensions were placed in a 24-well plate and incubated at 37 °C in a humidified, 5 % CO2 incubator for 2 h. Gentamicin (Life Technologies) was added to the media at a final concentration of 0, 100, 250, 500 and 750 µg ml-1, and the suspensions again incubated as above. Following incubation (2 or 3 h), the bacterial suspensions (1 ml) were removed and bacteria pelleted by centrifugation at 6000 g for 10 min. The supernatant fluids were discarded to remove the gentamicin and bacteria resuspended in 1 ml PBS. Suspensions were serially diluted and the number of viable bacteria assessed. Each isolate was tested at least three times in triplicate.
INT 407 binding and internalization assays.
A stock culture of INT 407 cells (human embryonic intestine, ATCC CCL 6) was obtained from the American Type Culture Collection. This cell line was cultured in MEM-10 % FBS at 37 °C in a humidified, 5 % CO2 incubator. For experimental assays, each well of a 24-well tissue culture tray was seeded with 1·4x105 cells per well and incubated for 18 h at 37 °C in a humidified, 5 % CO2 incubator. The cells were rinsed with MEM-1 % FBS and inoculated with approximately 5x107 c.f.u. of a bacterial suspension. Tissue culture trays were centrifuged at 600 g for 5 min, and incubated at 37 °C in a humidified, 5 % CO2 incubator. For binding, the infected monolayers were incubated for 2 h, rinsed three times with PBS and the epithelial cells lysed with a solution of 0·1 % (v/v) Triton X-100 (Calbiochem). The suspensions were serially diluted and the number of viable, adherent bacteria determined by counting the resultant colonies on MH/blood plates. To measure bacterial internalization, the infected monolayers were incubated for 2 h, rinsed three times with MEM-1 % FBS, and incubated for an additional 3 h in MEM-1 % FBS containing a bactericidal concentration of gentamicin. The number of internalized bacteria was determined as outlined above. Unless otherwise stated, the reported values represent the mean counts±SD derived from triplicate wells. All assays in this study using cytoskeletal inhibitors were performed at a m.o.i. ranging between 50 and 500 to ensure reproducibility, and repeated a minimum of three times. Regardless of whether a m.o.i. of 50 to 1 or 500 to 1 was used, the effect of the cytoskeletal inhibitory drugs was the same.
Competitive inhibition assays.
INT 407 cells were inoculated with a suspension containing the C. jejuni 81-176 (TetR) isolate and the C. jejuni cadF mutant (KanR) as well as the C. jejuni 81-176 (TetR) isolate and the C. jejuni F38011 (StrepR/NalR) isolate. Binding assays were performed as mentioned above except that a m.o.i. of approximately 10 was used for the C. jejuni 81-176 (TetR) wild-type isolate. To determine the number of bacteria of a particular isolate that became bound, serial dilutions of the suspensions were plated on MH/blood agar plates supplemented with the appropriate selective antibiotic. No evidence of horizontal gene transfer was apparent as judged by lack of recovery of StrepR/NalR/TetR isolates upon mixing experiments with the C. jejuni 81-176 (TetR) and C. jejuni F38011 (StrepR/NalR) isolates.
Inhibitor studies.
INT 407 cells were preincubated for 45 min in MEM-1 % FBS with nocodazole, cytochalasin D or mycalolide B at the concentrations indicated in the text. When performing experiments looking at the combined effects of inhibitors, nocodazole and cytochalasin D were both added to INT 407 cells during the preincubation step. Following incubation, cells were infected with approximately 5x107 c.f.u. of each isolate while maintaining indicated inhibitor concentrations. S. typhimurium was preincubated anaerobically at 37 °C for 3 h prior to infection. Binding and internalization assays were performed as outlined above. The values reported represent the percentage of adherent and internalized bacteria relative to the control (untreated sample) or the mean±SD of adherent and internalized bacteria. INT 407 cell viability, following inhibitor treatment, was assessed by rinsing the INT 407 cells twice with PBS and staining the cells for 5 min with 0·5 % trypan blue. The cells were then rinsed twice with PBS and counter-stained for 5 min with 0·5 ml 0·5 % phenol red (Humason, 1979). The INT 407 cells were visualized with an inverted microscope.
Reversibility of cytochalasin D.
INT 407 cells were pretreated with cytochalasin D and infected with bacteria as described above. To asses the reversibility of cytochalasin D, the INT 407 cell monolayers were rinsed after 2 h with MEM-1 % FBS. A set of the cytochalasin D-treated cells were then incubated for an additional 2 h in MEM-1 % FBS containing cytochalasin D or in the presence of medium alone. The number of adherent and internalized bacteria was determined as outlined above.
Preparation of the polyclonal antisera.
Female New Zealand White rabbits were subcutaneously injected with 100 µg of each bacterial whole-cell extract in TiterMax Gold (CytRx Corporation). A booster injection of 50 µg whole-cell extract in Freund's incomplete adjuvant (Sigma) was given after 4 weeks. Blood was collected prior to first and second immunizations, and 2 weeks after the second immunization. Sera were stored at -20 °C.
Confocal microscopy examination of C. jejuni infected cells.
INT 407 cells (5x104 cells) were cultured on 13 mm circular glass coverslips for 18 h at 37 °C in a humidified, 5 % CO2 incubator. The cells were infected by the addition of 0·5 ml of a bacterial suspension (1x106 c.f.u. per well) in MEM. Mock-infected cells were used in certain instances as a negative control. Prior to infection, cell monolayers were rinsed once with MEM. Following incubation (45 min), the cell monolayers were rinsed three times with PBS and fixed with 3·0 % glutaraldehyde or 3·0 % paraformaldehyde in 0·1 M phosphate buffer (pH 7·2). Cells were permeabilized with 0·1 % (v/v) Triton-X 100. Tubulin was stained using a 1 : 250 dilution of FITC-conjugated monoclonal antibody against tubulin (clone DM 1A; Sigma). Actin was stained using FITC-labelled phalloidin (Sigma) at a concentration of 0·4 µg ml-1. Primary antibodies directed towards bacteria were used at a 1 : 250 dilution followed by a secondary goat anti-rabbit IgG-rhodamine F(Ab')2 fragment antibody at a 1 : 500 dilution.
Immunoprecipitation.
INT 407 cells were cultured in six-well tissue culture trays as outlined above. INT 407 cell monolayers were rinsed once with MEM and inoculated with a suspension of the C. jejuni F38011 wild-type isolate and the isogenic cadF mutant (m.o.i. 100). Tissue culture trays were centrifuged and incubated as stated previously. At timepoints indicated, cell monolayers were rinsed three times with PBS and lysed by the addition of ice-cold lysis buffer (125 mM Tris/HCl pH 8·0, 137 mM NaCl, 10 % glycerol, 0·5 % sodium deoxycholate, 1 % NP-40, 2 mM EDTA, 1 mM PMSF, 1 mM Na3VO4 and 1·76 Trypsin Inhibitor Units aprotonin). The insoluble and soluble fractions were separated by centrifugation at 14 000 g for 10 min at 4 °C. Immunoprecipitation was performed by adding a mouse anti-paxillin antibody (Clone 349, Transduction Labs) to the soluble fraction and incubating at 4 °C for 2 h with end-over-end mixing. Following mixing, prewashed protein G agarose beads (Gibco-BRL) were added and samples mixed for an additional 2 h at 4 °C. The precipitate was rinsed four times in ice-cold lysis buffer and once with PBS. Samples were analysed by SDS-PAGE and immunoblot analysis as outlined above. Immunoblot detection of phosphorylated paxillin was performed using a 1 : 2000 dilution of a mouse anti-phosphotyrosine antibody (PT-66; Sigma) and a 1 : 80 000 dilution of a peroxidase-conjugated rabbit anti-mouse IgG by enhanced chemiluminescence (Renaissance; NEN Life Science Products). Detection of the total pool of paxillin was performed using a 1 : 10 000 dilution of a mouse anti-paxillin antibody and a 1 : 80 000 dilution of a peroxidase-conjugated rabbit anti-mouse IgG.
Other analytical procedures.
To determine the MIC of gentamicin sulphate, C. jejuni strains 81-176, 81116 and F38011 were cultured on MH/blood agar plates for 24 h at 37 °C under microaerophilic conditions. Bacteria were suspended in 3 ml PBS (OD540=0·17), and 10 µl of each cell suspension was placed onto a MH/blood agar plate containing gentamicin sulphate. Gentamicin concentrations ranged from 1 µg ml-1 to 512 µg ml-1. Before and after spotting the suspensions on the antibiotic containing plates, 10 µl of each cell suspension was also placed on a MH/blood agar plate without antibiotic to ensure that bacterial viability was maintained. MICs were assessed by determining the lowest concentration of gentamicin in which no growth was observed following a 24 h incubation.
To ensure that the phenotype associated with the C. jejuni CadF mutant was due specifically to the mutation of the cadF gene, a knockout was generated in Cj1477c. Cj1477c lies downstream of the cadF gene in C. jejuni F38011, and encodes a putative hydrolase (Cj1477c) (Lyngstadaas et al., 1999; Parkhill et al., 2000
). Cj1477c was disrupted by homologous recombination via a single crossover event between the putative hydrolase gene on the chromosome and an internal fragment of the hydrolase gene on a suicide vector using methods described previously (Konkel et al., 1997
). A 512 bp fragment, which is internal to the 648 bp coding region of Cj1477c, was amplified using the primers CJ1477cKOF (5'-GCA CTT TAA TTG ATT CTA CTG ATG-3') and CJ1477cRTR(KO) (5'-GGC CAA AGG AGT GAT ATT AGC AG-3'). The resultant fragment was ligated into the pCRII cloning vector (TA Cloning System; Invitrogen) and the ligation mixture used to transform E. coli MRF. Following plasmid purification, the cloned 512 bp fragment was then excised by restriction endonuclease digestion with EcoRI, gel-purified and ligated into pBluescript SK+ (pBSKII+) containing the aphA3 gene encoding kanamycin resistance. The pBSKII+ vector was digested with EcoRI and treated with calf intestinal alkaline phosphatase prior to ligation. The resultant plasmid was introduced into C. jejuni F38011 by electroporation. Potential insertional mutants were identified by the acquisition of kanamycin resistance. Disruption of the hydrolase gene in C. jejuni F38011 was confirmed by PCR using gene specific primers.
Plasmid carriage in C. jejuni 81-176 was confirmed by the polymerase chain reaction using the tetO (forward primer, 5'-TTG ACA AAT AAA GGG TTA AGG-3'; reverse primer, 5'-CCT TTC AAA TCT CAT TTT ATA CG-3') and virB11 (forward primer, 5'-GAA CAG GAA GTG GAA AAA CTA GC-3'; reverse primer, 5'-TTC CGC ATT GGG CTA TAT G-3') gene specific primers, followed by sequencing of the PCR-amplified products (Bacon et al., 2000).
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RESULTS |
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To determine if the C. jejuni 81-176 clinical isolate utilizes CadF as an adhesin for host cells, competitive inhibition assays were performed using the C. jejuni F38011 wild-type isolate and C. jejuni F38011 cadF mutant (Table 2). A significant decrease was noted in the number of C. jejuni 81-176 bound to the INT 407 cells in the presence of a 44- and 88-fold excess of the C. jejuni F38011 wild-type isolate. Moreover, increasing the number of competing organisms resulted in a dose-dependent decrease in the number of C. jejuni 81-176 bound. In contrast, a statistically significant difference was not observed in the number of C. jejuni 81-176 bound to the INT 407 cells in the presence of the C. jejuni cadF mutant. Based on these findings, we concluded that CadF serves as an adhesin for the C. jejuni F38011 and 81-176 clinical isolates.
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The effects of cytochalasin D on the C. jejuni F38011 and 81-176 clinical isolates, S. typhimurium, and C. freundii are shown in Table 3. Cytochalasin D resulted in a dose-dependent increase in binding and a dose-dependent decrease in internalization of both C. jejuni F38011 and 81-176 to INT 407 cells regardless of the m.o.i. used in an individual experiment. Consistent with previous reports (Bacon et al., 2000
; Biswas et al., 2000
; Hu & Kopecko, 1999
, 2000
; Oelschlaeger et al., 1993
), cytochalasin D also significantly inhibited the internalization of S. typhimurium and C. freundii by INT 407 cells. Confocal microscopy examination of the infected INT 407 cells did not reveal convincing evidence for the interaction of C. jejuni with cellular microfilaments. However, microfilament supported structures were observed in contact with C. jejuni 81-176 (Fig. 2
). Also noted was the expected interaction of S. typhimurium with a cellular structure supported by microfilaments.
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DISCUSSION |
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Two C. jejuni clinical isolates, F38011 (Lior serotype 90) and 81-176 (Lior serotype 5), were used in this study. The specificity of C. jejuni binding to Fn, via CadF, was demonstrated using antibodies reactive against Fn and CadF, as well as by the addition of exogenous Fn. The binding of C. jejuni F38011 and 81-176 to Fn was inhibited by 54 and 56 %, respectively, with the anti-CadF antibody. Based on these findings, the CadF protein was concluded to mediate the binding of both C. jejuni isolates to Fn. The adhesive nature of the CadF protein in C. jejuni 81-176 was further revealed upon performing competitive inhibition binding assays with the C. jejuni F38011 isolate and C. jejuni F38011 cadF mutant. Here, only the C. jejuni F38011 wild-type isolate was able to competitively inhibit the binding of C. jejuni 81-176 to the INT 407 cells. Because Fn is associated with microfilaments, the role of microfilaments in C. jejuni uptake was examined. Inhibitor studies revealed that the C. jejuni F38011 and 81-176 isolates require microfilament participation for efficient host cell entry. The reduction of C. jejuni uptake with cytochalasin D appeared specific as treatment of the INT 407 cells with this drug had no effect on INT 407 cell viability as judged by staining with trypan blue. Moreover, the effect of cytochalasin D, which inhibits actin polymerization and transient integrin-stimulated FAK activation (Schlaepfer et al., 1999), was reversible. Finally, treatment of the INT 407 cells with mycalolide B also inhibited C. jejuni uptake. Mycalolide B severs microfilaments and sequesters G-actin. The sequestering of G-actin inhibits microfilament polymerization. Consistent with the notion that C. jejuni uptake requires the stimulation of host cell signalling molecules, the amount of phosphorylated paxillin significantly increased 30 min after C. jejuni infection. The amount of phosphorylated paxillin returned to a level equivalent to that of a non-infected control at the 1 h time point. The increase in phosphorylated paxillin slightly preceeded and was concomitant with a sharp rise in the number of C. jejuni internalized, which occurs 3060 min post-infection (Konkel et al., 1993
).
The inhibitory effect of microtubule-depolymerizing agents on the entry of C. jejuni strain 81-176 has been noted previously (Bacon et al., 2000; Hu & Kopecko, 1999
, 2000
; Kopecko et al., 2001
; Oelschlaeger et al., 1993
). Based on this effect, the proposal has been put forth that C. jejuni are internalized via two pathways, one involving microtubules exclusively (considered a high efficiency uptake pathway) and the other involving microfilaments (considered a low efficiency uptake pathway) (Hu & Kopecko, 2000
). While we noted a decrease in the invasiveness of C. jejuni 81-176 with a microtubule inhibitor, a decrease was also noted in the number of intracellular bacteria with this organism in the presence of microfilament inhibitors. The discrepancy between our results and those reported earlier is most likely due to differences in assay protocols. More specifically, in previous work, a 2 h incubation with 100 µg gentamicin ml-1 was used to kill the extracellular bacteria. In our hands, treatment of C. jejuni 81-176 with 100 µg gentamicin ml-1 for 2 h typically resulted in the recovery of 312 % of the bacteria in the original suspension (n=4 individual experiments). Thus, even though C. jejuni 81-176 is gentamicin-sensitive (e.g. MIC of 4 µg ml-1), it was found necessary to increase both the time of exposure and the concentration of the antibiotic to ensure bacterial death. The protocol used by others to determine the bactericidal concentration of gentamicin was not reported (Bacon et al., 2000
; Hu & Kopecko, 1999
, 2000
; Oelschlaeger et al., 1993
). Also noteworthy is that an increase has been noted in the number of C. jejuni 81-176 internalized in the presence of cytochalasin D (Hu & Kopecko, 1999
, 2000
; Oelschlaeger et al., 1993
). One reason for the increase in C. jejuni 81-176 uptake noted by others with cytochalasin D may be due to an increase in the number of organisms bound to the host cells. Increasing the number of cell-associated bacteria while not using a bactericidal concentration of antibiotic would mask any effect of cytochalasin D on C. jejuni uptake. While this explanation seems possible, we do not know the number of cell-associated (adherent) bacteria in earlier studies in which the effect of cytochalasin D on C. jejuni 81-176 uptake was examined as it was not reported. Regardless, a sublethal concentration of gentamicin would not alter the net result of an internalization assay unless there was a significant increase or decrease in the number of adherent bacteria in the test sample versus the control.
To the best of our knowledge, C. jejuni 81-176 is the only isolate to date that has been reported to be internalized exclusively via a microtubule-dependent pathway (Kopecko et al., 2001; Oelschlaeger et al., 1993
). Using 250 µg gentamicin ml-1 for 3 h to kill the extracellular bacteria, Biswas et al. (2000)
observed a reduction in entry with both microfilament and microtubule inhibitors with every C. jejuni clinical isolate tested (n=9). Biswas et al. (2000)
concluded that the most invasive isolates examined in their study utilized microfilaments. Based on the data currently available, the most reasonable conclusion is that C. jejuni uptake involves cooperation of both microfilaments and microtubules. More specifically, uptake of C. jejuni 81-176 and F38011 by INT 407 cells was reduced from 56 to 66 %, respectively, in the presence of nocodazole and cytochalasin D relative to the untreated control (Table 7
). These results are consistent with those reported elsewhere, even though the experimental protocols vary (Biswas et al., 2000
; Oelschlaeger et al., 1993
). What is unclear is why in the presence of both inhibitors a significant number of C. jejuni are still internalized. In comparison, Salmonella invasion was reduced by greater than 99 % by cytochalasin D compared to untreated INT 407 cells. Comparable results for Salmonella have been reported by others (Bacon et al., 2000
; Biswas et al., 2000
; Hu & Kopecko, 1999
, 2000
; Oelschlaeger et al., 1993
).
The mechanism by which treatment of cells with microtubule inhibitors causes a reduction in C. jejuni-cell uptake is not known. However, it has been reported that microtubules regulate the turnover of focal adhesion contacts and modulate a cell's adhesive strength to the ECM (Ballestrem et al., 2000). In fact, treatment of cells with microtubule inhibitors leads to an increase in a cell's adherence to the ECM (Ballestrem et al., 2000
; Sastry & Burridge, 2000
). Thus, the effect of a microtubule inhibitor on C. jejuni-cell uptake could be indirect, as the turnover of the focal adhesion sites is retarded. Alternatively, following initial microfilament-dependent uptake at the level of the plasma membrane, microtubules may be required for subsequent trafficking of the endosome to the interior of the cell as has been proposed for C. freundii (Badger et al., 1999
). Regardless, there is clearly functional cooperation between host cytoskeletal elements (Ballestrem et al., 2000
; Goode et al., 2000
; Sastry & Burridge, 2000
). Because the effects of cytochalasin D and nocodazole on C. jejuni uptake were not additive when used together, it appears most likely that the C. jejuni isolates tested here utilize microfilaments and microtubules together.
C. jejuni strain 81-176 has recently been reported to harbour two plasmids, one of which harbours the tetracycline resistance gene (tetO) and the other harbouring a gene termed virB11 (Bacon et al., 2000). Moreover, in a recent review by Kopecko et al. (2001)
, the authors suggested a possible correlation between the plasmid-borne genes and the microtubule-dependent uptake pathway. To ensure that the strain of C. jejuni 81-176 used in this study harboured both plasmids, the isolate was subjected to PCR using tetO and virB11 gene-specific primers. The identity of the amplified products was subsequently confirmed upon sequencing of the PCR-amplified products (not shown).
Wooldridge et al. (1996) previously reported that C. jejuni uptake is reduced in the presence of protein tyrosine phosphorylation inhibitors. We chose to examine whether paxillin was phosphorylated upon infection of INT 407 cells with C. jejuni because protein tyrosine phosphorylation is one of the earliest events upon integrin stimulation (Clark & Brugge, 1995
). Consistent with the idea that the binding of C. jejuni leads to integrin stimulation, an increase in phosphorylated paxillin was observed 3045 min after C. jejuni F38011 infection. Noteworthy is that the increase in phosphorylated paxillin occurs just prior to and concomitant with an increase in C. jejuni internalization (Konkel et al., 1993
). In contrast to the C. jejuni wild-type isolate, an increase was not observed in phosphorylated paxillin over the course of the assay with cells inoculated with the C. jejuni cadF mutant at a m.o.i. of 100 to 1 (Fig. 4
). However, upon infection of the INT 407 cells with the C. jejuni cadF mutant at a m.o.i. of greater than 2000 to 1, the pattern of phosphorylated paxillin in cells inoculated with the C. jejuni cadF mutant mirrored that obtained with the C. jejuni wild-type isolate. A possible explanation for this finding is that C. jejuni adherence is multifactorial, and that several adhesins simultaneously function to promote host cell binding, after which cell signalling events are stimulated. Thus, the CadF protein does not appear to be required to induce host cell signalling events, but appears to promote signalling events by facilitating the organism's binding to appropriate host cells receptors. Noteworthy is that Watarai et al. (1996)
observed an increase in the tyrosine phosphorylation of FAK and paxillin 2030 min after infection of Chinese hamster ovary cells with Shigella flexneri.
In summary, the data suggest that CadF promotes C. jejunihost cell interactions. Consistent with the notion that bacterial uptake requires host cell signalling, an increase in tyrosine phosphorylated paxillin was observed upon infection of INT 407 cells with C. jejuni. Tyrosine phosphorylation of FAK and paxillin is a means of regulating host signal transduction events leading to actin rearrangement (Miyamoto et al., 1998; Tachibana et al., 1995
). Because paxillin is an integral component of focal adhesions, as are ECM components including Fn, we speculate that CadF is responsible for promoting the initial interaction of C. jejuni with the appropriate host cell receptors involved in uptake. Future studies will be directed toward the identification and biochemical characterization of the CadF Fn-binding domain(s).
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ACKNOWLEDGEMENTS |
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This work was supported by a grants from the NIH (DK58911) and USDA National Research Initiative Competitive Grants Program (USDA/NRICGP, 99-35201-8579) awarded to M. E. K.
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Received 19 June 2002;
revised 2 October 2002;
accepted 4 October 2002.
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