1 Department of Pathobiology, University of Washington, Seattle, WA 98195, USA
2 Department of Oral Biology, University of Florida, Gainesville, FL 32610, USA
Correspondence
Özlem Yilmaz
ozlem{at}u.washington.edu
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ABSTRACT |
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INTRODUCTION |
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The major fimbriae of P. gingivalis are involved in both adhesion to epithelial cells and in the subsequent signalling events associated with invasion (Weinberg et al., 1997; Njoroje et al., 1997
). Our previous studies (Yilmaz et al., 2002
) demonstrated that the P. gingivalis fimbriae mediate adhesion to GECs through integrin receptors. In vitro binding and immunochemical analysis revealed a direct physical association between
1 integrins and P. gingivalis fimbrillin (FimA). Moreover,
1 integrin antibodies inhibited P. gingivalis invasion into GECs. In separate studies, Nakagawa et al. (2002)
found that antibodies to
5
1 integrin blocked uptake of FimA-conjugated microspheres by epithelial (HEp-2) cells. At the subcellular level, infection of GECs by wild-type P. gingivalis induces phosphorylation of the focal adhesion protein, paxillin (Yilmaz et al., 2002
). Paxillin and focal adhesion kinase (FAK) have emerged as basic signal transducing components for integrin signalling. In many cell types, phosphorylation of paxillin and FAK is followed by the activation of other specific signalling molecules, promoting assembly of focal adhesion complexes subsequent to integrin activation (Clark & Brugge, 1995
; Yamada & Geiger, 1997
). Formation of these multi-component complexes generates forces that regulate actin and microtubule cytoskeleton dynamics and direct intracellular signals to specific targets in the cells. Therefore, the signalling events required for P. gingivalis uptake into GECs may emanate primarily from fimbriaeintegrin interactions, although other signal transduction pathways may be operational. In addition, recent findings have demonstrated the direct involvement of integrin-associated signalling and cytoskeletal molecules such as FAK, paxillin, vinculin and talin in the process of invasion of organisms such as uropathogenic Escherichia coli, Shigella flexneri, streptococci, Chlamydia pneumoniae and Yersinia spp. into host cells (Watarai et al., 1996
; Alrutz & Isberg, 1998
; Reddy et al., 2000
; Ozeri et al., 2001
; Martinez & Hultgren, 2002
; Coombes & Mahony, 2002
). The subcellular localization of focal adhesion constituents and cytoskeletal components in response to P. gingivalis invasion remains to be determined.
This study was directed towards testing the hypothesis that binding of P. gingivalis fimbriae to GECs results in the initiation of integrin-associated signalling pathways that ultimately converge on cytoskeletal architecture. As GECs can adapt to, and co-exist with, intracellular P. gingivalis, we investigated both short-term (30 min) and longer-term (24 h) responses to isogenic wild-type and fimbriae-deficient P. gingivalis strains.
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METHODS |
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Culture of GECs.
Low passage numbers of GEC cultures were generated as described previously (Lamont et al., 1995). Briefly, healthy gingival tissue was obtained after oral surgery and surface epithelium was separated by overnight incubation with 0·4 % dispase. Cells were cultured as monolayers in serum-free keratinocyte growth medium (KGM) (Clonetics) at 37 °C in 5 % CO2. GECs were used at passage four for experimentation and were at 7075 % confluence when reacted with bacterial cells in KGM.
Epithelial cell invasion assay.
P. gingivalis invasion of GECs was determined by the antibiotic protection assay described previously (Lamont et al., 1995). In brief, bacteria in KGM were incubated with GECs in 24-well plates for 30 min at 37 °C. After washing with PBS, remaining external bacteria were killed with metronidazole (200 µg ml-1) and gentamicin (300 µg ml-1) for 60 min. GECs were washed and lysed with sterile distilled water, and intracellular bacteria were enumerated by culture on blood agar supplemented with haemin and menadione.
Bacterial infection and preparation of GECs for fluorescence labelling.
GECs were seeded onto 4-well chambered coverglass slides (Nalge-Nunc International) at a density of 2x104 cells per well and cultured for 48 h. Cells were infected with bacteria at an m.o.i. of 100 at 37 °C in 5 % CO2/95 % air. After 30 min or 24 h incubation, the slides were washed four times with PBS containing 0·1 % Tween 20 to remove the non-adherent bacteria. Cells were fixed in 10 % neutral buffered formalin, rinsed in PBS and permeabilized for 15 min by 0·1 % Triton X-100 in PBS at room temperature. Samples were incubated for 30 min in a blocking solution of 5 % goat serum/0·1 % Tween 20 in PBS to mask non-specific binding sites prior to the fluorescence labelling.
Fluorescence microscopy.
Labelling of intracellular bacteria and the distribution of paxillin, FAK and microtubules was performed by indirect double immunofluorescence microscopy. Briefly, the coverglass slides were first co-incubated with rabbit polyclonal antibody to P. gingivalis 33277 at 1 : 5000 and mAbs to paxillin, or FAK antibodies (Transduction Laboratories), or monoclonal anti--tubulin antibody (Molecular Probes) at 1 : 200 for 1 h at room temperature. After washing three times with PBS and 0·1 % Tween 20, slides were then reacted simultaneously with fluorescein isothiocyanate (FITC)-conjugated Affini-Pure F(ab')2 fragment goat anti-rabbit IgG (H+L) (Jackson-Laboratories) and Rhodamine red-X-labelled goat anti-mouse IgG (H+L) (Molecular Probes) at 1 : 500 for 1 h in the dark at room temperature. Similarly, F-actin was labelled with phalloidintetramethylrhodamine B isothiocyanate (TRITC) (Sigma) at 1 : 100 for 45 min. All the antibodies and reagents used for labelling were diluted in the blocking buffer and each incubation step was followed by three washes with PBS and 0·1 % Tween 20. No fluorescence staining was observed when the primary antibodies were omitted, and primary P. gingivalis antibodies did not react with the uninfected GECs. For microscopy, samples were mounted in mounting medium containing anti-fade agent (Vector Laboratories) and examined using an epifluorescence microscope (Zeiss Axioskope) equipped with long pass optical filter sets appropriate for TRITC (RHODA), FITC and 4',6-diamidino-2-phenylindole (DAPI) dyes. Single exposure images were captured sequentially using a cooled CCD camera (Qimaging) and saved by QCAPTURE software version 1394. Collected image layers were superimposed into a single image using Adobe PHOTOSHOP 6.0 software. The images presented are a random representative of four separate experiments wherein at least 10 fields containing an average of 15 cells per sample were studied.
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RESULTS |
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DISCUSSION |
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To examine fimbriaeintegrin-based signalling events, we utilized a fimbriae-deficient mutant of P. gingivalis 33277, designated YPF1. Strain YPF1 contains an insertional inactivation of the fimA gene and hence is unable to produce FimA protein and lacks the major fimbriae (Love et al., 2000). In antibiotic-protection-based invasion assays, strain YPF1 showed a 10-fold reduction in internalization within GECs. These data, along with immunofluorescence of YPF1 within GECs, demonstrated that the mutant has impaired invasion capabilities; however, there remains a degree of residual, albeit less-efficient, invasion and the mutant is competent in non-fimbriae-dependent interactions with GECs.
Uptake of wild-type P. gingivalis into GECs is complete after approximately 20 min (Belton et al., 1999); therefore, 30 min P. gingivalis infection will reveal properties of GECs modulated concurrent with the invasion process. Visualization of the intracellular distribution of paxillin and FAK 30 min after P. gingivalis infection demonstrated a substantial amount of aggregation at the plasma membrane and the formation of microspikes and lamellipodial-like extensions along the edges of the cells. Paxillin activation and assembly into focal adhesions by P. gingivalis is consistent with our previous observations demonstrating phosphorylation of paxillin during P. gingivalis invasion (Yilmaz et al., 2002
). However, these earlier immunoprecipitation experiments did not demonstrate an increase in FAK activity triggered by P. gingivalis. This discrepancy is likely to originate from the use of only the Triton X-100 soluble cell lysate fractions in the prior study. Invasion by fimbriated P. gingivalis thus evokes the formation of integrin-associated focal adhesions and will likely stimulate the cascade of events that normally follows integrin activation (Alrutz & Isberg, 1998
; Metheniti et al., 2001
; Ozeri et al., 2001
; McGee et al., 2003
).
One downstream target of integrin signalling is the cellular cytoskeleton (Schoenwaelder & Burridge, 1999). Sensitivity of the invasion process to the inhibitors nocodazole and cytochalasin D signifies the necessity of both actin and microtubule cytoskeletal reassembly for the internalization of P. gingivalis (Lamont et al., 1995
). The present microscopic study demonstrated that wild-type P. gingivalis triggers distinct actin rearrangements during infection and promotes the formation of thin filamentous microspike-like structures emanating from the cell cortex. Microtubule rearrangements were less dramatic. However, microtubule dynamics can occur rapidly and may not be observable by a single technique (Waterman-Storer, 1998
). Therefore, our fixed point observations may not reflect the complete range of tubulin remodelling induced by P. gingivalis in GECs.
Following invasion, both P. gingivalis and epithelial cells remain viable for extended periods. Hence, GEC signal transduction pathways can be expected to be modulated both in the short term during the entry process and in the longer term as the bacteria and GECs adapt to their new conditions. After 24 h of P. gingivalis association with GECs, focal adhesions began to disassociate and paxillin and FAK redistributed back from the membrane and into the cytoplasm where there was a significant degree of co-localization with P. gingivalis cells in the perinuclear area. The early morphological alterations of both the actin filaments and the microtubules culminated in the significant amount of depolymerization and nucleation. Long-term stable changes in cell structure thus appear to accompany the adaptation of GECs to the burden of large numbers of intracellular P. gingivalis. This could also result in intracellular P. gingivalis interference with epithelial cell migration, proliferation and modulation of cell-matrix adhesion, along with disruption of cell-matrix remodelling over longer periods.
Collectively, the results presented here, along with our previous study demonstrating that integrin antibodies do not inhibit invasion of the non-fimbriated YPF1 (Yilmaz et al., 2002), show that P. gingivalis possesses mechanisms of entry into GECs that are independent of fimbriaeintegrin binding. While less efficient than fimbriae-dependent pathways, the YPF1 cells remain viable and accumulate in the perinuclear area. The GEC receptor for YPF1 invasins is currently under investigation. Although paxillin and FAK were only minimally redistributed during the fimbriae-independent invasion process, paxillin and FAK did co-localize with both parent and mutant strains after 24 h infection. This result indicates that redistribution of paxillin and FAK at later time points is a property of internalized bacteria and does not require the presence of fimbriae. Indeed, proteomic analysis (Wang et al., 2002
) has demonstrated that levels of FimA are reduced shortly after contact between P. gingivalis and GECs, thus internalized wild-type bacteria are likely to be only sparsely fimbriated. Actin rearrangements induced by invasion of the fimbriae-deficient mutant were also different from those of the parent. YPF1 induced actin condensation distributed throughout the cells as opposed to the filamentous structures induced by the wild-type. These findings are consistent with reports that bacterial invasion mediated through integrin binding is usually associated with minimal and transient cytoskeletal remodelling (Young et al., 1992
). YPF1 may, therefore, engage epithelial cells in a manner that requires major actin cytoskeletal rearrangements. As the fimA gene of P. gingivalis can be regulated by a number of environmental conditions (Xie et al., 1997
, 2000
), the ability to invade epithelial cells by fimbriae-independent mechanisms may be beneficial to the organism in situations where the fimbriae are down-regulated. Furthermore, the presence of fimbriae-dependent and independent invasion mechanisms may account, at least partially, for the differences in invasive pathways utilized by P. gingivalis in non-transformed GECs (Lamont et al., 1995
), transformed epithelial cells (Chen et al., 2001
) and endothelial cells (Dorn et al., 2000
).
This work demonstrates that invasive P. gingivalis modifies both the GEC membrane-associated structural and signalling protein arrangements and the cytoskeletal organization. Thus, P. gingivalis is capable of targeting specific epithelial cell pathways during invasion and adaptation to an intracellular lifestyle. Such complex and multi-threaded interactions point toward a long evolutionary relationship between P. gingivalis and host cells, resulting in a balanced association whereby the organism can survive within epithelial cells without causing excessive harm. P. gingivalis-induced diseases may then ensue from a disruption of this balance by factors that may trigger virulence or lead to host-immune-mediated tissue damage.
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ACKNOWLEDGEMENTS |
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Received 12 May 2003;
revised 18 June 2003;
accepted 19 June 2003.