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. 2004 Oct;72(10):5668–5675. doi: 10.1128/IAI.72.10.5668-5675.2004

Internalization of Staphylococcus aureus by Human Keratinocytes

Sompid Kintarak 1, Simon A Whawell 2, Paul M Speight 2, Samantha Packer 3, Sean P Nair 3,*
PMCID: PMC517534  PMID: 15385465

Abstract

Staphylococcus aureus is among the most important human pathogens and causes various superficial and systemic infections. The ability of S. aureus to be internalized by, and survive within, host cells, such as keratinocytes, may contribute to the development of persistent or chronic infections and may finally lead to deeper tissue infections or dissemination. To examine the mechanisms of internalization of S. aureus by keratinocytes, isogenic mutants lacking fibronectin-binding proteins (FnBPs), a recombinant protein consisting of the fibronectin-binding domain of S. aureus FnBPs, and an anti-α5β1 antibody were used in cocultures with immortalized keratinocytes and primary keratinocytes. We found that internalization of S. aureus by immortalized keratinocytes requires bacterial FnBPs and is mediated by the major fibronectin-binding integrin α5β1. In contrast to internalization by immortalized keratinocytes, internalization of S. aureus by primary keratinocytes could occur through FnBP-dependent and -independent pathways. S. aureus clumping factor B (ClfB), which was recently determined to bind to epithelial cells, was not involved in the uptake of this bacterium by keratinocytes. The identification of an alternate uptake pathway, which is independent of S. aureus FnBPs and host cell α5β1, has important implications for the design of therapies targeted to bacterial uptake by host cells.

Keratinocytes are the major cell type in the skin and oral mucosa, which serve as a primary barrier between the external environment and the internal tissues. They also provide a barrier to microorganisms, toxins, and various antigens. Staphylococcus aureus is among the most important human pathogens; this organism causes various superficial and systemic infections and is often implicated in oral mucositis, including angular cheilitis (24, 33) and denture-induced stomatitis (47). It is also a leading cause of bacterial keratitis (41) and has been implicated as a causative or exacerbating agent in a broad range of skin diseases, including atopic dermatitis, carbuncles, cellulitis, furuncles, follicles, Kawasaki syndrome, impetigo, psoriasis, and scalded skin syndrome (6, 10-12, 29, 34, 43, 50). Additionally, S. aureus is a major cause of wound infection and is thought to delay wound healing (3).

S. aureus is normally an extracellular pathogen; however, it can be internalized by a variety of nonphagocytic host cells in a fibronectin-binding protein (FnBP)-dependent manner. The host cells that have been studied to date include human umbilical vein endothelial cells (25), the human keratinocyte cell line HaCaT (28), corneal epithelial cells (20), osteoblasts (1), and epithelial 293 cells (a human embryonic kidney cell line) (42). In the previous studies the workers examined the interaction of host cells with S. aureus in vitro, but internalization of S. aureus by mammary gland epithelial cells has also been demonstrated in vivo (8). The uptake of S. aureus by host cells is a receptor-mediated process that has been hypothesized to involve host cell integrins and microbial surface components recognizing adhesive matrix molecules, MSCRAMMs (see reference 35 for a review of MSCRAMMs). Indeed, the internalization of S. aureus by endothelial cells (25), osteoblasts (1; Nair, unpublished data), and 293 cells (41) has been shown to require the host cell integrin α5β1 and the S. aureus fibronectin-binding MSCRAMM FnBPA and/or FnBPB. However, while there is little dispute that the S. aureus FnBPs are involved in bacterial uptake, studies with a variety of epithelial cells have provided conflicting evidence for an essential role for the FnBPs in the internalization process (8, 14, 19, 21). The differences may be due to the various sources of the epithelial cells and their nature (for example, immortalized cells versus normal primary cells).

The purpose of this study was to determine the mechanisms of internalization of S. aureus by primary human keratinocytes and keratinocyte cell lines derived from oral mucosa and skin.

We demonstrated in this study that internalization of S. aureus by human keratinocyte cell lines, like internalization by other cell types, requires bacterial FnBPs and is mediated by the major fibronectin-binding integrin α5β1. A second fibronectin-binding integrin found on keratinocytes, αvβ6, does not mediate internalization of S. aureus. In contrast to internalization by cell lines, in internalization of S. aureus by human primary keratinocytes both FnBP-dependent and -independent pathways are used. S. aureus clumping factor B (ClfB) has recently been shown to be a major adhesin involved in binding of the bacteria to epithelial cells (32). Using an isogenic mutant defective in ClfB, we found that this molecule is not involved in the internalization of S. aureus by primary keratinocytes.

MATERIALS AND METHODS

Chemical and reagents.

All chemicals and reagents were obtained from Sigma-Aldrich (Poole, United Kingdom) unless otherwise indicated. Culture media and phosphate-buffered saline without Ca2+ and Mg2+ (PBS) were obtained from Gibco, Invitrogen Ltd. (Paisley, United Kingdom). Function-blocking monoclonal antibody against α5β1 integrin (JBS5) was obtained from Chemicon Intl. Ltd. (Chandlers Ford, Hampshire, United Kingdom). Monoclonal antibody for cytokeratin 10 (DE-K10) was obtained from Dako UK Ltd. (Ely, United Kingdom). A function-blocking monoclonal antibody against av integrin was prepared in our laboratories from hybridoma cells (ATCC HB8449) obtained from the American Type Culture Collection (Rockville, Md.).

Human cells and cell culture.

UP cells are an immortalized, nontumorigenic, human keratinocyte cell line derived from human papilloma virus 16-transfected epidermal keratinocytes (36). Primary cultures of oral keratinocytes (NHK cells) were prepared as outgrowths from redundant normal mucosal tissues obtained, with permission, during minor oral surgical procedures. The oral tissues were cultivated as described by Freshney (16), with some modifications. Briefly, the epithelium was finely chopped into about 1-mm cubes after unwanted connective tissue was dissected away. Six epithelial pieces were transferred to a 25-cm2 tissue culture flask. Keratinocyte growth medium was gently added, and the cultures were incubated at 37°C in a humidified atmosphere consisting of 5% CO2 in air without disturbance for 1 week. After 1 week, substantial outgrowth of cells was observed, and the growth medium was changed at this time and then every 2 to 3 days until approximately 18 days, when the cells were almost confluent. The primary cultures of NHK cells were used at passage 2. H357 and H376 cells are oral squamous carcinoma cell lines (38). VB6 and C1 cells were generated from H357 (αv-negative) cells to express high and low levels of αvβ6 integrin, respectively (48). Primary human skin keratinocytes were a kind gift from Fiona Watt and Simon Broad, Keratinocyte Laboratory, CRUK, London, United Kingdom. Keratinocytes were grown and maintained in keratinocyte growth medium containing 3 parts of Dulbecco's modified Eagle's medium (DMEM) plus 1 part of Ham's F-12 nutrient mixture supplemented with 10% fetal calf serum (PAA Laboratories), 10 ng of epidermal growth factor per ml, 5 μg of insulin per ml, 0.5 μg of hydrocortisone per ml, 10−10 M cholera toxin, 1.8 × 10−4 M adenine, 100 IU of penicillin per ml, 100 μg of streptomycin per ml, and 2.5 μg of amphotericin B (Fungizone; Gibco) per ml. To harvest cells for experiments, the cells were lifted from a culture plate by trypsinization with 0.25% trypsin-0.05% EDTA (Gibco) at 37°C for 10 to 15 min and collected by centrifugation. The keratinocytes were subcultured in 24-well plates at a concentration of 7 × 104 cells/well and were grown for 2 days to confluence.

Bacterial strains and growth conditions.

The strains used in this study are listed in Table 1 All S. aureus strains were stored in Todd-Hewitt broth (THB) (Oxoid Ltd., Basingstoke, United Kingdom) containing glycerol (15%, vol/vol) at −80°C. An aliquot from a frozen stock of each S. aureus strain was streaked on a blood agar (Oxoid) plate and incubated overnight at 37°C before selection of a single colony for overnight growth in 10 ml of THB. The overnight broth cultures were then transferred (1:250) into 10 ml of THB and grown to the mid-exponential phase.

TABLE 1.

Bacterial strains

Strain Relevant characteristic(s) Reference(s)
NCTC6561 Laboratory strain from NCTCa
8325-4 NCTC8325 cured of prophages; FnBPA+ FnBPB+ RsbU− 31
DU5883 FnBPA− FnBPB−; isogenic mutant of 8325-4 (fnbA::TcrfnbB::Emr) 17
DU5961 ClfB−; isogenic mutant of 8325-4 (clfB::Emr) 26
LS-1 Isolated from a swollen joint of a spontaneously arthritic NZB/W mouse; FnBPA+ FnBPB+ RsbU+ 4, 5, 30
LSM(FnBP−) FnBPA− FnBPB−; isogenic mutant of LS-1 (fnbA::TcrfnbB::Emr) 1
E. coli M607/pREP4/pQE30-rfnbB[D1-D4] Nair, unpublished
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a

NCTC, National Collection of Type Cultures.

Preparation and purification of rFnBPB[D1-D4].

An E. coli clone (E. coli M607/pREP4/pQE30-fnbB[D1-D4]) that expresses a histidine-tagged recombinant protein consisting of the D1-D4 repeat region of the MSCRAMM FnBPB (rFnBPB[D1-D4]) was constructed previously in our laboratory (Nair, unpublished data) by using the pQE30 expression vector (QIAGEN Ltd., Crawley, United Kingdom). The E. coli clone was grown overnight in Luria-Bertani broth containing antibiotics (100 μg of ampicillin per ml, 25 μg of kanamycin per ml, 20 μg of spectinomycin per ml, and 20 μg of streptomycin per ml) at 30°C with constant shaking. Overnight cultures of E. coli were transferred (1:10) into 200 ml of Luria-Bertani broth containing antibiotics and incubated for 2 h. Protein expression was induced by adding 200 μl of 1 M isopropylthio-β-d-galactoside (IPTG) (VWR International Ltd., Poole, United Kingdom). After 4 h, the bacterial cells were harvested by centrifugation at 13,000 × g for 20 min at 4°C. The cell pellets were resuspended in lysis buffer consisting of the B-PER bacterial protein extraction reagent (Pierce, Perbio Science UK Ltd., Tattenhall, United Kingdom) plus 20 mM imidazole and protease inhibitors (Sigma-Aldrich), and cell debris was removed by centrifugation at 14,000 rpm at 4°C for 10 min. The histidine-tagged rFnBPB[D1-D4] was then purified by using Ni-nitrilotriacetic acid agarose (QIAGEN) as described by the manufacturer, except that a wash with 2 mg of polymyxin B per ml in PBS was used to remove endotoxin.

S. aureus adhesion and internalization assays.

Confluent keratinocytes in 24-well plates were incubated in DMEM for 3 h, blocked with 2% bovine serum albumin in PBS for 1 h, and then washed twice with PBS. The number of keratinocytes in each well was estimated to be between 1.5 × 105 and 1.7 × 105 by harvesting cells from five separate wells. A bacterial inoculum containing approximately 5 × 107 CFU suspended in DMEM was added to each well, and the plates were incubated at 37°C in a 5% CO2-95% air atmosphere for 90 min. Nonadherent bacteria were washed off; then the adherent bacteria plus intracellular bacteria were quantified (adhesion assay), or 1 ml of a solution containing 100 μg of gentamicin per ml in DMEM was added to each well to kill extracellular bacteria (internalization assay). The plates were incubated with gentamicin for 2 h at 37°C in a 5% CO2-95% air atmosphere and then washed twice with PBS. To determine the number of bacteria, the keratinocytes were trypsinized with trypsin-EDTA and lysed with 0.1% Triton X-100, and serial dilutions were plated on blood agar to determine bacterial viable counts.

Effect of rFnBPB[D1-D4] protein and anti-integrin antibodies on internalization of S. aureus by keratinocytes.

Internalization assays were performed as described above with the wild-type S. aureus strain 8325-4. Recombinant protein was used at a final concentration of 10 or 20 μg/ml. Mouse monoclonal antibodies recognizing the integrins α5β1(JBS5) and αv (L230) were used at final concentrations of 8 and 20 μg/ml, respectively. The recombinant protein or antibodies were preincubated with keratinocyte cultures for 30 min and remained present during internalization assays. Control wells were pretreated with DMEM alone.

Immunohistochemistry.

Keratinocytes were grown for 2 days in a 24-well plate on 13-mm glass coverslips. The cells were washed twice with PBS and fixed in 3% paraformaldehyde for 20 min at room temperature. After fixation, the cells were permeabilized with absolute methanol for 10 min before blocking with 1% bovine serum albumin in PBS for 1 h. Staining of cytokeratin 10 was performed by using a standard immunoperoxidase technique. Briefly, after two washes with PBS, the cells were incubated with an antibody to cytokeratin 10 (1:50) for 1 h at room temperature. Multilink secondary antibody (Biogenex) and horseradish peroxidase (Biogenex) were applied to the cells for 20 min. Color was developed in a freshly prepared diaminobenzidine substrate solution for 10 min, and then cells were counterstained with Mayer's hematoxylin. Negative controls were prepared by omitting the primary antibody.

Statistical analysis.

The data are expressed as means ± standard deviations. Statistical analysis was performed by using the nonparametric Mann-Whitney U test; P values of <0.05 were considered significant.

RESULTS

Role of S. aureus FnBPs in the interaction of bacteria with keratinocytes.

To determine the role of S. aureus FnBPs in the interaction with keratinocytes, S. aureus adhesion and internalization assays were performed with isogenic mutants with disruptions in the genes encoding these proteins. Evaluation of strain 8325-4 and its FnBP-deficient mutant DU5883 revealed reductions of approximately 68 and 83% (P < 0.01) in bacterial adherence to (Fig. 1A) and internalization by (Fig. 1B) the immortalized skin keratinocytes (UP cells). The ability of the S. aureus LS-1 isogenic mutant LSM(FnBP−) to adhere to UP cells was reduced by approximately 50% compared to the ability of the wild-type strain (Fig. 1A), and internalization was reduced by approximately 94% (P < 0.01) (Fig. 1B). These results demonstrate that S. aureus FnBPs play an important role in the adhesion and internalization processes that occur with UP cells. As previously reported for osteoblasts, UP cells took up larger numbers of S. aureus strain LS-1 cells than of strain 8325-4 cells (1). The viability of keratinocytes after incubation with bacterial strains was determined by propidium iodide staining and flow cytometry. There was no significant difference between the viability of keratinocytes incubated with 8325-4 (96.71% ± 0.93%) or LS-1 (95.51% ± 1.15%) and the viability of keratinocyte controls incubated with media alone (96.70% ± 1.58%), demonstrating that the differences in the capacities of these two strains to be internalized was not due to any effect on keratinocyte viability.

FIG. 1.

FIG. 1.

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Adherence to and internalization of S. aureus by UP keratinocytes. (A) Adherence of S. aureus strains 8325-4 and LS-1 and their isogenic mutants defective in FnBPA and FnBPB to UP keratinocytes. (B) Internalization of S. aureus strains 8325-4 and LS-1 and their isogenic mutants defective in FnBPA and FnBPB by UP keratinocytes. The results are the results of one representative experiment of at least three experiments performed; the data are the means and standard deviations for three replicate cultures. An asterisk indicates that the P value is <0.01.

In contrast to UP cells, no reduction in the adherence to (Fig. 2A) or internalization by (Fig. 2B) primary oral keratinocytes of the isogenic mutant DU5883 compared to the parental strain 8325-4 was observed in cells derived from three different individuals (NHK12, NHK15, and NHK22). However, there were reductions in the binding and internalization of the isogenic mutant LSM(FnBP−) compared to its parental strain, strain LS-1, with cells from one individual (NHK12). The levels of adherence to and internalization by NHK12 cells for LSM(FnBP−) were approximately 50% (P < 0.01) and 60% (P < 0.01) of the values for wild-type strain, respectively. Increased adherence of the isogenic mutant DU5883 compared to the parental strain was observed with cells from another individual (NHK22).

FIG. 2.

FIG. 2.

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Adherence to and internalization of S. aureus by primary oral keratinocytes from three separate individuals. (A) Adherence of S. aureus strains 8325-4 and LS-1 and their isogenic mutants defective in FnBPA and FnBPB to primary oral keratinocytes from three separate individuals. (B) Internalization of S. aureus strains 8325-4 and LS-1 and their isogenic mutants defective in FnBPA and FnBPB by primary oral keratinocytes from three separate individuals. The results are the results of one representative experiment of at least three experiments performed; the data are the means and standard deviations for three replicate cultures. An asterisk indicates that the P value is <0.01.

To determine whether the differences observed in adhesion and internalization of S. aureus by UP cells and primary oral keratinocytes were due to a difference in the tissue of origin (skin versus oral mucosa), the abilities of FnBP-deficient mutants to bind to and be internalized by oral squamous cell carcinoma line H376 were examined. The surface expression levels of the α5, β1, and αv integrin subunits on H376 cells are similar to those on normal primary oral keratinocytes (45). It was observed that the levels of adherence of FnBP-deficient strains DU5883 and LSM(FnBP−) to H376 were significantly decreased (P < 0.01) compared to those of the wild-type strains (Fig. 3). In addition, the FnBP-deficient strains DU5883 and LSM(FnBP−) were not internalized by H376 (P < 0.01). These findings are similar to those obtained with UP cells, suggesting that differences in the processes of internalization of S. aureus by primary oral keratinocytes and immortalized keratinocytes are unlikely to be due to a difference in the tissue from which the cells were derived.

FIG. 3.

FIG. 3.

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Adherence to and internalization of S. aureus by the oral squamous cell carcinoma line H376. (A) Adherence of S. aureus strains 8325-4 and LS-1 and their isogenic mutants defective in FnBPA and FnBPB to H376 cells. (B) Internalization of S. aureus strains 8325-4 and LS-1 and their isogenic mutants defective in FnBPA and FnBPB by H376 cells. The results are the results of one representative experiment of at least three experiments performed; the data are the means and standard deviations for three replicate cultures. An asterisk indicates that the P value is <0.01.

Effect of a recombinant protein consisting of the D1-D4 region of FnBPB on the internalization of S. aureus by keratinocytes.

To further investigate the role of S. aureus FnBPs in internalization by keratinocytes, the effect of a recombinant protein encompassing the D1-D4 repeat units of FnBPB (rFnBPB[D1-D4]) on internalization was determined. It has been demonstrated previously that the D1-D4 region of S. aureus FnBP can inhibit internalization of S. aureus by epithelial cells (42) and osteoblasts (Nair, unpublished). Internalization of S. aureus by UP keratinocytes was reduced by approximately 80% in the presence of 10 μg of rFnBPB[D1-D4] per ml (P < 0.01), whereas there was no effect on the internalization of S. aureus by primary oral keratinocytes even at higher concentrations (Fig. 4).

FIG. 4.

FIG. 4.

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Effect of rFnBPB[D1-D4] on the internalization of S. aureus by UP keratinocytes and primary oral keratinocytes (NHK15 cells). The results are the results of one representative experiment of at least three experiments performed; the data are the means and standard deviations for three replicate cultures. An asterisk indicates that the P value is <0.01.

Role of integrin α5β1 in the internalization of S. aureus by keratinocytes.

The presence of fibronectin and the fibronectin-binding integrins α5β1 and αvβ6 on the surface of UP cells and the primary oral keratinocytes was confirmed by immunofluorescent microscopy (data not shown). To examine the role of α5β1 in internalization of S. aureus by human keratinocytes, S. aureus internalization assays were performed after preincubation of keratinocytes with a function-blocking anti-α5β1 antibody (Fig. 5). Internalization of S. aureus by UP keratinocytes was reduced by approximately 54% in the presence of the antibody (P < 0.01). The anti-α5β1 antibody had no effect on the internalization of S. aureus by primary oral keratinocytes (NHK15); however, it did block the internalization of S. aureus by the oral squamous carcinoma cell line H357 (P < 0.01), which expresses high levels of α5β1 but not αv integrins.

FIG. 5.

FIG. 5.

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Effect of the anti-α5β1 antibody on the internalization of S. aureus strain 8325-4 by UP keratinocytes, primary oral keratinocytes (NHK15 cells), and oral squamous carninoma cell line H357. The results are the results of one representative experiment of at least three experiments performed; the data are the means and standard deviations for three replicate cultures. An asterisk indicates that the P value is <0.01.

Role of integrin αvβ6 in the internalization of S. aureus by human cells.

Integrin αvβ6, a fibronectin receptor, is expressed during wound healing, in oral squamous cell carcinomas, and in normal keratinocytes in culture. To determine the role of host cell αvβ6 integrin in S. aureus internalization, the internalization of S. aureus by VB6 cells and the internalization of S. aureus by C1 cells were compared. VB6 and C1 cells were generated from H357 (αv-negative) cells to express high and low levels of the αvβ6 integrin, respectively (48). The numbers of bacteria internalized by VB6 and C1 cells were similar for each strain, although the number of LS-1 cells taken up was higher than the number of 8325-4 cells taken up (Fig. 6). There were no differences in the numbers of bacteria adhering to VB6 and C1 cells (data not shown). Additionally, the uptake of S. aureus by NHK15 cells, which internalized the bacterium independent of the α5β1 integrin and bacterial FnBP (Fig. 2B and Fig. 5), could not be inhibited by the αv function-blocking antibody L230 (control cells internalized 0.482% ± 0.06% of the inoculum, while the cells treated with L230 internalized 0.457% ± 0.095% of the inoculum). These data suggest that the αvβ6 integrin is not involved in the process of internalization of S. aureus by keratinocytes.

FIG. 6.

FIG. 6.

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Role of the αvβ6 integrin in the internalization of S. aureus by human cells: internalization of S. aureus strains 8325-4 and LS-1 by VB6 and C1 cells, generated from H357 cells to express high and low levels of αvβ6 integrin, respectively. The results are the results of one representative experiment of at least three experiments performed; the data are the means and standard deviations for three replicate cultures.

Internalization of S. aureus by primary skin keratinocytes.

To further elucidate whether the differences in the mechanisms of uptake of S. aureus by keratinocytes seen in this study were due to differences between transformed and nontransformed cells or due to the different tissue sources from which the cells were derived, we examined the uptake of S. aureus by primary human skin keratinocytes. Primary human skin keratinocytes (F5 and M4) obtained from two separate individuals internalized S. aureus strain 8325-4 (Fig. 7). In contrast, the isogenic mutant DU5883, which was deficient in FnBPs, was efficiently internalized by only one set (M4) of primary skin keratinocytes (Fig. 7). These data suggest that uptake of S. aureus by primary skin keratinocytes can occur through FnBP-dependent and -independent processes.

FIG. 7.

FIG. 7.

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Internalization of S. aureus strain 8325-4 and its isogenic mutant defective in FnBPA and FnBPB by F5 and M4 primary skin keratinocytes obtained from two different individuals. The results are the results of one representative experiment of at least three experiments performed; the data are the means and standard deviations for three replicate cultures. An asterisk indicates that the P value is <0.01.

Role of ClfB in the process of internalization of S. aureus by keratinocytes.

To examine the role of possible interactions between S. aureus ClfB and cytokeratin 10 in the internalization of S. aureus by primary oral keratinocytes, the abilities of S. aureus strain 8325-4 and a ClfB-deficient mutant of this strain (DU5961) to be internalized by human keratinocytes were compared. There was no difference in the capacity of the 8325-4 ClfB− mutant to become internalized by primary oral keratinocytes compared to the wild-type strain (Fig. 8). In the case of UP keratinocytes, the 8325-4 ClfB− mutant had a greater capacity to be internalized by UP cells than the wild type (P < 0.01). Immunohistochemical staining demonstrated that UP cell cultures contained only occasional cytokeratin 10-positive cells, whereas primary oral keratinocyte cultures contained no cytokeratin 10-positive cells (data not shown).

FIG. 8.

FIG. 8.

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Internalization of S. aureus strain 8325-4 and its isogenic mutant defective in ClfB (DU5961) by primary oral keratinocytes and UP keratinocytes. The results are the results of one representative experiment of at least three experiments performed; the data are the means and standard deviations for three replicate cultures. An asterisk indicates that the P value is <0.01.

DISCUSSION

Although not generally considered an intracellular pathogen, S. aureus can be internalized by many host cells, including keratinocytes. Results with nonkeratinocyte cell types have shown that the process of internalization of S. aureus is dependent on bacterial FnBPs and the host cell integrin α5β1 (1, 15, 25, 42). In the present study we investigated the role of S. aureus FnBPs and host cell integrins in bacterial internalization by keratinocytes.

Our data show that internalization of S. aureus by immortalized skin keratinocytes (UP cells) requires bacterial FnBPs and the host cell integrin α5β1. Using isogenic mutants of S. aureus with disruptions in both fnb genes, we found that the capacities of the mutants to be internalized by UP cells were reduced by approximately 83 to 94% compared to the capacities of the parental strains. The reduced levels of internalization of FnBP-deficient mutants DU5883 and LS-1(FnBP−) by UP cells were probably partially due to decreases in the capacities of these strains to adhere to UP cells (50 to 68%). Similar reductions in the levels of adherence of S. aureus FnBP-deficient mutants to HaCaT keratinocytes (27) and bovine mammary epithelial (MAC-T) cells (14) have been reported, and the levels of adherence were reduced by 60 and 40%, respectively, compared to the parental strain. However, a recent report on the abilities of these same isogenic mutants to bind to osteoblasts (1) showed that there was a >90% reduction in adherence. The differences in adherence to different cell types are likely to be due to differences in the integrins and extracellular matrix molecules expressed by these host cells.

An antibody against integrin α5β1 significantly decreased the internalization of S. aureus by UP keratinocytes, suggesting that this fibronectin receptor plays an important role in the internalization process. However, in contrast to immortalized keratinocytes, the results obtained with primary oral keratinocytes (NHK cells) in the present study suggest that adhesion and internalization of S. aureus can occur by FnBP-dependent and -independent processes since adhesion and internalization of S. aureus by keratinocytes from two of three donors were not affected by expression of FnBPs. In addition, the presence of the rFnBPB[D1-D4] protein or the anti-integrin α5β1 antibody in the internalization assay mixture failed to block the uptake of S. aureus by primary oral keratinocytes. The different roles of FnBPs in the uptake by immortalized skin keratinocytes (UP cells) and primary oral keratinocytes (NHK cells) were not due to the source of the cells (skin versus oral mucosa), as demonstrated by the fact that there was reduced adherence and internalization of FnBP-deficient mutants of S. aureus by the oral squamous carcinoma cell line H376. Internalization of S. aureus by H357 cells was also significantly reduced in the presence of an anti-α5β1 antibody. Additionally, internalization of S. aureus by primary skin keratinocytes could occur through FnBP-dependent or -independent pathways depending on the donor from which the skin was obtained. Although this is the first report that S. aureus can be internalized by keratinocytes through an FnPB-independent pathway, Cho et al. (11) recently reported that adherence of S. aureus to atopic and normal skin sections can occur through FnBP-dependent and -independent mechanisms, respectively.

Keratinocytes also express αvβ6, another fibronectin-binding integrin. αvβ6 is not detectable on normal epithelium, but high levels are expressed in migrating basal keratinocytes during wound healing (7, 13, 18). A possible role for αvβ6 in the adhesion and/or internalization of S. aureus by keratinocytes was therefore investigated by using oral squamous carcinoma cell lines expressing high (VB6) and low (C1) levels of αvβ6. There was no difference in the adhesion or the internalization of S. aureus by VB6 and C1 cells. Furthermore, an αv function-blocking antibody had no effect on the uptake of S. aureus by keratinocytes (NHK15 cells) that internalized this bacterium independent of any interaction between the α5β1 integrin and the bacterial FnBP. These data suggest that the integrin αvβ6 is not an important mediator of internalization of S. aureus by keratinocytes.

It should be noted that primary keratinocytes and immortalized or transformed keratinocytes are different in many ways. Immortalized keratinocytes commonly have a reduced degree of stratification, a lower proportion of differentiating cells, and altered integrin expression (22, 36, 45). Alteration of cytokeratin (K) expression in UP keratinocytes has also been reported (37), with reduced expression of K16 but increased expression of K18 (associated with simple epithelia) and K13 (associated with nonkeratinizing stratified epithelia) compared to the normal parental keratinocytes. The abilities of UP keratinocytes and primary oral keratinocytes to respond to stimuli have been reported to be different since, for example, the levels of both matrix metalloproteinase 2 (MMP-2) and MMP-9 are increased in primary oral keratinocytes in response to scatter factor, but only MMP-9 is inducible in UP cells (2). Our data from the studies with primary skin keratinocytes demonstrate that cells from different individuals can utilize either FnBP-dependent or -independent pathways to internalize S. aureus. Together, these data demonstrate that there is more than one pathway for the uptake of S. aureus by keratinocytes. We hypothesize that the differences in the mechanisms of internalization of S. aureus between the keratinocytes from the different sources may be a result of differential expression of integrins or other keratinocyte cell surface receptors, although this remains to be established.

There is increasing evidence that host cell cytokeratins may act as important mediators of bacterial adhesion and/or invasion (9, 23, 39, 44, 46, 49). K18 has been found to mediate Salmonella invasion of HEp-2 cells (9). K13 has been reported to promote adhesion and/or invasion of Burkholderia cepacia in squamous epithelial cells (39, 40). Recently, it has been shown that ClfB of S. aureus is an important factor for mediating attachment of the bacterium to desquamated epithelial cells and HPV-G keratinocytes, possibly through an interaction with K10 (32). Thus, in this study we investigated the possibility that this bacterial MSCRAMM may be involved in the internalization of S. aureus by normal keratinocytes and found that this is not the case. Cytokeratin 10 was not detectable in the primary oral keratinocyte cell cultures, while very low levels were detected in immortalized UP keratinocytes. A possible explanation for the increased internalization of the S. aureus ClfB mutant by UP keratinocytes could be competition for bacterial binding to α5β1 via fibronectin by binding to K10 and that the latter interaction did not result in uptake of bacteria by keratinocytes.

In conclusion, the S. aureus FnBPs and host cell integrin α5β1 were important for internalization of S. aureus by immortalized keratinocytes. With primary keratinocytes, S. aureus internalization could occur through FnBP-dependent or -independent pathways. The differences in the bacterial and host cell receptors utilized for internalization of S. aureus by immortalized and primary keratinocytes and indeed by keratinocytes from different individuals have important implications for the interpretation and translation of in vitro studies in order to predict in vivo responses. Indeed, it has recently been demonstrated that an FnBP-deficient isogenic mutant of S. aureus is internalized by mammary epithelial cells in a mouse model of mastitis (8).

Acknowledgments

We thank S. S. Prime, University of Bristol, for supplying the H357 and H376 cell lines, Gareth Thomas, Eastman Dental Institute, University College London, for supplying the VB6 and C1 cell lines, and Timothy J. Foster, Trinity College Dublin, for supplying S. aureus strains DU5883 and DU5961.

We also thank the government of Thailand for support of S.K. and the Arthritis Research Campaign for support of S.P.N. (grant H0600).

Editor: J. B. Bliska

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