FimH is sufficient to mediate bacterial internalization

The ability of FimH to mediate directly internalization into bladder epithelial cells was tested using adhesin-coated polystyrene latex beads. Similar amounts of purified FimC–FimH chaperone–adhesin complexes, FimC alone or bovine serum albumin (BSA) were covalently attached to latex beads (~1.17 µm in diameter). FimH, when bound to the FimC chaperone, can be purified easily in a native state that is able to bind mannose-containing host receptors (Choudhury et al., 1999). The ability of the protein-coated beads to be internalized by 5637 cells was examined by SEM and TEM. Following a 1 h incubation with FimCH-coated beads, SEM revealed numerous beads just beneath the bladder cell surface and in the process of internalization (Figure 3A and B). TEM indicated that the bladder cell membrane could ‘zipper’ around and eventually internalize attached FimCH-coated beads (Figure 3D–G). This zippering process appeared to be morphologically identical to that leading to the internalization of type 1-piliated E.coli (see Figure 1A; Mulvey et al., 1998). In contrast, BSA-coated beads and FimC-coated beads rarely adhered to the 5637 bladder cells and did not induce any apparent changes in the host cell membrane (Figure 3C and data not shown).

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Fig. 3. Internalization of FimCH-coated beads. 5637 cells were incubated with FimCH- or BSA-coated beads for 30–60 min and then processed for SEM (A–C) or TEM (D–G). (A and B) FimCH-coated beads were internalized efficiently by 5637 cells, while the few adherent BSA-coated beads (C) did not induce any appreciable alterations in the 5637 cell membrane. (D–G) The host cell membrane appears to zipper around and eventually envelope FimCH-coated beads during the internalization process. Scale bars represent 5.0 µm (A and E), 0.5 µm (B–D) and 1.0 µm (F and G).

The ability of FimCH-coated beads to be internalized by 5637 cells was quantified using an immunofluorescence-based assay (see Materials and methods). As shown in Figure 4, significant numbers of adherent FimCH-coated beads were internalized into 5637 cells within 60 min after addition to the cell culture wells. In contrast, the few BSA- and FimC-coated beads that occasionally adhered to 5637 cells were rarely internalized (Figure 4A). Like type 1-piliated bacteria, internalization of the FimCH-coated beads was inhibited by soluble d-mannose. However, d-mannose had no effect on the number of BSA- or FimC-coated beads that were internalized (Figure 4A). These results indicate that the type 1 pilus adhesin, FimH, cannot only mediate adherence to host cells, but is also sufficient to trigger internalization into human bladder cells in the absence of other bacterial factors.

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Fig. 4. FimH is necessary and seemingly sufficient to mediate internalization. (A) 5637 cells, grown on sterile coverslips, were incubated with equal amounts of FimCH-, FimC- or BSA-coated beads ±2.5% d-mannose for 30–60 min. FimCH-coated beads were internalized in a d-mannose-inhibitable manner. In contrast, few control FimC- or BSA-coated beads were internalized and their uptake was not inhibited by d-mannose. Numbers of intracellular beads were determined as described in Materials and methods. Data are presented as an invasion index (total number of internalized beads divided by the total number of host cell-associated beads) and are representative of at least four independent assays. (B and C) Representative images of the internalization assay demonstrating the internalization of FimCH-coated beads into 5637 cells. (C) Fluorescent images (TRITC filter setting) were superimposed onto the corresponding phase contrast images such that extracellular beads appear red (arrow). The scale bar represents 5 µm.

FimH-mediated invasion is dependent upon host actin polymerization

The envelopment of type 1-piliated bacteria and FimCH-coated beads by 5637 cells appeared to involve substantial host cytoskeletal alterations, probably requiring actin rearrangements at sites of FimH-mediated contact with host cells. The role of actin in the invasion process was investigated using a specific inhibitor of F-actin polymerization, cytochalasin D. 5637 bladder cells were incubated with increasing concentrations of cytochalasin D for 30 min prior to infection with either NU14 or AAEC185/pSH2 (type 1⁺, fimH⁺). Cytochalasin D dramatically reduced FimH-mediated invasion (>99% inhibition; Figure 5A) but had no effect on bacterial adherence to the bladder cells (data not shown). Removal of the drug prior to infection made the host cells susceptible once again to type 1 pilus-mediated bacterial invasion, indicating that the effects of cytochalasin D were reversible (Figure 5A). The treatment of 5637 cells with cytochalasin D also inhibited the internalization of FimCH-coated beads (~90% reduction; Figure 5B).

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Fig. 5. Host cell actin polymerization plays an important role in the invasion process. (A) 5637 cells were pre-treated with cytochalasin D for 30 min prior to infection with AAEC185/pSH2. Gentamicin protection assays demonstrate that cytochalasin D (1 µg/ml) can completely abolish invasion. The effects of cytochalasin D are reversible if the host cells are washed with PBS prior to infection in fresh medium without drug. Similar results were obtained using NU14. (B) Cytochalasin D also inhibits the internalization of FimCH-coated beads.

The effects of FimH-mediated adherence on the host cell actin cytoskeletal network were visualized using 5637 cells that were infected with green fluorescent protein (GFP)-expressing AAEC185/pSH2 or NU14. Following infection, 5637 cells were stained with Texas red-labeled phalloidin, a toxin that specifically binds to host F-actin. Confocal microscopy revealed that type 1-piliated bacteria (green) induced localized rearrangement of actin (red) at points of bacterial attachment (arrows, Figure 6B). Such localized alterations of the actin cytoskeleton were not observed in uninfected cells (Figure 6A). Like type 1-piliated E.coli, FimCH-coated beads, but not control BSA-coated beads, also induced localized changes of the actin cytoskeleton (data not shown). No large-scale, global changes, such as the formation of large actin stress fibers, were induced by FimH-mediated bacterial or bead attachment. In addition, type 1-piliated bacteria lacking the FimH adhesin did not bind 5637 cells and failed to induce any cytoskeletal changes (data not shown). These data suggest that FimH-mediated attachment is sufficient to induce localized actin polymerization.

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Fig. 6. Actin rearrangements leading to internalization of type 1-piliated E.coli. (A) Texas red–phalloidin staining of uninfected 5637 cells highlighting cortical actin (red). (B) Localized actin polymerization is induced at points of contact (arrows) with AAEC185/pSH2/pcomGFP (type 1⁺, GFP⁺). (C and D) Images from (B) (arrows) and (E) are enlarged to show detail. The scale bar represents 2 µm.

Invasion requires host protein tyrosine phosphorylation and PI 3-kinase activation

Modification of the host actin cytoskeletal network can be influenced directly by the induction of specific host signaling events, including protein tyrosine phosphorylation and the activation of phosphoinositide 3-kinase (PI 3-kinase) (Guinebault et al., 1995; Hartwig et al., 1995; Carpenter and Cantley, 1996; Parsons, 1996). The involvement of host protein tyrosine phosphorylation in FimH-mediated bacterial invasion was tested using a potent general tyrosine kinase inhibitor, genistein, and a specific inhibitor of Src-family tyrosine kinases, PP1 (Gerwien et al., 1999). Treatment with genistein greatly inhibited the ability of both NU14 (Figure 7A) and AAEC185/pSH2 (data not shown) to invade 5637 bladder cells in a concentration-dependent fashion. In contrast, the inhibition of Src-family tyrosine kinases using PP1 had no effect on the ability of type 1-piliated E.coli to bind to and subsequently invade 5637 cells (Figure 7B). These data indicate that tyrosine kinases, other than members of the Src-family of kinases, are involved in FimH-mediated bacterial invasion.

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Fig. 7. Inhibition of host protein tyrosine phosphorylation and PI 3-kinase activation diminishes invasion. (A) Gentamicin protection assays show that genistein prevents FimH-mediated bacterial internalization into 5637 cells in a concentration-dependent manner (>90% inhibition at 250 µM). (B) PP1, a Src-family tyrosine kinase inhibitor, had no effect on the internalization process. 5637 cells were treated with PP1 at concentrations that are known to inhibit Src-family kinase activity effectively (Gerwein et al., 1999). (C and D) Wortmannin and LY294002, two compounds that inhibit PI 3-kinase activity, can block FimH-mediated bacterial invasion of 5637 cells (~90% inhibition at 100 nM and 50 µM, respectively). (E) Internalization of FimCH-coated beads into 5637 cells is also inhibited by wortmannin (~80% inhibition at 100 nM).

The role of PI 3-kinase in FimH-mediated bacterial invasion was investigated using two structurally unrelated compounds, wortmannin and LY294002, both of which inhibit PI 3-kinase activity (Vlahos et al., 1994). Treatment of 5637 cells with either 100 nM wortmannin or 50 µM LY294002 reduced FimH-mediated invasion of 5637 cells by ~90% when compared with the control cells treated with solvent (dimethylsulfoxide; DMSO) alone (Figure 7C and D). Wortmannin also greatly inhibited the internalization of FimCH-coated beads by 5637 cells (Figure 7E). Neither wortmannin nor LY294002 had any effect on FimH-mediated bacterial adherence (data not shown).

Adherence and gentamicin protection (invasion) assays

Adherence and invasion assays were performed essentially as described (Elsinghorst, 1994). Briefly, host epithelial cells were seeded into 24-well plates and grown to confluence. Just before infection, the cell culture medium in each well was replaced with 960 µl of fresh, pre-warmed medium. In three sets of triplicate wells, bladder cells were infected with a multiplicity of infection (m.o.i.) of 5–10 bacteria per host cell (20 µl of bacteria diluted in LB broth, A₆₀₀ ~0.5). Bacterial contact with host cells was expedited by centrifugation of plates at 600 g for 5 min. After 2 h of incubation at 37°C, one set of triplicate wells was lysed by the addition of 20 µl of 5% Triton X-100. Bacteria present in these lysates, representing the total number of bacteria present both intra- and extracellularly, were titered. For adherence assays, a second set of wells was washed five times in PBS (containing Mg²⁺/Ca²⁺), lysed in 0.1% Triton X-100 in ddH₂O, and then plated on LB-agar plates. Adherence frequencies were calculated as the number of bacteria recovered after PBS washes divided by the total number of bacteria present in each well. To determine invasion frequencies, after the initial 2 h incubation, an additional set of wells was washed twice with PBS (with Mg²⁺/Ca²⁺) and then incubated for another 2 h in medium containing 100 µg/ml membrane-impermeable bactericidal antibiotic gentamicin (Sigma) to kill any extracellular bacteria. Cells were then washed three times with PBS, lysed in 1 ml of 0.1% Triton X-100 in ddH₂O, and plated on LB-agar plates. Invasion frequencies were calculated as the number of bacteria surviving incubation with gentamicin divided by the total number of bacteria present just before addition of gentamicin.

To test the effects of specific inhibitors on bacterial invasion, genistein (100 mM stock in DMSO) was added 15 min prior to addition of bacteria, while LY294002 (100 mM stock) and wortmannin (0.1 mM stock in DMSO) were added 10 min before infection. Cytochalasin D (0.1 or 1 µg/ml) was added 30 min prior to infection. As a control, DMSO was added to cells to a final concentration of 0.1%. None of the inhibitors or DMSO alone had any effect on bacterial attachment or host cell viability during the course of the experiments (data not shown).

Immunoprecipitations

Subconfluent monolayers of 5637 cells, grown in six-well plates, were infected with 100 µl of a bacterial suspension in PBS (OD₆₀₀ ~1.0). Plates were centrifuged for 5 min at 600 g and then incubated at 37°C for 0, 5, 15, 30 and 60 min. Bacteria and inhibitors were added directly to the conditioned medium in which the host cells had been grown. At the end of each time point, cells were washed three times with ice-cold PBS, lysed with 1% NP-40 lysis buffer [20 mM Tris pH 8.0, 137 mM NaCl, 1% NP-40, 10% glycerol and 1× complete protease inhibitor cocktail (Boehringer Mannheim, Germany)] and then centrifuged at 12 000 g. Cleared lysates were adjusted for equal protein content using the BCA reagent system (Pierce) and immunoprecipitated overnight at 4°C with anti-FAK antiserum (1:100 rabbit polyclonal; Santa Cruz Biotech. Inc.) or anti-vinculin antiserum (2 µg/ml, clone V284 Upstate Biotechnology Inc.). Immune complexes were captured with 50 µl of 50% protein A–Sepharose for 1 h at 4°C, washed three times with ice-cold lysis buffer and then boiled for 8 min in SDS sample buffer. Proteins were resolved by SDS–PAGE using 7.5% polyacrylamide gels, transferred to nitrocellulose and immunoblotted with anti-PI 3-kinase antiserum (1 µg/ml rabbit polyclonal; UBI), rabbit phospho-specific anti-FAKPY397 (0.5 µg/ml; Biosource Inc.), anti-α-actinin (2 µg/ml, clone AT6/172; UBI), anti-tensin (2 µg/ml, clone 5B9; UBI) or anti-talin (4 µg/ml; UBI). Proteins were visualized with the Super Signal WestDura enhanced chemiluminescence system (Pierce) and analyzed using the Fluor-S MultiImager system equipped with Quantity One analysis software (Bio-Rad). In some cases, blots were stripped with IgG elution buffer (Pierce) according to the manufacturer’s instructions and re-probed with the appropriate antisera to demonstrate that similar amounts of proteins were present in each lane.

Protein-coated bead preparation and internalization assays

Carboxylate-modified non-fluorescent beads (1.17 µm diameter; IDC Microspheres, Portland, OR) were incubated with ~250 µg of purified FimCH complex, FimC or BSA (Sigma) as described (Braun et al., 1998). The presence of FimC in the FimCH complex is solely to stabilize the FimH adhesin in a mannose-binding-competent conformation (Choudhury et al., 1999). Equal numbers of FimC-, FimCH- and BSA-coated beads (±2.5% d-mannose) were added to 5637 cells grown on 12 mm sterile glass coverslips in 24-well plates. Plates were then centrifuged for 5 min at 600 g and incubated for 30–60 min at 37°C. Cells were then washed three times with PBS ± 2.5% d-mannose and fixed for 20 min at room temperature in fresh 2.5% paraformaldehyde in PBS.

To stain extracellular beads, cells were blocked with RPMI/10% FBS for 10 min at room temperature, washed with RPMI and then incubated for 1 h at room temperature with a 1:500 dilution of either mouse anti-FimCH antiserum (MedImmune Inc., Gaithersburg, MD) or rabbit anti-BSA antiserum (Sigma) in RPMI/1% FBS. Cells were washed in PBS and incubated with either anti-mouse IgG–Cy3 or anti-rabbit IgG–Cy3 conjugate (1:500) (Amersham) for 45 min at room temperature. After additional washes, coverslips were mounted in anti-fade mounting medium (kind gift of R.Rosqvist, Umea, Sweden). Samples were examined with an Olympus BX60 fluorescent microscope using an 80× objective. Images were captured using IP LabSpectrum software. Random fields of cells were viewed in phase contrast and fluorescent modes and the images were superimposed using Adobe Photoshop. Beads that were inaccessible to the staining procedure were scored as intracellular. Data are presented as the number of beads internalized divided by the number of adherent beads. Data shown are representative of at least four separate assays.

Examination of the actin cytoskeleton

5637 cells, seeded onto 12 mm sterile glass coverslips, were infected with GFP-expressing bacteria or incubated with FimCH-, FimC- or BSA-coated beads as described above. After 30–60 min, cells were washed, fixed with 2.5% paraformaldehyde/PBS and blocked with RPMI/10% FBS. Cells were then permeabilized using 0.2% Triton X-100 in RPMI/1% FBS and incubated for 20 min at room temperature with Texas red-labeled phalloidin (Molecular Probes, Eugene, OR). FimCH-coated beads were stained with mouse anti-FimCH antiserum followed by anti-mouse IgG–Cy3 conjugate (Amersham) prior to host cell permeabilization. Samples were examined using a Bio-Rad MRC 1024 dual laser scanning confocal microscope system equipped with a Zeiss Axioplan fluorescent microscope, and images were analyzed using the LaserSharp (Bio-Rad) and Confocal Assistant software packages.