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Neisseria meningitidis, the causative agent of meningitis and septicemia, is able to attach to and invade a variety of cell types. In a previous study we showed that entry of N. meningitidis into human brain microvascular endothelial cells (HBMEC) is mediated by fibronectin bound to the outer membrane protein Opc, which forms a molecular bridge to α5β1-integrins. This interaction results in cytoskeletal remodeling and uptake of the bacteria. In this study we identified and characterized the intracellular signals involved in integrin-initiated uptake of N. meningitidis. We determined that the Src protein tyrosine kinases (PTKs) are activated in response to contact with N. meningitidis. Inhibition of Src PTK activity by the general tyrosine kinase inhibitor genistein and the specific Src inhibitor PP2 reduced Opc-mediated invasion of HBMEC and human embryonic kidney (HEK) 293T cells up to 90%. Moreover, overexpression of the cellular Src antagonist C-terminal Src kinase (CSK) also significantly reduced N. meningitidis invasion. Src PTK-deficient fibroblasts were impaired in the ability to internalize N. meningitidis and showed reduced phosphorylation of the cytoskeleton and decreased development of stress fibers. These data indicate that the Src family PTKs, particularly the Src protein, along with other proteins, are important signal proteins that are responsible for the transfer of signals from activated integrins to the cytoskeleton and thus mediate the endocytosis of N. meningitidis into brain endothelial cells.
Neisseria meningitidis is a common colonizing bacterium in the human nasopharynx, and it is found in 8 to 20% of healthy individuals (5). In a small percentage of carriers, N. meningitidis can cross the epithelial barrier and enter the bloodstream. Following bacteremia, N. meningitidis may bind and subsequently cross the blood-cerebrospinal fluid (B-CSF) barrier to enter the subarachnoidal space, resulting in acute and purulent meningitis (29). To overcome this barrier, N. meningitidis has evolved the ability to invade and pass through the host cells.
N. meningitidis binds to endothelial cells using a variety of microbial structures and proteins, including type IV pili, the Opa and Opc proteins, the newly identified minor adhesion or adhesion-like proteins Neisseria Hia homologue A (NhhA) and adhesion penetration protein (App), the two-partner secretion system hemagglutinin/hemolysis-related protein A (HrpA)-HrpB, neisserial adhesion A (NadA), and meningococcal serine protease A (MspA) (6, 40, 42, 46, 48). The primary meningococcal invasins that facilitate bacterial uptake by endothelial cells are the Opa and Opc proteins.
In particular, outer membrane protein Opc allows tight association of the bacteria with the extracellular matrix (ECM) proteins, such as vitronectin and fibronectin (47, 52). Both vitronectin and fibronectin are also abundant in human serum (37, 39), and interaction of Opc with these serum factors leads to binding to endothelial αVβ3-integrin (the vitronectin receptor) and α5β1-integrin (the fibronectin receptor) (47, 48, 52). We recently showed that Opc-expressing meningococci bind to fibronectin, which acts as a molecular bridge, linking N. meningitidis to α5β1-integrins on the host cell surface of human brain microvascular endothelial cells (HBMEC) (47). This interaction promotes uptake of N. meningitidis by the endothelial cell (44, 47), and uptake requires rearrangement of an active actin cytoskeleton, as shown by cytochalasin D treatment (44). Further, we recently showed that tyrosine kinases are likely to play an important role in invasion of mammalian cells by N. meningitidis, since inhibitors of tyrosine kinases prevented uptake of this bacterium by HBMEC (44). However, the kinases involved in the intracellular signaling pathways initiated after binding to α5β1-integrin are still unclear.
The integrins are a family of heterodimeric cell surface receptors that mediate adhesion to extracellular matrix ligands and cell surface ligands (20). Protein tyrosine kinases (PTKs) are key components of a multiprotein complex that assembles at the cytoplasmic face of ligand-bound integrins (14, 17). This complex is responsible for connecting integrins to the actin cytoskeleton and transferring signals into the cell.
Among the tyrosine kinases, the Src family PTKs play a particularly important role downstream of integrin adhesion receptors. The Src PTKs include the ubiquitously expressed proteins c-Src, c-Yes, and Fyn, as well as Hck, Lck, Lyn, Yrk, Blk, and Fgr, which are expressed in hematopoietic tissues (45). Src PTKs are 52- to 62-kDa intracellular proteins that contain an N-terminal myristylation site that enables membrane association, Src homology 2 (SH2) and SH3 domains that mediate protein-protein interactions, a catalytic domain, and a C-terminal domain that includes a tyrosine residue (Y529 in humans), which is important in the regulation of the catalytic activity (55). When Y529 is phosphorylated by the C-terminal Src kinase (CSK), the Src PTK is kept in an inactive form through an intramolecular interaction between the SH2 domain and the C-terminal domain. This conformation blocks phosphorylation of the catalytic domain residue (Y419 in humans), thereby preventing Src activation. Dephosphorylation of Y529 alters the conformation of the protein and opens the kinase domain, resulting in autophosphorylation of Y418, which is required for full activation of the kinase (38, 55).
In this study, we analyzed the intracellular signals leading to β1-integrin-mediated uptake of N. meningitidis. We demonstrated that host cell PTKs and the actin cytoskeleton have essential roles and identified Src family PTKs as signal proteins that are important in the invasion process. We showed that Src kinases are activated in response to Opc-expressing N. meningitidis. Furthermore, pharmacological blocking of Src familiy PTKs, as well as overexpression of the specific inhibitor CSK, reduced bacterial uptake. In addition, Src-deficient fibroblasts were resistant to N. meningitidis uptake, confirming the essential role of Src family PTKs in endocytosis.
N. meningitidis serogroup B strain MC58 (B:15:P1.7,16b) is a clinical isolate belonging to the sequence type (ST-32) complex, which was isolated from an outbreak of meningococcal infections in Stroud, Gloucestershire, United Kingdom, in 1981 and 1982 (26) and was kindly provided by E. R. Moxon. Unencapsulated mutant strain N. meningitidis MC58 siaD and unencapsulated opc-deficient strain MC58 siaD opc have been described previously (47). All strains were routinely cultured in proteose-peptone medium (PPM+) with 1% Polyvitex (bioMerieux, Marcy l'Etoile, France). For invasion assays, bacteria were diluted in RPMI 1640 medium (Biochrom AG, Berlin, Germany) supplemented with 10% heat-inactivated (30 min, 56°C) human serum (HS) as described recently (47).
Human brain microvascular endothelial cells (HBMEC) were kindly provided by K. S. Kim (Baltimore, MD) and were cultured as described recently (44). Human embryo kidney (HEK) cell line 239T was cultured in Dulbecco modified Eagle medium (DMEM) (Biochrom, Berlin, Germany) with 10% fetal calf serum (FCS) at 37°C in the presence of 5% CO2. Fibroblasts derived from Src, Yes, Fyn triple-knockout mouse embryos (SYF cells ) were kindly provided by P. Soriano (Fred Hutchinson Cancer Research Center, Seattle, WA). SYF cells expressing c-Src (SYF+c-Src cells) were used as a control. Cells were grown in gelatin-coated (0.1% in phosphate-buffered saline [PBS]) cell culture dishes or flasks in DMEM with 10% FCS supplemented with 1% nonessential amino acids. All cell cultures were incubated at 37°C with 5% CO2.
The tyrosine kinase inhibitors genistein and PP2, as well as the actin filament function inhibitor cytochalasin D, were purchased from Calbiochem (La Jolla, CA). Inhibitors were reconstituted in dimethyl sulfoxide (DMSO) and stored according to the manufacturer's instructions. For Western blot and immunofluorescence analyses the following antibodies were used: purified monoclonal antibody (MAb) against CSK (clone 52; BD Biosciences Technology), polyclonal Src antibody ab7950 (abcam, Cambridge, MA), anti-Src[pY418] (Invitrogen, Camarillo, CA), phosphotyrosine mouse MAb p-Tyr-100 9411 (Cell Signaling Technology, Danvers, MA), tetramethyl rhodamine isocyanate (TRITC)-conjugated antibody 115-025-003 (Dianova, Hamburg, Germany), and Alexa Fluor 488 phalloidin (Molecular Probes/Invitrogen).
For invasion assays, HBMEC, 293T cells, or fibroblasts were seeded onto 24-well tissue culture plates (Corning Costar) at a density of 5 × 104 cells per well and were grown to a concentration of ~1 × 105 prior to infection. Cells were infected with bacteria at a multiplicity of infection (MOI) of 30 either in the presence of RPMI 1640 medium with 10% HS (HBMEC) or in the presence of DMEM with 10% HS (293T and fibroblasts). After 4 h of infection, the number of adherent bacteria in each supernatant was determined by lysis of HBMEC with 1% saponin for 15 min and subsequent determination of the number of CFU by plating appropriate dilutions of the lysates of blood agar (bioMérieux, France). The numbers of intracellular bacteria were determined after 2 h of incubation with cell culture medium containing gentamicin (Biochrom, Berlin, Germany) at a concentration of 200 μg ml−1. The proportion of invasive bacteria was calculated by determining the ratio of the number of intracellular bacteria to the number of total cell-associated bacteria. The number of adherent bacteria was determined by determining the difference between the number of CFU before gentamicin treatment and the number of CFU after gentamicin treatment. All samples were tested in duplicate, and experiments were repeated at least three times.
Human cytomegalovirus promoter-driven expression constructs for wild-type Src, inactive Src(K297M) with a point mutation, and wild-type CSK were kindly provided by David D. Schlaepfer (University of California at San Diego, La Jolla, CA). pcDNA3.1 was purchased from Invitrogen (Carlsbad, CA) and used as a negative control in transfection experiments. 293T cells were transfected using the calcium phosphate coprecipitation protocol and 1 μg plasmid DNA. In brief, 293T cells were seeded onto 24-well tissue culture plates and grown to semiconfluence. Cells were then incubated with 1 μg plasmid DNA in 2× BBS [50 mM N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid [BES] [Calbiochem, La Jolla, CA], 180 mM NaCl, 1.5 mM Na2HPO4·H2O] with 220 mM CaCl2. After 24 h of incubation, the DMEM was replaced. SYF cells were transfected with Lipofectamine (Invitrogen, Carlsbad, CA) used according to the manufacturer's instructions. The transfection efficiencies ranged from 70 to 80% and were monitored by parallel transfection with a green fluorescent actin-expressing construct (pTagGFP2-actin vector; Evrogen, Moscow, Russia) and quantification of fluorescent cells. Cells were used in infection experiments 48 h after transfection.
Src and CSK expression levels were examined by using cell extracts prepared parallel to cells used for invasion assays. Four hours postinfection, cells were washed three times in ice-cold phosphate-buffered saline (PBS) (Biochrome AG, Berlin, Germany) and lysed in 2× SDS sample buffer (SDS obtained from Sigma Chemie GmbH, Steinheim, Germany). Samples were boiled, and proteins were separated by SDS-PAGE. After electrotransfer onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany), the membranes were blocked in PBS-0.1% Tween containing 6% dry milk (Bio-Rad, Munich, Germany). Membranes were probed with the anti-Src, anti-Src[pY418], and anti-CSK antibodies at 4°C overnight. After three washes with PBS-Tween, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000 in PBS-Tween containing 6% dry milk) for 1 h. Immunoreactivity was detected using the enhanced chemiluminescence (ECL) reagent (Pierce, Rockford, IL).
At time points indicated below, infected cells were washed twice with ice-cold PBS and lysed in modified RIPA2 buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 24 mM sodium deoxycholate, 50 mM NaF, 0.2 mM sodium orthovanadate, 1 mM 1,10-phenantroline monohydrate, protease inhibitor cocktail tablet [Roche Diagnostics, Mannheim, Germany]). After mechanical disruption of cells and incubation on ice for 30 min, the lysates were spun down, and each supernatant was supplemented with 1 μg rabbit IgG and 20 μl G Plus agarose (both obtained from Santa Cruz Biotechnology, Santa Cruz, CA). After 30 min the probes were spun down again, and equal amounts of supernatant were incubated with Src antibody at a final dilution at 1:1,000 and then 30 min later with 20 μl G Plus agarose. The probes were incubated overnight and spun down, and the pellets were washed twice in PBS containing a protease inhibitor cocktail tablet, 0.2 mM sodium orthovanadate, and 1 mM 1,10-phenantroline monohydrate. For Western blot analysis, the precipitates were resuspended in reducing 2× SDS sample buffer and analyzed as described above. All preparations were incubated at 4°C. The protein contents of the supernatants were quantified by using the Lowry method with bovine serum albumin (BSA) as the standard.
For flow cytometric analyses, HBMEC and 293T cells were released from confluent monolayer cultures with trypsin, washed, and resuspended in fluorescence-activated cell sorting (FACS) buffer (5% fetal calf serum [FCS] and 0.1% sodium azide in PBS). Then 1 × 106 cells were added to each vial and incubated with MAb P5D2 to β1 for 90 min on ice and washed twice with FACS buffer. Then goat anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC) in 0.3 ml (total volume) of FACS buffer was added, and the cells were incubated for 30 min on ice. After two washes with FACS buffer, the cells were used for analysis. The analysis was performed using CellQuest Pro software (version 5.2) with a FACSCalibur (Becton Dickinson). Positive fluorescence was determined using a 4-decade log scale, and FITC fluorescence (FL1) was expressed as the mean channel number for 10,000 cells.
For indirect immunofluorescence analysis, 293T cells and fibroblasts were seeded on glass coverslips in 24-well plates and grown to semiconfluence. Cells were infected with fluorescently labeled Neisseria using an MOI of 10 as described elsewhere (2). In brief, bacteria (1 × 109 ml−1 cells) were washed twice with sterile PBS, suspended in 0.4 μg ml−1 5-(6)-carboxyfluorescein-succinylester (Molecular Probes/Invitrogen) dissolved in PBS (FITC-PBS), and incubated for 15 min with constant shaking at room temperature. After infection, cells were washed once with PBS and fixed with 3.7% paraformaldehyde-PBS for 20 min at room temperature. Paraformaldehyde-fixed cells were washed three times with PBS and permeabilized by incubation with 1% Triton X-100-PBS for 10 min. Samples were incubated with phosphotyrosine mouse MAb p-Tyr-100 at a final dilution of 1:200 and Alexa Fluor 488 phalloidin (Molecular Probes/Invitrogen) at a final dilution at 1:40 in blocking buffer (PBS, 2% FCS) for 1 h at room temperature. Cells were washed three times with PBS for 5 min and then incubated with secondary TRITC-conjugated antibody (diluted 1:100 in blocking buffer) for 60 min at room temperature.
After three washes with PBS, coverslips were mounted in quick-hardening mounting medium (Fluka, Steinheim, Germany). All incubations were performed in a wet chamber at room temperature. Fluorescence microscopy was performed using a Zeiss Axio Imager.Z1 fluorescence microscope (Zeiss, Heidelberg, Germany). Images were photographed using an AxioCam digital camera and AxioVision software and were documented using Adobe Photoshop CS.
Two-tailed Student's t test was used to calculate statistical significance (P values).
We previously found that N. meningitidis invades human nonprofessional phagocytes, such as endothelial cells, by endocytosis (47). This process is mediated by fibronectin bound to the outer membrane protein Opc, which forms a molecular bridge to α5β1-integrins (47). As the cytoplasmic tails of integrin molecules lack intrinsic enzymatic activity, regulation of integrin signaling is dependent on adaptor proteins like protein tyrosine kinases (PTKs) that possess enzymatic activity or are linkers that connect the integrin tail and the actin cytoskeleton (14). To examine the involvement of tyrosine kinases during the entry of N. meningitidis into endothelial cells, invasion assays were performed in the presence of various tyrosine kinase inhibitors. Infection assays were carried out using N. meningitidis serogroup B strain MC58 (sequence type 32 [ST-32] complex) and the unencapsulated but more invasive strain MC58 siaD (47). The general PTK inhibitor genistein blocked invasion by N. meningitidis strains MC58 and MC58 siaD efficiently in a dose-dependent manner (>60% inhibition with 50 μM genistein for strain MC58 siaD and >80% inhibition with 50 μM genistein for the N. meningitidis wild-type strain MC58 [P < 0.05]) (Fig. 1A and B). To exclude the possibility that the observed inhibition was due to inefficient adhesion of N. meningitidis to genistein-treated HBMEC, the total number of cell-associated bacteria was determined. Genistein treatment did not have any effect on the total number of cell-associated bacteria. Furthermore, the growth of bacteria was not affected in the presence of genistein (data not shown).
Several PTKs have been shown to be activated in response to integrin engagement by fibronectin or ECM proteins (for an overview, see reference 14). To examine whether particular PTKs are involved in N. meningitidis invasion, we blocked Src family PTKs with the specific inhibitor PP2 (Fig. (Fig.1).1). Pretreatment of HBMEC with PP2 significantly decreased invasion by N. meningitidis (Fig. 1A and B). At a concentration of 10 μmol/ml, invasion by both isolates was reduced by about 90% (Fig. 1A and B). As observed for genistein treatment, the PP2 inhibitor had no effect on the total number of cell-associated bacteria or the growth of bacteria (data not shown).
Furthermore, phosphorylation of cytoskeleton components during N. meningitidis infection was confirmed by immunofluorescence analysis. As shown in Fig. Fig.1C,1C, N. meningitidis induced cytoskeletal changes and development of stress fibers (data shown for invasive strain MC58 siaD). Further staining with antiphosphotyrosine antibody p-Tyr-100 revealed that N. meningitidis caused enhanced tyrosine phosphorylation of proteins in focal adhesions present at the tips of actin stress fibers. Accumulation of stress fibers and tyrosine phosphorylation of proteins were observed at the site of bacterial adhesion (Fig. 1C and D). Addition of the general PTK inhibitor genistein and the specific inhibitor PP2 resulted in decreased stress fiber formation and tyrosine phosphorylation (data not shown). Together with the results of the inhibition studies, these results suggested that Src family PTKs are involved in integrin-mediated uptake of N. meningitidis.
Having demonstrated that tyrosine kinases are required for N. meningitidis invasion, we next examined the tyrosine phosphorylation pattern of HBMEC after infection with N. meningitidis. Confluent HBMEC monolayers were infected with N. meningitidis wild-type strain MC58 and the invasive unencapsulated mutant strain N. meningitidis MC58 siaD for 8 h, and cell lysates were prepared at the time points indicated below. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with antiphosphotyrosine antibody p-Tyr-100. As shown in Fig. Fig.2,2, both isolates induced tyrosine phosphorylation of several proteins with apparent masses of 125 kDa and 65 kDa. Phosphorylation peaked between 120 and 240 min and declined thereafter, indicating that N. meningitidis invasion is associated with specific tyrosine phosphorylation.
Src family PTKs have been shown to have roles in integrin signaling and control of the actin cytoskeleton in many types of cells. Since the size of the 60-kDa protein present in infected HBMEC was in range of the sizes of Src PTKs, we next determined the role of Src PTKs in the invasion process. To confirm the role of Src kinases in N. meningitidis invasion, 293T cells were transfected with mammalian expression vectors containing the specific cellular c-Src inhibitor CSK. CSK is a cellular PTK that has been shown to specifically regulate the activity of c-Src at the carboxy-terminal domain (30). 293T cells were used for this genetic interference analysis because HBMEC could not be sufficiently transfected transiently. Both the HBMEC and 293T cell lines express endogenous c-Src (23; data not shown).
N. meningitidis had similar invasion kinetics and Opc-dependent uptake mechanisms in 293T cells and HBMEC; however, the absolute number of invasive bacteria was about 1 log lower in 293T cells (Fig. 3A and B), probably because of the lower levels of β1-integrin expression in 293T cells than in HBMEC, as demonstrated by flow cytometry analysis. As shown in Fig. Fig.3C3C HBMEC bound three times more MAb P5D2 than 293T HEK cells.
We first established that PTKs are also involved in uptake of N. meningitidis by 293T cells. Indeed, invasion by N. meningitidis was significantly impaired in the presence of genistein and PP2 (Fig. (Fig.4A).4A). 293T cells were then transiently transfected with mammalian expression vectors containing wild-type CSK. Overexpression of CSK significantly (>80%) inhibited invasion of 293T cells by N. meningitidis MC58 siaD compared to the invasion of 293T cells transfected with a control vector (pcDNA) (Fig. (Fig.4B4B).
To confirm the role of Src, we employed mammalian expression vectors containing either wild-type Src or an inactivated version of Src with a point mutation [Src(K297M)]. Overexpression of wild-type Src resulted in a 4-fold increase in N. meningitidis uptake compared to the results for 293T cells transfected with the control vector, while overexpression of Src(K297M) resulted in a prominent decrease in the uptake of bacteria (Fig. (Fig.4C).4C). The higher number of intracellular bacteria in Src(K297M)-expressing 293T cells than in cells transfected with the control vector (pcDNA) was due to the minor rest activity of the protein (see the results of the Western blot analysis with anti-Src[pY418] in Fig. Fig.4C).4C). Together, these results confirmed the critical role of Src in N. meningitidis internalization and supported the results obtained for pharmacological inhibition with the general PTK inhibitor genistein and the more selective inhibitor PP2 in HBMEC.
The results described above suggested that Src has an important role involving activation of cytoskeleton remodeling in N. meningitidis invasion. Thus, cells lacking Src should be resistant to invasion by N. meningitidis. To test this hypothesis, we used fibroblasts (SYF cells) which were derived from Src-, Yes-, and Fyn-deficient mouse embryos (21). SYF cells lack these three members of the Src PTK family that are normally expressed in this type of cells (21). SYF cells expressing c-Src (SYF+c-Src cells) were used as a control.
To test whether N. meningitidis is able to invade embryonic mouse SYF+c-Src fibroblasts, we infected SYF+c-Src cells with strain MC58 siaD in the presence of human serum. As shown in Fig. Fig.5A,5A, maximal invasion was detected 240 min postinfection, which was similar to the invasion kinetics observed for 293T cells and HBMEC. When infection assays were carried out for more than 240 min, increased detachment of SYF+c-Src cells was observed, suggesting that there were cytotoxic effects on the cultured cells. To confirm the essential role of Opc in endocytosis, an opc-deficient mutant (47) was employed in gentamicin protection assays. Importantly, the opc-deficient mutant N. meningitidis MC58 siaD opc was internalized less by SYF+c-Src cells, demonstrating that there is an Opc-dependent invasion mechanism for mouse fibroblasts, as previously shown for HBMEC (47).
Src PTK-deficient SYF cells were infected with N. meningitidis, which resulted in a significant decrease in the number of invasive bacteria (Fig. (Fig.6A).6A). In addition, Src activity could be restored in SYF cells by transiently transfecting SYF cells with mammalian expression vectors containing the specific cellular c-Src protein. In Src-expressing SYF cells, an increase in bacterial internalization was observed (Fig. (Fig.6B).6B). It is important that the average transfection efficiency, as measured by cotransfection with a green fluorescent actin-expressing construct (pTagGFP2-actin vector), was between 75 and 80% of the total cell population (data not shown). Therefore, the increase in the invasion rate to 65% in Src-expressing SYF cells corresponded to the results for invasion of SYF+c-Src cells. As observed with 293T cells, cells transfected with the inactive version of Src [Src(K297M)] contained significantly fewer invasive bacteria than SYF+c-Src cells (Fig. (Fig.6B).6B). As observed with 293T cells, the higher number of intracellular bacteria in Src(K297M)-expressing fibroblasts than in cells transfected with the control vector (pcDNA) was due to the minor resting activity of the protein (Fig. (Fig.4C4C).
To further examine the role of Src in stress fiber formation and tyrosine phosphorylation of proteins in focal adhesions in response to N. meningitidis infection, SYF cells and Src-expressing SYF+c-Src cells were infected with N. meningitidis MC58 siaD, fixed, and analyzed by using immunofluorescence as described above. As shown in Fig. Fig.6C,6C, in cells lacking Src activity there were decreases in stress fiber formation and tyrosine phosphorylation of HBMEC proteins compared to the results for the control.
The results of the PP2 inhibition experiments, the overexpression of Src and Src(K297M), and the lack of N. meningitidis invasion of SYF cells suggested that Src kinase activity might drive the integrin-mediated uptake of N. meningitidis. To analyze if Src kinase activity is altered upon infection of cells, Src activity in HBMEC was measured by using a phospho-specific antibody (anti-Src[pY418], recognizing phosphorylated Y419 in human c-Src) that detects phosphorylation of Src at its regulatory tyrosine residues (22). HBMEC were serum starved and plated on poly-l-lysine-coated dishes to minimize integrin engagement by the cell culture substrate (1). Cells were infected with N. meningitidis MC58 siaD or not infected either in the presence of 10% human serum (HS) or without HS. After cell lysis samples were immunoprecipitated with an Src-specific monoclonal antibody and analyzed by Western blotting with the phospho-specific antibody mentioned above (anti-Src[pY418]). As shown in Fig. Fig.7,7, the phosphorylation of Src was greater in N. meningitidis MC58 siaD-infected cells than in uninfected cells and cells cultured in the presence of 10% HS. In addition, the opc-deficient unencapsulated mutant MC58 siaD opc was employed in this assay, and it did not induce Src activation (Fig. (Fig.7B).7B). HBMEC were treated with 10% HS as a control to exclude the possibility that there was Src activation due to serum-dependent effects. Densitometric analysis revealed a 1.7-fold increase in activity after infection with N. meningitidis MC58 siaD. This small but significant increase in Src activity upon infection is in line with the view that integrin-bound meningococci provide a locally confined and transient stimulus.
The pathogenesis of meningococcal meningitis involves the crossing of two cellular barriers by the microorganism, one in the nasopharynx and one in the brain. Adherence of bacteria to the host cells that form the barriers is often a prerequisite for successful invasion of deeper tissues by bacteria. In N. meningitidis the type IV pili and the outer membrane proteins Opa and Opc are the major adhesins which enable anchoring of the meningococcus to host tissues (48-51). We recently showed that Opc-expressing meningococci bind to the extracellular matrix (ECM) protein fibronectin, which acts as a molecular bridge linking N. meningitidis to α5β1-integrin of the host cell surface (47). This interaction promotes uptake of the bacteria by the endothelial cells (44, 47). Our previous results also showed that cytochalasin D, an inhibitor of actin polymerization, blocked Opc-dependent N. meningitidis invasion of HBMEC, indicating that an intact actin cytoskeleton is required.
Integrins are the major cell surface adhesion receptors for ligands in the extracellular matrix (20). Once integrins bind to ligands of the ECM, they become clustered in the plane of the cell membrane and associate with a cytoskeletal and signaling complex that promotes assembly of actin filaments. Among the signaling molecules, the tyrosine kinase focal adhesion kinase (FAK), Src family kinases, abl, and the serine-threonine kinase integrin-linked kinase (ILK) are activated (8, 15, 16, 22, 41, 53, 54). Since Src family PTKs have functions in integrin signaling and control of the actin cytoskeleton in many types of cells, we focused on the role of Src family PTKs in the present study.
Our initial data obtained using the general tyrosine kinase inhibitor genistein indicated that PTKs have a significant role in invasion of HBMEC by N. meningitidis. The observed effect of genistein on N. meningitidis invasion was not the result of diminished adhesion to HBMEC as genistein-treated and untreated cells did not differ in the number of total cell-associated bacteria. Further pharmacological blocking with PP2 indicated that specific Src family PTKs, including c-Src, Hck, Fck, and Lyn, have a significant role in the invasion process. In this study we found that c-Src PTK is essential for this process. In particular, Src kinase activity is enhanced by infection with N. meningitidis. Genetic interference with this nonreceptor kinase decreased bacterial uptake, and Src-deficient fibroblasts were shown to be resistant to N. meningitidis invasion, demonstrating the essential role of this kinase in integrin-triggered uptake of this bacterium. Furthermore, immunofluorescence analyses demonstrated that the Src PTKs regulate bacterial internalization by cytoskeleton remodeling, probably by activating cytoskeleton-regulating proteins. The conceivable integrin-associated actin-regulating proteins are talin, paxillin, α-actinin, filamin, vinculin, and cortactin, which are recruited to clustered, ligand-bound integrins in a hierarchical manner (9, 27). Several of these actin-regulating proteins are substrates for the tyrosine kinase activity of Src (18, 34), and their role in cytoskeletal rearrangements induced after infection with N. meningitidis in endothelial cells is a topic for further investigation.
Src PTKs are likely to function in concert with the nonreceptor PTK focal adhesion kinase (FAK) in response to ligand-induced integrin clustering (36). This occurs as the result of FAK autophosphorylation of tyrosine residue 397 (Y397), which provides a docking site for the SH2 domain of Src PTKs. The c-Src-FAK complex propagates integrin signals, in part because it supports Src PTK-dependent phosphorylation of additional tyrosine residues in FAK. When we analyzed the tyrosine phosphorylation pattern of HBMEC after infection with N. meningitidis, we observed an additional phosphorylated protein with an apparent molecular mass of about 125 kDa, which is in the range of the molecular masses of FAK. The role of FAK in invasion, however, remains to be determined.
Involvement of c-Src in bacterial uptake has been observed for other pathogens (1, 7, 10-12, 25). c-Src can be activated downstream of either integrin engagement or other receptors (21). Staphylococcus aureus and Yersinia spp. induce activation of Src PTKs as a downstream result of integrin engagement (1, 3). Integrins are used by the pathogens either as primary attachment receptors or as coreceptors in the entry process (35). While Yersinia directly enters host cells by an integrin-dependent mechanism, S. aureus has been shown to engage integrins indirectly by binding to components of the ECM (13, 31-33, 43). In each case, integrin ligation results in assembly of focal adhesion complexes at sites of bacterial attachment and the activation of integrin-dependent signaling cascades, facilitating bacterial internalization. However, ligation of integrins also triggers a wide variety of signal transduction events that modulate a multitude of cellular responses, including proliferation, survival and apoptosis, cell shape and motility, and gene expression (19). It is quite probable that binding of N. meningitidis to integrins also stimulates further cellular processes in addition to bacterial uptake.
Besides transferring integrin signals into the cell, c-Src also plays a significant role in signal transduction downstream of growth factor receptors and G protein-coupled receptors. It is interesting that epidermal growth factor (EGF) and its receptor (EGFR) have also been shown to couple with Src kinase (for example, to regulate cancer progression) (4). Recent work has demonstrated that integrin-mediated adhesion can promote activation of growth factor receptors independent of growth factor binding (28). Such cross-activation of EGFR following integrin ligation is involved in the induction of Rac1 activity and promotes formation of lamellipodia and membrane ruffling (24). It is tempting to speculate that activation of integrins acts synchronously with EGFR to mediate invasion of host cells by bacteria.
We are indebted to P. Soriano (FHCRC, Seattle, WA) for providing the SYF cells and to D. Schlaepfer (University of California at San Diego, La Jolla, CA) for providing cDNA constructs. We thank Biju Joseph for critical reading of the manuscript.
This work was supported by grant SCHU 2394/1-1 from the Deutsche Forschungsgemeinschaft.
Editor: J. N. Weiser
Published ahead of print on 22 February 2010.