Entry of Neisseria meningitidis into mammalian cells requires the Src family protein tyrosine kinases

Abstract

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 alpha 5 beta 1-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.

FIG. 1.

N. meningitidis internalization by HBMEC requires protein tyrosine kinase activity. (A) HBMEC were preincubated with the concentrations of genistein and the more specific Src PTK inhibitor PP2 indicated and infected for 4 h with unencapsulated N. meningitidis strain MC58 siaD. The numbers of intracellular bacteria were determined by the gentamicin protection assay. The data are the means and standard deviations of three independent experiments performed in duplicate. *, P < 0.05 compared to cells infected without an inhibitor. (B) Prior to infection with encapsulated wild-type strain MC58, HBMEC were preincubated with genistein and PP2. After 4 h of infection, the numbers of intracellular N. meningitidis bacteria were determined by the gentamicin protection assay. The data are the means and standard deviations of three independent experiments performed in duplicate. *, P < 0.05 compared to cells infected without inhibitor. (C) Immunofluorescence analysis of HBMEC infected with FITC-labeled N. meningitidis MC58 siaD. Infected cells were fixed and double labeled with antibody to p-Tyr-100 (red fluorescence) and phalloidin (FITC-phalloidin, green fluorescence). A considerable increase in the amount of tyrosine-phosphorylated proteins was observed in infected cells. (D) Aggregation of f-actin close to attached bacteria (TRITC labeled, red fluorescence), which is indicated by arrowheads. N. m., N. meningitidis.

FIG. 2.

N. meningitidis induces increased tyrosine phosphorylation of HBMEC proteins. Confluent monolayers of HBMEC were infected with N. meningitidis wild-type strain MC58 and unencapsulated mutant MC58 siaD for the times indicated. Cell lysates were prepared, separated by SDS-PAGE, and immunoblotted with antiphosphotyrosine antibody p-Tyr-100. Two distinct bands at 125 kDa and 60 kDa were produced by infected cells. Sizes are indicated on the right. The control was uninfected HBMEC. WCL, whole-cell lysate.

FIG. 3.

Opc-mediated uptake of N. meningitidis by 293T cells. (A) 293T cells were infected with invasive strain N. meningitidis MC58 siaD at an MOI of 30 in the presence of RPMI cell culture medium supplemented with 10% human serum (HS). The numbers of intracellular bacteria were determined after gentamicin treatment at 60, 120 and 240 min postinfection. (B) Cells were infected with strain MC58 siaD (opc+) and the isogenic Opc-deficient mutant strain MC58 siaD opc (opc−) at an MOI of 30. Four hours postinfection, the numbers of invasive bacteria were determined by using gentamicin protection assays. The data are the means and standard deviations of three independent experiments performed in duplicate. *, P < 0.05. (C) FACS analyses of 293T cells and HBMEC to determine β1-integrin expression. Cells were labeled with MAb P5D2 and were detected with FITC-conjugated goat anti-mouse IgG. The fluorescence intensity obtained with the MAb (filled area) was compared with that of the controls (solid line). MFI, mean fluorescence intensity.

FIG. 4.

Interference with the Src PTK family function results in decreased uptake of N. meningitidis by host cells. (A) 293T cells were preincubated with genistein and PP2 and infected for 4 h with the unencapsulated strain N. meningitidis MC58 siaD. The numbers of intracellular bacteria were determined by using the gentamicin protection assay. The data are the means and standard deviations of three independent experiments performed in duplicate. *, P < 0.05 compared to cells infected without the inhibitor. (B) 293T cells were transfected with a control plasmid (pcDNA) and a plasmid encoding the C-terminal Src kinase (CSK). Transfected cells were infected with invasive strain MC58 siaD, and the numbers of intracellular bacteria were estimated 4 h postinfection by using gentamicin protection assays. The data in the graphs are the means and standard deviations of three independent experiments performed in duplicate. *, P < 0.05 compared to cells transfected with the control vector. In parallel, whole-cell lysate (WCL) extracts were prepared and subjected to Western blot analysis. Staining with an anti-CSK monoclonal antibody demonstrated that there was overexpression of CSK in transfected cells. (C) 293T cells were transfected with the empty control plasmid (pcDNA), a plasmid encoding wild-type c-Src (Src), and a vector encoding inactive c-Src kinase (Src K297M). Transfected cells were used in gentamicin protection assays with N. meningitidis strain MC58 siaD. The data in the graphs are the means and standard deviations of three independent experiments performed in duplicate. Western blot analysis of WCL with anti-c-Src monoclonal antibody showed that there was expression of transfected Src and inactive c-Src (Src K297M). In parallel, samples were analyzed by Western blotting with the phospho-specific anti-Src[pY418] antibody to demonstrate the activity of Src in transfected cells.

FIG. 5.

N. meningitidis invades Src-expressing mouse fibroblasts (SYF+c-Src) in an Opc-dependent manner. (A) Syf+c-Src fibroblasts were infected with N. meningitidis invasive strain MC58 siaD at an MOI of 30 in the presence of RPMI cell culture medium supplemented with 10% human serum (HS). The numbers of intracellular were determined after gentamicin treatment at 60, 120, and 240 min postinfection, and the results demonstrated that the invasion kinetics were similar to those of 293T cells and HBMEC. (B) Cells were infected with strain MC58 siaD (opc+) and the isogenic opc-deficient mutant strain MC58 siaD opc (opc−) at an MOI of 30. Four hours postinfection, the numbers of invasive bacteria were determined by using gentamicin protection assays. The data are the means and standard deviations of three independent experiments performed in duplicate. *, P < 0.05.

FIG. 6.

Src-deficient cells are resistant to N. meningitidis uptake. (A) Src-expressing (SYF+c-Src) and c-Src-deficient (SYF) fibroblasts were infected with invasive strain MC58 siaD in HS-supplemented RPMI cell culture medium. The numbers of invasive bacteria were determined at 4 h postinfection. The data are the means and standard deviations of three independent experiments performed in duplicate. *, P < 0.05. (B) To confirm the role of c-Src in triple-knockout SYF fibroblasts, SYF cells were transfected with control DNA (pcDNA), a plasmid encoding wild-type c-Src (Src wt), and a plasmid encoding inactive c-Src kinase (Src K297M). The transfection efficiencies ranged from 70 to 80%. Transfected cells were infected with strain MC58 siaD, and the numbers of intracellular bacteria were determined after gentamicin treatment. The data in the graph are the means and standard deviations of three independent experiments performed in duplicate. *, P < 0.05. Western blotting of whole-cell lysates (WCL) with anti-c-Src monoclonal antibody demonstrated that there was overexpression of transfected Src. (C) Immunofluorescence analyses of Src-expressing SYF (SYF+c-Src) cells and triple-knockout SYF cells after infection with FITC-labeled N. meningitidis MC58 siaD (panels B and D). Infected cells were fixed and double stained with antibody to p-Tyr-100 (red fluorescence) and FITC-phalloidin (green fluorescence). Immunofluorescence confirmed the central role of Src activity in stress fiber formation and tyrosine phosphorylation of HBMEC proteins in response to bacterial infection. Uninfected Src-expressing SYF+c-Src and SYF cells were stained with p-Tyr-100 and FITC-phalloidin as controls (panels A and C). N. m., N. meningitidis.

FIG. 7.

Src kinase activity is enhanced during infection with N. meningitidis. Serum-starved HBMEC were grown to confluence and infected with N. meningitidis strains MC58 siaD (A) and MC58 siaD opc (B) in the presence of 10% HS for 4 h. Control cells were not infected in either cell culture medium or cell culture medium supplemented with 10% HS. After lysis, Src was immunoprecipitated (Src-IP), and samples were analyzed by Western blotting with the phospho-specific anti-Src[pY418] antibody. The amount of phosphorylated Src was quantified by densitometric analysis, and the increase was estimated by comparison with uninfected cells without HS.