Cell Invasion by Neisseria meningitidis Requires a Functional Interplay between the Focal Adhesion Kinase, Src and Cortactin

Entry of Neisseria meningitidis (the meningococcus) into human brain microvascular endothelial cells (HBMEC) is mediated by fibronectin or vitronectin bound to the surface protein Opc forming a bridge to the respective integrins. This interaction leads to cytoskeletal rearrangement and uptake of meningococci. In this study, we determined that the focal adhesion kinase (FAK), which directly associates with integrins, is involved in integrin-mediated internalization of N. meningitidis in HBMEC. Inhibition of FAK activity by the specific FAK inhibitor PF 573882 reduced Opc-mediated invasion of HBMEC more than 90%. Moreover, overexpression of FAK mutants that were either impaired in the kinase activity or were not capable of autophosphorylation or overexpression of the dominant-negative version of FAK (FRNK) blocked integrin-mediated internalization of N. meningitidis. Importantly, FAK-deficient fibroblasts were significantly less invaded by N. meningitidis. Furthermore, N. meningitidis induced tyrosine phosphorylation of several host proteins including the FAK/Src complex substrate cortactin. Inhibition of cortactin expression by siRNA silencing and mutation of critical amino acid residues within cortactin, that encompass Arp2/3 association and dynamin binding, significantly reduced meningococcal invasion into eukaryotic cells suggesting that both domains are critical for efficient uptake of N. meningitidis into eukaryotic cells. Together, these results indicate that N. meningitidis exploits the integrin signal pathway for its entry and that FAK mediates the transfer of signals from activated integrins to the cytoskeleton. A cooperative interplay between FAK, Src and cortactin then enables endocytosis of N. meningitidis into host cells

Differential Activation of Acid Sphingomyelinase and Ceramide Release Determines Invasiveness of Neisseria meningitidis into Brain Endothelial Cells

The interaction with brain endothelial cells is central to the pathogenicity of Neisseria meningitidis infections. Here, we show that N. meningitidis causes transient activation of acid sphingomyelinase (ASM) followed by ceramide release in brain endothelial cells. In response to N. meningitidis infection, ASM and ceramide are displayed at the outer leaflet of the cell membrane and condense into large membrane platforms which also concentrate the ErbB2 receptor. The outer membrane protein Opc and phosphatidylcholine-specific phospholipase C that is activated upon binding of the pathogen to heparan sulfate proteoglycans, are required for N. meningitidis-mediated ASM activation. Pharmacologic or genetic ablation of ASM abrogated meningococcal internalization without affecting bacterial adherence. In accordance, the restricted invasiveness of a defined set of pathogenic isolates of the ST-11/ST-8 clonal complex into brain endothelial cells directly correlated with their restricted ability to induce ASM and ceramide release. In conclusion, ASM activation and ceramide release are essential for internalization of Opc-expressing meningococci into brain endothelial cells, and this segregates with invasiveness of N. meningitidis strains. Author Summary Neisseria meningitidis, an obligate human pathogen, is a causative agent of septicemia and meningitis worldwide. Meningococcal infection manifests in a variety of forms, including meningitis, meningococcemia with meningitis or meningococcemia without obvious meningitis. The interaction of N. meningitidis with human cells lining the blood vessels of the blood-cerebrospinal fluid barrier is a prerequisite for the development of meningitis. As a major pathogenicity factor, the meningococcal outer membrane protein Opc enhances bacterial entry into brain endothelial cells, however, mechanisms underlying trapping of receptors and signaling molecules following this interaction remained elusive. We now show that Opc-expressing meningococci activate acid sphingomyelinase (ASM) in brain endothelial cells, which hydrolyses sphingomyelin to cause ceramide release and formation of extended ceramide-enriched membrane platforms wherein ErbB2, an important receptor involved in bacterial uptake, clusters. Mechanistically, ASM activation relied on binding of N. meningitidis to its attachment receptor, HSPG, followed by activation of PC-PLC. Meningococcal isolates of the ST-11 clonal complex, which are reported to be more likely to cause severe sepsis, but rarely meningitis, barely invaded brain endothelial cells and revealed a highly restricted ability to induce ASM and ceramide release. Thus, our results unravel a differential activation of the ASM/ceramide system by the species N. meningitidis determining its invasiveness into brain endothelial cells

Comparison of the inflammatory response of brain microvascular and peripheral endothelial cells following infection with Neisseria meningitidis.

International audienceThe interaction of Neisseria meningitidis with both peripheral and brain endothelial cells is a critical event in the development of invasive meningococcal disease. In this study, we used in vitro models based on human brain microvascular endothelial cells (HBMEC), and peripheral endothelial EA.hy926 cells, to investigate their roles in the inflammatory response towards meningococcal infection. Both cell lines were infected with two pathogenic N. meningitidis isolates and secretion of the cytokine interleukin-6 (IL-6), the CXC chemokine IL-8 and the monocyte chemoattractant protein-1 (MCP-1) were estimated by ELISA. Neisseria meningitidis was able to stimulate the production of IL-6 and IL-8 by HBMEC and EA.hy926 cells in a time- and concentration-dependent manner. Interestingly, HBMEC released significant higher amounts of IL-6 and IL-8. Moreover, we observed that heat-killed bacteria stimulated high levels of IL-8. In addition, capsule expression had an inhibitory effect on IL-8 release. We extended our study and included serogroup C strains belonging to sequence type 11 clonal complex (cc) from a recent outbreak in France, as well as isolates belonging to the hypervirulent clonal complexes cc8, cc18, cc32 and cc269 and analyzed their ability to induce the secretion of IL-8 from both cell lines. Although individual variations were observed among different isolates, no clear correlations were observed between strain origin, clinical presentation and IL-8 levels

Entry of Neisseria meningitidis into Mammalian Cells Requires the Src Family Protein Tyrosine Kinases

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}5β1-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 Induces Brain Microvascular Endothelial Cell Detachment from the Matrix and Cleavage of Occludin: A Role for MMP-8

Disruption of the blood-brain barrier (BBB) is a hallmark event in the pathophysiology of bacterial meningitis. Several inflammatory mediators, such as tumor necrosis factor alpha (TNF-a), nitric oxide and matrix metalloproteinases (MMPs), contribute to this disruption. Here we show that infection of human brain microvascular endothelial cells (HBMEC) with Neisseria meningitidis induced an increase of permeability at prolonged time of infection. This was paralleled by an increase in MMP-8 activity in supernatants collected from infected cells. A detailed analysis revealed that MMP-8 was involved in the proteolytic cleavage of the tight junction protein occludin, resulting in its disappearance from the cell periphery and cleavage to a lower-sized 50-kDa protein in infected HBMEC. Abrogation of MMP-8 activity by specific inhibitors as well as transfection with MMP-8 siRNA abolished production of the cleavage fragment and occludin remained attached to the cell periphery. In addition, MMP-8 affected cell adherence to the underlying matrix. A similar temporal relationship was observed for MMP activity and cell detachment. Injury of the HBMEC monolayer suggested the requirement of direct cell contact because no detachment was observed when bacteria were placed above a transwell membrane or when bacterial supernatant was directly added to cells. Inhibition of MMP-8 partially prevented detachment of infected HBMEC and restored BBB permeability. Together, we established that MMP-8 activity plays a crucial role in disassembly of cell junctio

FAK-deficient cells are impaired in their ability to internalize <i>N. meningitidis.</i>

<p>(A) FAK^+/+ fibroblasts were infected with MC58 <i>siaD</i> at an MOI of 30 in presence of RPMI cell culture medium, supplemented with 10% human serum (HS). Intracellular bacteria were defined after gentamicin treatment at 60, 120 and 240 min post-infection (p.i.) demonstrating an invasion kinetic similar to 293T cells and HBMEC. The graphs represent mean value ± S.D. of three different independent experiments done in duplicate. * <i>P</i><0.05. (B) FAK re-expressing (FAk^+/+) and FAK-deficient (FAK^−/−) fibroblasts were infected with invasive strain MC58 <i>siaD</i> in HS-supplemented RPMI cell culture medium. Intracellular bacteria were estimated at 4 h p.i. by gentamicin protection assays. The graph represents mean value ± S.D. of three different independent experiments done in duplicate. * <i>P</i><0.05. (C) FAK^+/+ and FAK^−/− fibroblasts were infected with <i>N. meningitidis</i> strain MC58 <i>siaD</i> for 4 h and analyzed by immunofluorescence microscopy. Extracellular bacteria (arrowhead) stain positive with both TRITC (red fluorescence) and AMCA (blue fluorescence), whereas intracellular bacteria (arrow) are labeled with TRITC only. Cell actin was stained with Alexa Fluor® 488 phalloidin (green fluorescence).</p

Interference with FAK functions results in decreased uptake of <i>N. meningitidis.</i>

<p>(A) 293T cells were transfected with a control plasmid (pcDNA) and a plasmid encoding wildtype FAK (HA-FAK WT) or a plasmid encoding a kinase inactive mutant (HA-FAK K454M) or a mutant that was not capable of autophosphorylation (HA-FAK Y397F). Transfected cell were infected with invasive strain MC58 <i>siaD</i> and intracellular bacteria were estimated 4 h post-infection by gentamicin protection assays. The graph represents mean values ± S.D. of three independent experiments done in duplicate. * <i>P</i><0.05, relative to cells transfected with the control plasmid, ^#<i>P</i> ≤ 0.05, relative to cells transfected FAK wildtype were considered significant. In parallel, Western blotting of WCL extracts with anti-HA-tag antibody demonstrates expression of HA-tagged FAK constructs. (B) 293T cells were transfected with HA-FAK WT or a plasmid encoding the FAK related non-kinase (HA-FRNK) and infected with MC58 <i>siaD</i> as described above. The graph represents mean values ± S.D. of three independent experiments done in duplicate. * <i>P</i><0.05. WCL were analyzed by Western blotting using an anti-HA-tag antibody and demonstrated overexpression of FRNK in transfected cells.</p

<i>N. meningitidis</i> induces recruitment of cortactin.

<p>(A) 293T cells were infected for 4 h with FITC-labeled meningococci (green fluorescence). Infected cells were fixed and stained with an anti-cortactin antibody followed by secondary α-rabbit TRITC-conjugated antibody (red fluorescence). Immunofluorescence demonstrated local recruitment of cortactin to cell-associated bacteria. Scale bars represent 25 µm. (B) For the close-up view, cells were infected with <i>N. meningitidis</i> for 4 h, fixed and incubated with DAPI (blue fluorescence), Alexa Fluor® 488 phalloidin (green fluorescence), cortactin goat α-rabbit/TRITC-conjugated goat α-rabbit (red fluorescence) and α-meningococcal serum/Alexa Fluor® 594 goat α-mouse (pseudocolored in gray). The immunofluorescence confirmed an accumulation of cortactin and the formation of stress fibers adjacent of bacterial attachment. (C) To exclude that bacterial attachment occurred solely on dead cells, cells were either infected with FITC-labeled bacteria or left uninfected and were incubated with MitoTracker (red fluorescence) and DAPI. No signs of apoptosis could be observed before or after 4 h of infection.</p

<i>N. meningitidis</i> induced increased tyrosine phosphorylation of cellular proteins.

<p>(A) FAK^+/+ cells were serum starved and plated on poly-L-lysine coated dishes. Fibroblasts were left uninfected or infected with <i>N. meningitidis</i> MC58 <i>siaD</i> for a 8 h period and cell lysated were collected at from uninfected control cells and at 30 min, 60 min, 120 min, 240 min and 480 min post-infection and analyzed by Western blotting using the anti-phosphotyrosine antibody p-Tyr-100. (B) The increase of the phosphorylated protein was quantified by densitometric analysis and increase was estimated by comparison to the uninfected control. Densitometric analysis was performed as described in Experimental procedures. Staining of the samples with anti-actin antibody was used as loading control. (C) FAK^+/+ cells were infected for 4 h as described above and samples were immunoprecipitated (IP) with an anti-cortactin antibody and immunoprecipitates were analyzed with α-p-Tyr-100 antibody (upper panel). Membranes were stripped and reprobed with polyclonal anti-cortactin antibody (lower panel). (D) Src re-expressing (SYF + c-Src) and c-Src-deficient (SYF) fibroblasts were infected with invasive strain MC58 <i>siaD</i> for 4 h as described above. Cell lysates were collected and analyzed by Western blotting using antibody p-Tyr-100. After stripping membranes were re-probed with polyclonal anti-cortactin antibody (lower panel). (E) SYF + c-Src and SYF cells were infected with strain MC58 <i>siaD</i> for 4 h and samples were immunoprecipitated either with an α-FAK antibody (Fig. 5E, upper panel) or α-cortactin antibody (Fig. 5E, lower panel) and immunoprecipitates were analyzed with α-p-tyr-100 antibody. (F) 293T cells were transfected with the empty control vector (pcDNA), a plasmid encoding wild-type c-Src and a vector encoding the inactive version of Src [Src K297M] to prove that Src kinase activity is required FAK/cortactin phosphorylation. Transfected cells were infected as described above, followed by an IP with an α-FAK antibody or α-cortactin antibody, respectively. Western blot analysis with α-p-tyr-100 demonstrated that Src kinase activity is required FAK/cortactin phosphorylation.</p

<i>N. meningitidis</i> internalization by HBMEC requires focal adhesion kinase (FAK) activity.

<p>(A and B) HBMEC were pre-incubated with indicated concentrations of the specific FAK inhibitor PF573228 and infected for 4 h and 8 h with the unencapsulated <i>N. meningitidis</i> strain MC58 <i>siaD</i>. Intracellular bacteria were defined by gentamicin protection assay. The graph shows mean values +/− S.D. of three independent experiments done in duplicate. ** <i>P</i><0.01, relative to cells infected without inhibitor. (C) HBMEC were pre-incubated with the indicated concentrations of the FAK inhibitor PF 573228 for 1 h and infected with <i>N. meningitidis</i> MC58 <i>siaD</i> for 4 h. Cell lysates were resolved by SDS-PAGE and blotted with α-phospho-FAK Tyr³⁹⁷ demonstrating that PF 573228 blocked FAK Tyr³⁹⁷ phosphorylation in a dose-dependent manner. (D) 293T cells were transfected with indicated concentration of a commercial siRNA specific for FAK to limit FAK protein expression or transfected with unspecific control siRNA. siRNA transfected cells were infected with mutant strain MC58 <i>siaD</i> and internalized bacteria were measured by gentamicin protection assay at 4 h p.i. The graph represents mean values ± S.D. of three independent experiments done in duplicate. * <i>P</i><0.05 and ** <i>P</i><0.01, relative to cells transfected with the control siRNA.</p