RESUMO
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.
Assuntos
Encéfalo/irrigação sanguínea , Ceramidas/metabolismo , Endotélio Vascular/microbiologia , Interações Hospedeiro-Patógeno , Neisseria meningitidis/patogenicidade , Esfingomielina Fosfodiesterase/metabolismo , Regulação para Cima , Aderência Bacteriana/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/microbiologia , Linhagem Celular Transformada , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/enzimologia , Microdomínios da Membrana/metabolismo , Meningite Meningocócica/enzimologia , Meningite Meningocócica/metabolismo , Meningite Meningocócica/microbiologia , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Microvasos/microbiologia , Mutação , Neisseria meningitidis/fisiologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Propriedades de Superfície/efeitos dos fármacos , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Neisseria meningitidis, the causative agent of meningitis and septicemia, attaches to and invades various cell types. Both steps induce and/or require tyrosine phosphorylation of host cell proteins. Here, we used a phospho array platform to identify active receptor tyrosine kinases (RTKs) and key signaling nodes in N. meningitidis-infected brain endothelial cells to decipher RTK-dependent signaling pathways necessary for bacterial uptake. We detected several activated RTKs, including the ErbB family receptors epidermal growth factor receptor (EGFR), ErbB2, and ErbB4. We found that pharmacological inhibition and genetic ablation of ErbB receptor tyrosine phosphorylation and expression resulted in decreased bacterial uptake and heterologous expression of EGFR, ErbB2, or ErbB4 in Chinese ovary hamster (CHO-K1) cells, which do not express of EGFR and ErbB4; the decrease caused a significant increase in meningococcal invasion. Activation of EGFR and ErbB4 was mediated by transactivation via the common ligand HB-EGF (heparin-binding EGF-like ligand), which was significantly elevated in infected cell culture supernatants. We furthermore determined that N. meningitidis induced phosphorylation of EGFR at Tyr845 independent of ligand binding, which required c-Src activation and was involved in mediating uptake of N. meningitidis into eukaryotic cells. Increased uptake was repressed by expression of EGFR Y845F, which harbored a point mutation in the kinase domain. In addition, activation of ErbB4 at its autophosphorylation site, Tyr1284, and phosphorylation of ErbB2 Thr686 were observed. Altogether, our results provide evidence that EGFR, ErbB2, and ErbB4 are activated in response to N. meningitidis infection and shed new light on the role of ErbB signaling in meningococcal infection biology.
Assuntos
Células Endoteliais/metabolismo , Receptores ErbB/metabolismo , Meningite Meningocócica/metabolismo , Neisseria meningitidis/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/microbiologia , Células CHO , Linhagem Celular , Cricetulus , Células Endoteliais/microbiologia , Humanos , Meningite Meningocócica/microbiologia , Fosforilação/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptor ErbB-4 , Transdução de Sinais/fisiologiaRESUMO
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-alpha), 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 junction components and cell adhesion during meningococcal infection.
Assuntos
Encéfalo/metabolismo , Células Endoteliais/metabolismo , Metaloproteinase 8 da Matriz/metabolismo , Proteínas de Membrana/metabolismo , Meningite Meningocócica/metabolismo , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Western Blotting , Encéfalo/patologia , Permeabilidade Capilar/fisiologia , Adesão Celular/fisiologia , Linhagem Celular , Células Endoteliais/patologia , Humanos , Imunoprecipitação , Meningite Meningocócica/patologia , Microscopia Eletrônica de Varredura , Neisseria meningitidis , OcludinaRESUMO
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.
Assuntos
Endocitose , Células Endoteliais/microbiologia , Interações Hospedeiro-Patógeno , Neisseria meningitidis/patogenicidade , Proteínas Tirosina Quinases/metabolismo , Linhagem Celular , Células Cultivadas , Citoesqueleto/metabolismo , Genisteína/farmacologia , Humanos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidoresRESUMO
The 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.
Assuntos
Quimiocina CCL2/metabolismo , Células Endoteliais/imunologia , Interações Hospedeiro-Patógeno , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Neisseria meningitidis/imunologia , Células Cultivadas , Quimiocina CCL2/análise , Surtos de Doenças , Ensaio de Imunoadsorção Enzimática , França/epidemiologia , Humanos , Interleucina-6/análise , Interleucina-8/análise , Meningite Meningocócica/epidemiologia , Meningite Meningocócica/microbiologia , Neisseria meningitidis/isolamento & purificaçãoRESUMO
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.
Assuntos
Cortactina/metabolismo , Células Endoteliais/enzimologia , Células Endoteliais/microbiologia , Quinase 1 de Adesão Focal/metabolismo , Neisseria meningitidis/fisiologia , Quinases da Família src/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Encéfalo/patologia , Endocitose , Fibroblastos/enzimologia , Fibroblastos/microbiologia , Fibroblastos/patologia , Quinase 1 de Adesão Focal/deficiência , Interações Hospedeiro-Patógeno , Humanos , Integrinas/metabolismo , Camundongos , Microvasos/patologia , Neisseria meningitidis/citologia , Fosforilação , Fosfotirosina/metabolismoRESUMO
Despite antibiotic therapy, infections with Neisseria meningitidis still demonstrate a high rate of morbidity and mortality even in developed countries. The fulminant septicaemic course, named Waterhouse-Friderichsen syndrome, with massive haemorrhage into the adrenal glands and widespread petechial bleeding suggest pathophysiological inhibition of platelet function. Our data show that N. meningitidis produces the important physiological platelet inhibitor and cardiovascular signalling molecule nitric oxide (NO), also known as endothelium-derived relaxing factor (EDRF). N. meningitidis -derived NO inhibited ADP-induced platelet aggregation through the activation of soluble guanylyl cyclase (sGC) followed by an increase in platelet cyclic nucleotide levels and subsequent activation of platelet cGMP- and cAMP- dependent protein kinases (PKG and PKA). Furthermore, direct measurement of horseradish peroxidase (HRP) passage through a vascular endothelial cell monolayer revealed that N. meningitidis significantly increased endothelial monolayer permeability. Immunfluorescence analysis demonstrated NO dependent disturbances in the structure of endothelial adherens junctions after co-incubation with N. meningitidis . In contrast to platelet inhibition, the NO effects on HBMEC were not mediated by cyclic nucleotides. Our study provides evidence that NO plays an essential role in the pathophysiology of septicaemic meningococcal infection.