Involvement of human CD44 during Cryptococcus neoformans infection of brain microvascular endothelial cells

Pathogenic yeast Cryptococcus neoformans causes devastating cryptococcal meningoencephalitis. Our previous studies demonstrated that C. neoformans hyaluronic acid was required for invasion into human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. In this report, we demonstrate that C. neoformans hyaluronic acid interacts with CD44 on HBMEC. Our results suggest that HBMEC CD44 is a primary receptor during C. neoformans infection, based on the following observations. First, anti-CD44 neutralizing antibody treatment was able to significantly reduce C. neoformans association with HBMEC. Second, C. neoformans association was considerably impaired using either CD44-knock-down HBMEC or C. neoformans hyaluronic acid-deficient strains. Third, overexpression of CD44 in HBMEC increased their association activity towards C. neoformans. Fourth, confocal microscopic images showed that CD44 was enriched at and around the C. neoformans association sites. Fifth, upon C. neoformans and HBMEC engagement, a subpopulation of CD44 and actin translocated to the host membrane rafts. Our results highlight the interactions between C. neoformans hyaluronic acid and host CD44 and the dynamic results of these interactions, which may represent events during the adhesion and entry of C. neoformans at HBMEC membrane rafts.     

SBi4211阻断HIV-1 gp41促进新生隐球菌对人脑微血管内皮细胞的黏附作用

【背景】目前艾滋病和新型隐球菌性脑膜炎共病因素导致其高发病率和死亡率的机制尚不明确。【目的】探索S100B抑制剂SBi4211对HIV-1 gp41促进新生隐球菌黏附人脑微血管内皮细胞的影响和可能机制。【方法】黏附实验分析SBi4211是否能阻断HIV-1 gp41诱导下新生隐球菌黏附人脑微血管内皮细胞。使用免疫印迹方法进一步检测在此过程中SBi4211对脑微血管内皮细胞上新生隐球菌透明质酸受体CD44表达的影响。【结果】SBi4211可显著抑制HIV-1gp41对新生隐球菌黏附脑微血管内皮细胞的增强作用,且呈时间、剂量效应(P0.05);免疫印迹结果显示SBi4211可抑制新生隐球菌和/或HIV-1 gp41增加脑微血管内皮细胞上新生隐球菌透明质酸受体CD44的表达。【结论】SBi4211可通过下调受体CD44来阻断HIV-1 gp41对新生隐球菌黏附人脑微血管内皮细胞的增强效应,这为了解HIV-1与新生隐球菌共病机制及其防治策略提供了新思路。     

Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells requires protein kinase C-alpha activation

Pathogenic fungus Cryptococcus neoformans has a predilection for the central nervous system causing devastating meningoencephalitis. Traversal of C. neoformans across the blood–brain barrier (BBB) is a crucial step in the pathogenesis of C. neoformans . Our previous studies have shown that the CPS1 gene is required for C. neoformans adherence to the surface protein CD44 of human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In this report, we demonstrated that C. neoformans invasion of HBMEC was blocked in the presence of G109203X, a protein kinase C (PKC) inhibitor, and by overexpression of a dominant-negative form of PKCα in HBMEC. During C. neoformans infection, phosphorylation of PKCα was induced and the PKC enzymatic activity was detected in the HBMEC membrane fraction. Our results suggested that the PKCα isoform might play a crucial role during C. neoformans invasion. Immunofluorescence microscopic images showed that induced phospho-PKCα colocalized with β-actin on the membrane of HBMEC. In addition, cytochalasin D (an F-filament-disrupting agent) inhibited fungus invasion into HBMEC in a dose-dependent manner. Furthermore, blockage of PKCα function attenuated actin filament activity during C. neoformans invasion. These results suggest a significant role of PKCα and downstream actin filament activity during the fungal invasion into HBMEC.     

Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells is mediated through the lipid rafts-endocytic pathway via the dual specificity tyrosine phosphorylation-regulated kinase 3 (DYRK3)

Cryptococcus neoformans is a neurotropic fungal pathogen, which provokes the onset of devastating meningoencephalitis. We used human brain microvascular endothelial cells (HBMEC) as the in vitro model to investigate how C. neoformans traverses across the blood-brain barrier. In this study, we present several lines of evidence indicating that C. neoformans invasion is mediated through the endocytic pathway via lipid rafts. Human CD44 molecules from lipid rafts can directly interact with hyaluronic acid, the C. neoformans ligand. Bikunin, which perturbs CD44 function in the lipid raft, can block C. neoformans adhesion and invasion of HBMEC. The lipid raft marker, ganglioside GM1, co-localizes with CD44 on the plasma membrane, and C. neoformans cells can adhere to the host cell in areas where GM1 is enriched. These findings suggest that C. neoformans entry takes place on the lipid rafts. Upon C. neoformans engagement, GM1 is internalized through vesicular structures to the nuclear membrane. This endocytic redistribution process is abolished by cytochalasin D, nocodazole, or anti-DYRK3 (dual specificity tyrosine-phosphorylation-regulated kinase 3) siRNA. Concomitantly, the knockdown of DYRK3 significantly reduces C. neoformans invasion across the HBMEC monolayer in vitro. Our data demonstrate that the lipid raft-dependent endocytosis process mediates C. neoformans internalization into HBMEC and that the CD44 protein of the hosts, cytoskeleton, and intracellular kinase-DYRK3 are involved in this process.     

Hyaluronic acid receptor CD44 deficiency is associated with decreased Cryptococcus neoformans brain infection

Cryptococcus neoformans is a pathogenic yeast that can invade the brain and cause meningoencephalitis. Our previous in vitro studies suggested that the interaction between C. neoformans hyaluronic acid and human brain endothelial CD44 could be the initial step of brain invasion. In this report, we used a CD44 knock-out (KO or CD44(-/-)) mouse model to explore the importance of CD44 in C. neoformans brain invasion. Our results showed that C. neoformans-infected CD44 KO mice survived longer than the infected wild-type mice. Consistent with our in vitro results, the brain and cerebrospinal fluid fungal burden was reduced in CD44-deficient mice. Histopathological studies showed smaller and fewer cystic lesions in the brains of CD44 KO mice. Interestingly, the cystic lesions contained C. neoformans cells embedded within their polysaccharide capsule and were surrounded by host glial cells. We also found that a secondary hyaluronic acid receptor, RHAMM (receptor of hyaluronan-mediated motility), was present in the CD44 KO mice. Importantly, our studies demonstrated an in vivo blocking effect of simvastatin. These results suggest that the CD44 and RHAMM receptors function on membrane lipid rafts during invasion and that simvastatin may have a potential therapeutic role in C. neoformans infections of the brain.     

Lipid raft/caveolae signaling is required for Cryptococcus neoformans invasion into human brain microvascular endothelial cells

Background

Cryptococcus neoformans has a predilection for central nervous system infection. C. neoformans traversal of the blood brain barrier, composed of human brain microvascular endothelial cells (HBMEC), is the crucial step in brain infection. However, the molecular mechanism of the interaction between Cryptococcus neoformans and HBMEC, relevant to its brain invasion, is still largely unknown.

Methods

In this report, we explored several cellular and molecular events involving the membrane lipid rafts and caveolin-1 (Cav1) of HBMEC during C. neoformans infection. Immunofluorescence microscopy was used to examine the roles of Cav1. The knockdown of Cav1 by the siRNA treatment was performed. Phosphorylation of Cav1 relevant to its invasion functions was investigated.

Results

We found that the host receptor CD44 colocalized with Cav1 on the plasma membrane, and knockdown of Cav1 significantly reduced the fungal ability to invade HBMEC. Although the CD44 molecules were still present, HBMEC membrane organization was distorted by Cav1 knockdown. Concomitantly, knockdown of Cav1 significantly reduced the fungal crossing of the HBMEC monolayer in vitro. Upon C. neoformans engagement, host Cav1 was phosphorylated in a CD44-dependent manner. This phosphorylation was diminished by filipin, a disrupter of lipid raft structure. Furthermore, the phosphorylated Cav1 at the lipid raft migrated inward to the perinuclear localization. Interestingly, the phospho-Cav1 formed a thread-like structure and colocalized with actin filaments but not with the microtubule network.

Conclusion

These data support that C. neoformans internalization into HBMEC is a lipid raft/caveolae-dependent endocytic process where the actin cytoskeleton is involved, and the Cav1 plays an essential role in C. neoformans traversal of the blood-brain barrier.     

The ABC transporter-encoding gene AFR1 affects the resistance of Cryptococcus neoformans to microglia-mediated antifungal activity by delaying phagosomal maturation

The pathogenic yeast Cryptococcus neoformans has evolved several strategies to survive within phagocytes. Recently, it has been demonstrated that upregulation of the ATP binding cassette transporter-encoding gene antifungal resistance 1 ( AFR1 ) is important not only for determining the resistance of C. neoformans to fluconazole but also in influencing fungal virulence. In the present study, we showed that the fluconazole-resistant AFR1- overexpressing mutant strain was not sensitive to microglia-mediated anticryptococcal activity, as compared with the fluconazole-susceptible isogenic strains, the wild type and the afr1 Δ mutant. Interestingly, although the three strains were phagocytosed to a similar extent, reduced acidification and delayed maturation were observed in phagosomes containing the AFR1 -overexpressing strain with respect to the others. These findings provide the first evidence that upregulation of the AFR1 gene affects C. neoformans –microglia interplay, adding insights to the complexity of cryptococcal virulence and to its unexpected link with azole resistance.     

Escherichia coli K1 induces IL-8 expression in human brain microvascular endothelial cells

Microbial penetration of the blood-brain barrier (BBB) into the central nervous system is essential for the development of meningitis. Considerable progress has been achieved in understanding the pathophysiology of meningitis, however, relatively little is known about the early inflammatory events occurring at the time of bacterial crossing of the BBB. We investigated, using real-time quantitative PCR, the expression of the neutrophil chemoattractants alpha-chemokines CXCL1 (Groalpha) and CXCL8 (IL-8), and of the monocyte chemoattractant beta-chemokine CCL2 (MCP-1) by human brain microvascular endothelial cells (HBMEC) in response to the meningitis-causing E. coli K1 strain RS218 or its isogenic mutants lacking the ability to bind to and invade HBMEC. A nonpathogenic, laboratory E. coli strain HB101 was used as a negative control. CXCL8 was shown to be significantly expressed in HBMEC 4 hours after infection with E. coli K1, while no significant alterations were noted for CXCL1 and CCL2 expression. This upregulation of CXCL8 was induced by E. coli K1 strain RS218 and its derivatives lacking the ability to bind and invade HBMEC, but was not induced by the laboratory strain HB101. In contrast, no upregulation of CXCL8 was observed in human umbilical vein endothelial cells (HUVEC) after stimulation with E. coli RS218. These findings indicate that the CXCL8 expression is the result of the specific response of HBMEC to meningitis-causing E. coli K1.     

On the mechanism of oleate transport across human brain microvessel endothelial cells

The blood–brain barrier formed by the brain capillary endothelial cells provides a protective barrier between the systemic blood and the extracellular environment of the CNS. As most fatty acids in the brain enter from the blood, we examined the mechanism of oleate (C18:1) transport across primary human brain microvessel endothelial cells (HBMEC). The permeability of [1-¹⁴C]oleate was determined using confluent cells grown on Transwell® inserts in both the absence or presence of bovine serum albumin in the basolateral media, and following inhibition of various fatty acid transporters. The passage of [1-¹⁴C]oleate across confluent HBMEC monolayers was significantly enhanced when fatty acid free albumin was present in the basolateral media. The presence of the non-specific fatty acid uptake inhibitor phloretin significantly decreased [1-¹⁴C]oleate uptake by HBMEC and the subsequent release of [1-¹⁴C]oleate into the basolateral medium. Knockdown of fatty acid transport protein-1 or fatty acid translocase/CD36 significantly decreased [1-¹⁴C]oleate transport across the HBMEC monolayer from either apical as well as basolateral sides. The findings indicate that a fatty acid acceptor is a requirement for oleate transport across HBMEC monolayers. In addition, transport of oleate across HBMEC is, in part, a transcellular process mediated by fatty acid transport proteins.     

Comparison of serological and chemical characteristics of capsular polysaccharides of Cryptococcus neoformans var. neoformans serotype A and Cryptococcus albidus var. albidus

The antigenic formula and chemical structure of capsular polysaccharide (CPS) of Cryptococcus albidus var. albidus (C. albidus) were studied in relation to those of C. neoformans var. neoformans serotype A (C. neoformans A). The results of slide agglutination tests with factor sera and reciprocal adsorption experiments showed that antigenic formula of C. albidus was the same as that of C. neoformans A. The soluble CPSs from the two species were obtained from culture supernatants by precipitation with ethanol followed by purification by chromatography on DEAE-cellulose column. The structural analyses of such CPSs from the two species showed that the antigenic CPS fractions consisted of a backbone of alpha(1-3)-linked D-mannopyranosyl residues with a single branch of beta(1-2)-xylose or glucuronic acid, and mostly with O-acetyl groups, in which side chains and O-acetyl groups were responsible for antigenic specificity. It was found that there was a minor difference between the CPS of C. neoformans A and that of C. albidus; in the former, unsubstituted mannose residues existed in a low frequency, but in the latter none. Moreover, the 1H-nuclear magnetic resonance spectra of partially hydrolyzed acidic fragments of the two CPSs indicated that two xylose side chains were present between glucuronic acid side chains. Taken together, it was suggested that these two species of C. neoformans A and C. albidus are closely related to each other in their CPSs.     

Identification and characterization of the Cryptococcus neoformans phosphomannose isomerase-encoding gene, MAN1, and its impact on pathogenicity

The polysaccharide capsule surrounding Cryptococcus neoformans comprises manose, xylose and glucuronic acid, of which mannose is the major constituent. The GDP-mannose biosynthesis pathway is highly conserved in fungi and consists of three key enzymes: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP). The MAN1 gene, encoding for the PMI enzyme, was isolated and sequenced from C. neoformans, and a disruption of the MAN1 gene was generated. One MAN1 disruption mutant, man1, which showed poor capsule formation, reduced polysaccharide secretion and morphological abnormalities, was chosen for virulence studies. In both the rabbit and the mouse models of invasive cryptococcosis, man1 was shown to be severely impaired in its virulence, with complete elimination of the yeast from the host. A reconstituted strain of man1 was constructed using gene replacement at the native locus. The wild-type and reconstituted strains were significantly more virulent than the knock-out mutant in both animal models. Our findings reveal that PMI activity is essential for the survival of C. neoformans in the host. The fact that the man1 mutant was not pathogenic suggests that blocking mannose synthesis could be fungicidal in the mammalian host and thus an excellent target for antifungal drug development.     

脑膜炎大肠杆菌K1株ppk1基因致病机制初探

【目的】构建脑膜炎大肠杆菌K1(Escherichia coli,E.coli K1)株E44的聚磷酸盐激酶1(Polyphosphate kinase 1,PPK1)基因敲除株,并对其生物学功能进行初步研究,为明确ppk1基因在E.coli K1株致脑膜炎机制中的作用奠定基础。【方法】利用自杀质粒pCVD442及基因同源重组技术敲除E.coli K1株E44中的ppk1基因,构建ppk1缺失突变株Δppk1;体外比较野生株和突变株在低营养及氧化压力情况下的生存能力;考察二者对人脑微血管内皮细胞(Human brain microvascular endothelial cells,HBMEC)的黏附能力;通过测定乳酸脱氢酶(Lactic dehydrogenase,LDH)释放活性,比较野生株和突变株对HBMEC的损伤效应。【结果】PCR及序列分析证实,突变株缺失全长ppk1基因。与野生株E44相比,ppk1突变株Δppk1在低营养环境中和氧化刺激条件下的生存能力明显降低。相对于E44,Δppk1对HBMEC的黏附能力减弱。与HBMEC孵育后,突变株孵育组HBMEC的LDH释放活性明显低于野生株孵育组。【结论】ppk1对E.coli K1株E44在低营养环境中的生存、抵抗氧化压力,以及黏附HBMEC和对细胞的毒性损伤有重要作用。     

cPLA2α-mediated actin rearrangements downstream of the Akt signaling is required for Cronobacter sakazakii invasion into brain endothelial cells

Cronobacter sakazakii (C. sakazakii) is an opportunistic pathogen that causes sepsis and meningitis in neonate. The molecular mechanism involved in the pathogenesis of C. sakazakii meningitis remains unclear. In this study, we found that C. sakazakii invasion was significantly decreased in human brain microvascular endothelial cells (HBMEC) treated with cytosolic phospholipases A(2)α (cPLA(2)α) inhibitor. Increased phosphorylation of cPLA(2)α was observed in HBMEC infected with C. sakazakii, which was prevented by treatment with cPLA(2)α inhibitor. cPLA(2)α knockdown in HBMEC significantly attenuated C. sakazakii invasion into HBMEC. Immunofluorescence demonstrated that the rearrangements of actin filaments in HBMEC induced by C. sakazakii were effectively blocked by either treatment with cPLA(2)α inhibitor or transfection with cPLA(2)α siRNA. Interestingly, we found that C. sakazakii infection promoted the aggregation of phosphorylated cPLA(2)α, which was associated with depolymerized actin filaments in HBMEC. Furthermore, our data revealed that cPLA(2)α acts downstream of Akt signaling pathway in HBMEC stimulated with C. sakazakii. Taken together, our results illustrated that cPLA(2)α-mediated actin filament rearrangements downstream of Akt activation is required for C. sakazakii invasion into brain endothelial cells.     

Perfluorooctane sulfonate triggers tight junction "opening" in brain endothelial cells via phosphatidylinositol 3-kinase

Perfluorooctane sulfonate (PFOS), an environmental pollutant, is widely distributed in humans and wildlife. Accumulation of PFOS in the brain and its neurotoxicity has been reported. Whether PFOS has any effect on the blood–brain barrier (BBB) remains unknown. In this study, human brain microvascular endothelial cells (HBMEC), which are the major components of BBB, were treated with PFOS and indicators of endothelial permeability were measured. Disassembly of endothelial tight junction (TJ) and increase of permeability were observed in response to PFOS. The PFOS-induced TJ disassembly in HBMEC was attenuated by pretreatment with PI3K inhibitors, whereas Rho kinase inhibitor had no such effect. Further results demonstrated that PFOS promoted the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling in HBMEC. We found that overexpression of PI3K dominant-negative mutant in HBMEC abolished the PFOS-induced TJ disassembly. These data demonstrated that PFOS can trigger the “opening” of tight junction in brain endothelial cells through PI3K signaling pathway.     

Cryptococcal xylosyltransferase 1 (Cxt1p) from Cryptococcus neoformans plays a direct role in the synthesis of capsule polysaccharides

The opportunistic yeast Cryptococcus neoformans causes serious disease in humans and expresses a prominent polysaccharide capsule that is required for its virulence. Little is known about how this capsule is synthesized. We previously identified a beta1,2-xylosyltransferase (Cxt1p) with in vitro enzymatic activity appropriate for involvement in capsule synthesis. Here, we investigate C. neoformans strains in which the corresponding gene has been deleted (cxt1Delta). Loss of CXT1 does not affect in vitro growth of the mutant cells or the general morphology of their capsules. However, NMR structural analysis of the two main capsule polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM), showed that both were missing beta1,2-xylose residues. There was an approximately 30% reduction in the abundance of this residue in GXM in mutant compared with wild-type strains, and mutant GalXM was almost completely devoid of beta1,2-linked xylose. The GalXM from the mutant strain was also missing a beta1,3-linked xylose residue. Furthermore, deletion of CXT1 led to attenuation of cryptococcal growth in a mouse model of infection, suggesting that the affected xylose residues are important for normal host-pathogen interactions. Cxt1p is the first glycosyltransferase with a defined role in C. neoformans capsule biosynthesis, and cxt1Delta is the only strain identified to date with structural alterations of the capsule polysaccharide GalXM.     

Cryptococcus neoformans phospholipase B1 activates host cell Rac1 for traversal across the blood-brain barrier

Cryptococcus neoformans penetration into the central nervous system (CNS) requires traversal of the blood–brain barrier that is composed of a single layer of human brain microvascular endothelial cells (HBMEC), but the underlying mechanisms of C. neoformans traversal remain incompletely understood. C. neoformans transcytosis of HBMEC monolayer involves rearrangements of the host cell actin cytoskeleton and small GTP‐binding Rho family proteins such as Rac1 are shown to regulate host cell actin cytoskeleton. We, therefore, examined whether C. neoformans traversal of the blood–brain barrier involves host Rac1. While the levels of activated Rac1 (GTP‐Rac1) in HBMEC increased significantly upon incubation with C. neoformans strains, pharmacological inhibition and down‐modulation of Rac1 significantly decreased C. neoformans transcytosis of HBMEC monolayer. Also, Rac1 inhibition was efficient in preventing C. neoformans penetration into the brain. In addition, C. neoformans phospholipase B1 (Plb1) was shown to contribute to activating host cell Rac1, andSTAT3 was observed to associate with GTP‐Rac1 in HBMEC that were incubated with C. neoformans strain but not with its Δplb1 mutant. These findings demonstrate for the first time that C. neoformans Plb1 aids fungal traversal across the blood–brain barrier by activating host cell Rac1 and its association with STAT3, and suggest that pharmacological intervention of host–microbial interaction contributing to traversal of the blood–brain barrier may prevent C. neoformans penetration into the brain.     

Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells

The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.     

FimH-mediated Escherichia coli K1 invasion of human brain microvascular endothelial cells

Adhesion to brain microvascular endothelial cells, which constitute the blood-brain barrier is considered important in Escherichia coli K1 bacterial penetration into the central nervous system. Type 1 fimbriae are known to mediate bacterial interactions with human brain microvascular endothelial cells (HBMEC). Here, we demonstrate that type 1 fimbriae, specifically FimH adhesin is not only an adhesive organelle that provides bacteria with a foothold on brain endothelial cells but also triggers signalling events that promote E. coli K1 invasion in HBMEC. This is shown by our demonstrations that exogenous FimH increases cytosolic-free-calcium levels as well as activates RhoA. Using purified recombinant mannose-recognition domain of FimH, we identified a glycosylphosphatidylinositol-anchored receptor, CD48, as a putative HBMEC receptor for FimH. Furthermore, E. coli K1 binding to and invasion of HBMEC were blocked by CD48 antibody. Taken together, these findings indicate that FimH induces host cell signalling cascades that are involved in E. coli K1 invasion of HBMEC and CD48 is a putative HBMEC receptor for FimH.     

Interaction of Neisseria meningitidis with human brain microvascular endothelial cells: role of MAP- and tyrosine kinases in invasion and inflammatory cytokine release

Neisseria meningitidis traversal across the blood-cerebrospinal fluid barrier is an essential step in the pathogenesis of bacterial meningitis. We have previously shown that invasion of human brain microvascular endothelial cells (HBMEC) by meningococci is mediated by bacterial outer membrane protein Opc that binds fibronectin, thereby anchoring the bacterium to the integrin alpha 5 beta 1-receptor on the endothelial cell surface. However, subsequent signal transduction mechanisms essential for or regulated by N. meningitidis adhesion and invasion, or HBMEC responses to N. meningitidis are unknown. In this report we investigated the role of c-Jun N-terminal kinases 1 and 2 (JNK1 and JNK2), p38 mitogen-activated (MAP) kinase and protein tyrosine kinases in endothelial-N. meningitidis interaction. Binding of meningococci to HBMEC phosphorylated and activated JNK1 and JNK2 and p38 MAPK as well as their direct substrates c-Jun and MAP kinase activated kinase-2 (MAPKAPK-2), respectively. Non-invasive meningococcal strains lacking opc gene (opc mutants and sequence type 11 complex meningococci) still activated p38 MAPK, however, failed to activate JNK. Inhibition of JNK1 and JNK2 significantly reduced internalization of N. meningitidis by HBMEC without affecting its adherence. Blocking the endothelial integrin alpha 5 beta 1 also decreased N. meningitidis-induced JNK activation in HBMEC. These findings indicate the crucial role of JNK signalling pathway in N. meningitidis invasion in HBMEC. In contrast, p38 MAPK pathway was important for the control of interleukin-6 (IL-6) and IL-8 release by HBMEC. Genistein, a protein tyrosine kinase inhibitor, decreased both invasion of N. meningitidis into HBMEC and IL-6 and IL-8 release, indicating that protein tyrosine kinases, which link signals from integrins to intracellular signalling pathways are essential for both bacterial internalization and cytokine secretion by HBMEC.