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1.
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.  相似文献   

2.
Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) requires the reorganization of host cytoskeleton at the sites of bacterial entry. Both actin and myosin constitute the cytoskeletal architecture. We have previously shown that myosin light chain (MLC) phosphorylation by MLC kinase is regulated during E. coli invasion by an upstream kinase, p21-activated kinase 1 (PAK1), which is an effector protein of Rac and Cdc42 GTPases, but not of RhoA. Here, we report that the binding of only Rac1 to PAK1 decreases in HBMEC upon infection with E. coli K1, which resulted in increased phosphorylation of MLC. Overexpression of a constitutively active (cAc) form of Rac1 in HBMEC blocked the E. coli invasion significantly, whereas overexpression of a dominant negative form had no effect. Increased PAK1 phosphorylation was observed in HBMEC expressing cAc-Rac1 with a concomitant reduction in the phosphorylation of MLC. Immunocytochemistry studies demonstrated that the inhibition of E. coli invasion into cAc-Rac1/HBMEC is due to lack of phospho-MLC recruitment to the sites of E. coli entry. Taken together the data suggest that E. coli modulates the binding of Rac1, but not Cdc42, to PAK1 during the invasion of HBMEC.  相似文献   

3.
Escherichia coli K1 invasion of microvascular endothelial cells of human brain (HBMEC) is required for E. coli penetration into the central nervous system, but the microbial-host interactions that are involved in this invasion of HBMEC remain incompletely understood. We have previously shown that FimH, one of the E. coli determinants contributing to the binding to and invasion of HBMEC, induces Ca2+ changes in HBMEC. In the present study, we have investigated in detail the role of cellular calcium signaling in the E. coli K1 invasion of HBMEC, the main constituents of the blood-brain barrier. Addition of the meningitis-causing E. coli K1 strain RS218 (O18:K1) to HBMEC results in transient increases of intracellular free Ca2+. Inhibition of phospholipase C with U-73122 and the chelating of intracellular Ca2+ by BAPTA/AM reduces bacterial invasion of HBMEC by approximately 50%. Blocking of transmembrane Ca2+ fluxes by extracellular lanthanum ions also inhibits the E. coli invasion of HBMEC by approximately 50%. In addition, E. coli K1 invasion is significantly inhibited when HBMEC are pretreated by the calmodulin antagonists, trifluoperazine or calmidazolium, or by ML-7, a specific inhibitor of Ca2+/calmodulin-dependent myosin light-chain kinase. These findings indicate that host intracellular Ca2+ signaling contributes in part to E. coli K1 invasion of HBMEC. This work was supported by the American Heart Association (grant SDG 0435177N to Y.K.) and by NIH grants (to K.S.K.).  相似文献   

4.
5.
Invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for successful crossing of the blood-brain barrier by Escherichia coli K1. We have previously demonstrated the requirement of cytoskeletal rearrangements and activation of focal adhesion kinase (FAK) in E. coli K1 invasion of human BMEC (HBMEC). The current study investigated the role of phosphatidylinositol 3-kinase (PI3K) activation and PI3K interaction with FAK in E. coli invasion of HBMEC. PI3K inhibitor LY294002 blocked E. coli K1 invasion of HBMEC in a dose-dependent manner, whereas an inactive analogue LY303511 had no such effect. In HBMEC, E. coli K1 increased phosphorylation of Akt, a downstream effector of PI3K, which was completely blocked by LY294002. In contrast, non-invasive E. coli failed to activate PI3K. Overexpression of PI3K mutants Deltap85 and catalytically inactive p110 in HBMEC significantly inhibited both PI3K/Akt activation and E. coli K1 invasion of HBMEC. Stimulation of HBMEC with E. coli K1 increased PI3K association with FAK. Furthermore, PI3K/Akt activation was blocked in HBMEC-overexpressing FAK dominant-negative mutants (FRNK and Phe397FAK). These results demonstrated the involvement of PI3K signaling in E. coli K1 invasion of HBMEC and identified a novel role for PI3K interaction with FAK in the pathogenesis of E. coli meningitis.  相似文献   

6.
7.
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.  相似文献   

8.
Escherichia coli K1 traversal of the human brain microvascular endothelial cells (HBMEC) that constitute the blood-brain barrier (BBB) is a complex process involving E. coli adherence to and invasion of HBMEC. In this study, we demonstrated that human transforming growth factor-beta-1 (TGF-beta1) increases E. coli K1 adherence, invasion, and transcytosis in HBMEC. In addition, TGF-beta1 increases RhoA activation and enhances actin condensation in HBMEC. We have previously shown that E. coli K1 invasion of HBMEC requires phosphatidylinositol-3 kinase (PI3K) and RhoA activation. TGF-beta1 increases E. coli K1 invasion in PI3K dominant-negative HBMEC, but not in RhoA dominant-negative HBMEC, indicating that TGF-beta1-mediated increase in E. coli K1 invasion is RhoA-dependent, but not PI3K-dependent. Our findings suggest that TGF-beta1 treatment of HBMEC increases E. coli K1 adherence, invasion, and transcytosis, which are probably dependent on RhoA.  相似文献   

9.
Eukaryotic cells utilize multiple endocytic pathways for specific uptake of ligands or molecules, and these pathways are commonly hijacked by pathogens to enable host cell invasion. Escherichia coli K1, a pathogenic bacterium that causes neonatal meningitis, invades the endothelium of the blood‐brain barrier, but the entry route remains unclear. Here, we demonstrate that the bacteria trigger an actin‐mediated uptake route, stimulating fluid phase uptake, membrane ruffling and macropinocytosis. The route of uptake requires intact lipid rafts as shown by cholesterol depletion. Using a variety of perturbants we demonstrate that small Rho GTPases and their downstream effectors have a significant effect on bacterial invasion. Furthermore, clathrin‐mediated endocytosis appears to play an indirect role in E. coli K1 uptake. The data suggest that the bacteria effect a complex interplay between the Rho GTPases to increase their chances of uptake by macropinocytosis into human brain microvascular endothelial cells.   相似文献   

10.
Escherichia coli K1 strains are predominant in causing neonatal meningitis. We have shown that invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for E. coli K1 crossing of the blood-brain barrier. BMEC invasion by E. coli K1 strain RS218, however, has been shown to be significantly greater with stationary-phase cultures than with exponential-phase cultures. Since RpoS participates in regulating stationary-phase gene expression, the present study examined a possible involvement of RpoS in E. coli K1 invasion of BMEC. We found that the cerebrospinal fluid isolates of E. coli K1 strains RS218 and IHE3034 have a nonsense mutation in their rpoS gene. Complementation with the E. coli K12 rpoS gene significantly increased the BMEC invasion of E. coli K1 strain IHE3034, but failed to significantly increase the invasion of another E. coli K1 strain RS218. Of interest, the recovery of E. coli K1 strains following environmental insults was 10-100-fold greater on Columbia blood agar than on LB agar, indicating that growing medium is important for viability of rpoS mutants after environmental insults. Taken together, our data suggest that the growth-phase-dependent E. coli K1 invasion of BMEC is affected by RpoS and other growth-phase-dependent regulatory mechanisms.  相似文献   

11.
12.
Escherichia coli K1 is the most common gram-negative bacterium causing neonatal meningitis. The outer membrane protein A (OmpA) assembles a beta-barrel structure having four surface-exposed loops in E. coli outer membrane. OmpA of meningitis-causing E. coli K1 is shown to contribute to invasion of the human brain microvascular endothelial cells (HBMEC), the main cellular component of the blood-brain barrier (BBB). However, the direct evidence of OmpA protein interacting with HBMEC is not clear. In this study, we showed that OmpA protein, solubilized from the outer membrane of E. coli, adhered to HBMEC surface. To verify OmpA interaction with the HBMEC, we purified N-terminal membrane-anchoring beta-barrel domain of OmpA and all surface-exposed loops deleted OmpA proteins, and showed that the surface-exposed loops of OmpA were responsible for adherence to HBMEC. These findings indicate that the OmpA is the adhesion molecule with HBMEC and the surface-exposed loops of OmpA are the determinant of this interaction.  相似文献   

13.
Escherichia coli K1 is the most common gram-negative bacteria that cause meningitis during the neonatal period. The ibeA gene product in E. coli K1 has been characterized as a virulence factor that contributes to the binding to and invasion of brain microvascular endothelial cells (BMEC). Here, we identified a surface protein on human BMEC, vimentin, that interacts with the E. coli invasion protein IbeA. The binding sites of the IbeA-vimentin interaction are located in the 271-370 residue region of IbeA and the vimentin head domain. The regulatory protease factor Xa is able to cleave IbeA between R297 and K298 residues, and this cleavage abolishes the IbeA-vimentin interaction.  相似文献   

14.
Although epidemiological studies have shown that exposure to tobacco smoking significantly increases the risk of bacterial meningitis, heretofore the pathogenic effects of smoking on this disease have been poorly understood. In order to dissect this issue, we have investigated the effects of nicotine, the major component of tobacco, on E. coli invasion of human brain microvascular endothelial cells (HBMEC). Our studies showed that E. coli invasion of HBMEC was significantly enhanced by nicotine in a dose-dependent manner. The nicotine-mediated enhancement was associated with actin cytoskeleton rearrangement and morphological changes in the eukaryotic host cell that are essential for bacterial entry. The recombinant IbeA protein and alpha-bungarotoxin (a nicotinic acetylcholine receptor antagonist) were able to efficiently block the nicotine-mediated cellular effects, suggesting the involvement of the IbeA and nicotinic receptors. Blocking of phosphatidylinositol 3-kinase (PI3K) by LY294002 abolished the entry of E. coli in HBMECs treated with nicotine in a dose-dependent manner. Inhibition of PI3K was associated with decreased phosphorylation of Akt and actin cytoskeletal rearrangement. In contrast to PI3K, blockage of Rho kinase (ROCK) by Y27632 upregulated both nicotine- and E. coli-mediated cellular responses. Thus, this study provides experimental evidence for the first time that the major component of tobacco, nicotine, enhances meningitic E. coli invasion of HBMEC through modulation of cytoskeleton.  相似文献   

15.
Escherichia coli K1 meningitis is a serious central nervous system disease with unchanged mortality and morbidity rates for last few decades. Intercellular adhesion molecule 1 (ICAM-1) is a cell adhesion molecule involved in leukocyte trafficking toward inflammatory stimuli at the vascular endothelium; however, the effect of E. coli invasion of endothelial cells on the expression of ICAM-1 is not known. We demonstrate here that E. coli K1 invasion of human brain microvascular endothelial cells (HBMEC) selectively up-regulates the expression of ICAM-1, which occurs only in HBMEC invaded by the bacteria. The interaction of outer membrane protein A (OmpA) of E. coli with its receptor, Ecgp, on HBMEC was critical for the up-regulation of ICAM-1 and was depend on PKC-alpha and PI3-kinase signaling. Of note, the E. coli-induced up-regulation of ICAM-1 was not due to the cytokines secreted by HBMEC upon bacterial infection. Activation of NF-kappaB was required for E. coli mediated expression of ICAM-1, which was significantly inhibited by over-expressing the dominant negative forms of PKC-alpha and p85 subunit of PI3-kinase. The increased expression of ICAM-1 also enhanced the binding of THP-1 cells to HBMEC. Taken together, these data suggest that localized increase in ICAM-1 expression in HBMEC invaded by E. coli requires a novel interaction between OmpA and its receptor, Ecgp.  相似文献   

16.
Escherichia coli K1 is the most common Gram-negative organism causing meningitis, and its invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for penetration into the central nervous system. We have reported previously that cytotoxic necrotizing factor 1 (CNF1) contributes to E. coli K1 invasion of HBMEC and interacts with 37-kDa laminin receptor precursor (37LRP) of HBMEC, which is a precursor of 67-kDa laminin receptor (67LR). In the present study, we examined the role of 67LR in the CNF1-expressing E. coli K1 invasion of HBMEC. Immunofluorescence microscopy and ligand overlay assays showed that 67LR is present on the HBMEC membrane and interacts with CNF1 protein as well as the CDPGYIGSR laminin peptide. 67LR was up-regulated and clustered at the sites of E. coli K1 on HBMEC in a CNF1-dependent manner. Pretreatment of CNF1+ E. coli K1 with recombinant 37-kDa laminin receptor precursor reduced the invasion rate to the level of Deltacnf1 mutant, and the invasion rate of CNF1+ E. coli K1 was enhanced in 67LR-overexpressing HBMEC, indicating 67LR is involved in the CNF1+ E. coli K1 invasion of HBMEC. Coimmunoprecipitation analysis showed that, upon incubation with CNF1+ E. coli K1 but not with Deltacnf1 mutant, focal adhesion kinase and paxillin were recruited and associated with 67LR. When immobilized onto polystyrene beads, CNF1 was sufficient to induce internalization of coupled beads into HBMEC through interaction with 67LR. Taken together, this is the first demonstration that E. coli K1 invasion of HBMEC occurs through the ligand-receptor (CNF1-67LR) interaction, and 67LR promotes CNF1-expressing E. coli K1 internalization of HBMEC.  相似文献   

17.
The IbeA (ibe10) gene is an invasion determinant contributing to E. coli K1 invasion of the blood-brain barrier. This gene has been cloned and characterized from the chromosome of an invasive cerebrospinal fluid isolate of E. coli K1, strain RS218 (018:K1: H7). In the present study, a genetic island of meningitic E. coli containing ibeA (GimA) has been identified. A 20.3-kb genomic DNA island unique to E. coli K1 strains has been cloned and sequenced from an RS218 E. coli K1 genomic DNA library. Fourteen new genes have been identified in addition to the ibeA. The DNA sequence analysis indicated that the ibeA gene cluster was localized to the 98 min region and consisted of four operons, ptnIPKC, cglDTEC, gcxKRCI and ibeRAT. The G+C content (46.2%) of unique regions of the island is substantially different from that (50.8%) of the rest of the E. coli chromosome. By computer-assisted analysis of the sequences with DNA and protein databases (GenBank and PROSITE databases), the functions of the gene products could be anticipated, and were assigned to the functional categories of proteins relating to carbon source metabolism and substrate transportation. Glucose was shown to enhance E. coli penetration of human brain microvascular endothelial cells and exogenous cAMP was able to block the stimulating effect of glucose, suggesting that catabolic regulation may play a role in control of E. coli K1 invasion gene expression. Our data suggest that this genetic island may contribute to E. coli invasion of the blood-brain barrier through a carbon-source-regulated process. Electronic Publication  相似文献   

18.
The transcellular entry of Escherichia coli K1 through human brain microvascular endothelial cells (HBMEC) is responsible for tight junction disruption, leading to brain oedema in neonatal meningitis. Previous studies demonstrated that outer membrane protein A (OmpA) of E. coli K1 interacts with its receptor, Ecgp96, to induce PKC‐α phosphorylation, adherens junction (AJ) disassembly (by dislodging β‐catenin from VE‐cadherin), and remodelling of actin in HBMEC. We report here that IQGAP1 mediates β‐catenin dissociation from AJs to promote actin polymerization required for E. coli K1 invasion of HBMEC. Overexpression of C‐terminal truncated IQGAP1 (IQΔC) that cannot bind β‐catenin prevents both AJ disruption and E. coli K1 entry. Of note, phospho‐PKC‐α interacts with the C‐terminal portion of Ecgp96 as well as with VE‐cadherin after IQGAP1‐mediated AJ disassembly. HBMEC overexpressing either C‐terminal truncated Ecgp96 (Ecgp96Δ200) or IQΔC upon infection with E. coli showed no interaction ofphospho‐PKC‐α with Ecgp96. These data indicate that the binding of OmpA to Ecgp96 induces PKC‐α phosphorylation and association of phospho‐PKC‐α with Ecgp96, and then signals IQGAP1 to detach β‐catenin from AJs. Subsequently, IQGAP1/β‐catenin bound actin translocates to the site of E. coli K1 attachment to promote invasion.  相似文献   

19.
Invasion of brain microvascular endothelial cells is a prerequisite for successful crossing of the blood-brain barrier by Escherichia coli (E. coli), but the underlying mechanism remains unclear. Here we showed activation of Src tyrosine kinase in E. coli K1 invasion of human brain microvascular endothelial cells (HBMEC). E. coli invasion of HBMEC and the E. coli-induced rearrangement of actin filaments were blocked by Src inhibitors. Overexpression of dominant-negative Src in HBMEC significantly attenuated E. coli invasion and the concomitant actin filaments rearrangement. Furthermore, E. coli K1-triggered phosphatidylinositol 3-kinase (PI3K) activation in HBMEC was effectively blocked by Src inhibitors and dominant-negative Src. These results demonstrated the involvement of Src and its interaction with PI3K in E. coli K1 invasion of HBMEC.

Structured summary

MINT-7296127, MINT-7296136: Src (uniprotkb:P12931) physically interacts (MI:0915) with p85 (uniprotkb:P27986) by anti bait coimmunoprecipitation (MI:0006)MINT-7296149: F-actin (uniprotkb:P60709) and Src-DN (uniprotkb:P12931) colocalize (MI:0403) by fluorescence microscopy (MI:0416)  相似文献   

20.
Two thermostable phytases were identified from Thai isolates of Aspergillus japonicus BCC18313 (TR86) and Aspergillus niger BCC18081 (TR170). Both genes of 1404 bp length, coding for putative phytases of 468 amino acid residues, were cloned and transferred into Pichia pastoris . The recombinant phytases, r-PhyA86 and r-PhyA170, were expressed as active extracellular, glycosylated proteins with activities of 140 and 100 U mL−1, respectively. Both recombinant phytases exhibited high affinity for phytate but not for p -nitrophenyl phosphate. Optimal phytase activity was observed at 50 °C and pH 5.5. High thermostability, which is partly dependent on glycosylation, was demonstrated for both enzymes, as >50% activity was retained after heating at 100 °C for 10 min. The recombinant phytases also exhibited broad pH stability from 2.0 to 8.0 and are resistant to pepsin. In vitro digestibility tests suggested that r-PhyA86 and r-PhyA170 are at least as efficient as commercial phytase for hydrolyzing phytate in corn-based animal feed and are therefore suitable sources of phytase supplement.  相似文献   

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