Strain-dependent disruption of blood-cerebrospinal fluid barrier by Streptoccocus suis in vitro

Streptococcus suis capsular type 2 is an important agent of diseases including meningitis among pigs worldwide, and is also a zoonotic agent. The barrier function of the choroid plexus epithelium that constitutes the structural basis for the blood-cerebrospinal fluid (CSF) barrier has not been elucidated yet in bacterial meningitis. We investigated the influence of various S. suis isolates on the barrier function of cultured porcine choroid plexus epithelial cells with respect to the transepithelial resistance and paracellular [(3)H]-mannitol flux. Preferentially apical application of S. suis isolates significantly decreased transepithelial resistance and significantly increased paracellular [(3)H]-mannitol flux in a time-, dose- and strain-dependent manner. Viable S. suis isolates caused cytotoxicity determined by lactate dehydrogenase assay and electron microscopy, whereas S. suis sonicates and UV-inactivated S. suis did not cause cytotoxicity. The observed effects on porcine choroid plexus epithelial cells barrier function could not exclusively be ascribed to known virulence factors of S. suis such as suilysin. In conclusion, S. suis isolates induce loss of blood-cerebrospinal fluid barrier function in an in vitro model. Thus, S. suis may facilitate trafficking of bacteria and leucocytes across the blood-cerebrospinal fluid barrier. The underlying mechanisms for the barrier breakdown have yet to be determined.     

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.     

Phospholipase activities in clinical and environmental isolates of Acanthamoeba

The pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Phospholipases are known to cleave phospholipids, suggesting their possible involvement in the host cell plasma membrane disruption leading to host cell penetration and lysis. The aims of the present study were to determine phospholipase activities in Acanthamoeba and to determine their roles in the pathogenesis of Acanthamoeba. Using an encephalitis isolate (T1 genotype), a keratitis isolate (T4 genotype), and an environmental isolate (T7 genotype), we demonstrated that Acanthamoeba exhibited phospholipase A(2) (PLA(2)) and phospholipase D (PLD) activities in a spectrophotometry-based assay. Interestingly, the encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, but the environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PLD activities compared with the PLA(2). Acanthamoeba exhibited optimal phospholipase activities at 37℃ and at neutral pH indicating their physiological relevance. The functional role of phospholipases was determined by in vitro assays using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that a PLD-specific inhibitor, i.e., compound 48/80, partially inhibited Acanthamoeba encephalitis isolate cytotoxicity of the host cells, while PLA(2)-specific inhibitor, i.e., cytidine 5'-diphosphocholine, had no effect on parasite-mediated HBMEC cytotoxicity. Overall, the T7 exhibited higher phospholipase activities as compared to the T4. In contract, the T7 exhibited minimal binding to, or cytotoxicity of, HBMEC.     

Balamuthia mandrillaris stimulates interleukin-6 release in primary human brain microvascular endothelial cells via a phosphatidylinositol 3-kinase-dependent pathway

Balamuthia mandrillaris is an emerging protozoan parasite that can cause fatal granulomatous encephalitis. Haematogenous spread is a likely route prior to entry into the central nervous system (CNS), but it is not clear how circulating amoebae cross the blood-brain barrier. Using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, we determined HBMEC inflammatory response to B. mandrillaris and the underlying mechanisms associated with this response. We demonstrated that HBMEC incubated with B. mandrillaris released significantly higher levels of interleukin-6 (IL-6) (>400 pg/ml) as compared with less than 50 pg/ml in HBMEC incubated alone. Western blotting assays determined that B. mandrillaris specifically activates phosphatidylinositol 3-kinase (PI3K). By using LY294002, a PI3K inhibitor, as well as by using HBMEC expressing dominant-negative PI3K, we have identified PI3K as an important mediator of B. mandrillaris-mediated IL-6 release. We conclude that B. mandrillaris induces HBMEC signalling pathways, which lead to IL-6 release. This is the first time PI3K has been shown to play a crucial role in B. mandrillaris-mediated IL-6 release in HBMEC.     

Streptococcus suis stimulates ICAM-1 shedding from microvascular endothelial cells

In this study, we hypothesized that Streptococcus suis induces the shedding of adhesion molecules from the surface of human brain microvascular endothelial cells (HBMEC), which may contribute to the ongoing pathophysiological processes of meningitis. When HBMEC were stimulated with whole cells of S. suis S735, significantly larger amounts of soluble intercellular adhesion molecule-1 (sICAM-1) were shed into conditioned medium while basal levels of soluble E-cadherin and P-selectin were unaffected. At a multiplicity of infection of 1 and 10, S. suis increased the concentration of sICAM-1 3.5- and 5-fold, respectively. A capsule-deficient mutant of S. suis induced more shedding than the parental strain. In addition, an S. suis cell wall preparation dose-dependently stimulated ICAM-1 shedding. Specific inhibitors of tyrosine kinase, mitogen-activated extracellular kinase 1, 2, and c-JUN N-terminal kinase significantly reduced S. suis-mediated ICAM-1 release. ICAM-1 shedding was also inhibited by a specific inhibitor of matrix metalloproteinases. The capacity of S. suis to induce ICAM-1 shedding has many functional implications that may contribute to the pathophysiological process of meningitis.     

Ca2+/calmodulin-dependent invasion of microvascular endothelial cells of human brain by Escherichia coli K1

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.).     

Polar invasion and translocation of Neisseria meningitidis and Streptococcus suis in a novel human model of the blood-cerebrospinal fluid barrier

Acute bacterial meningitis is a life-threatening disease in humans. Discussed as entry sites for pathogens into the brain are the blood-brain and the blood-cerebrospinal fluid barrier (BCSFB). Although human brain microvascular endothelial cells (HBMEC) constitute a well established human in vitro model for the blood-brain barrier, until now no reliable human system presenting the BCSFB has been developed. Here, we describe for the first time a functional human BCSFB model based on human choroid plexus papilloma cells (HIBCPP), which display typical hallmarks of a BCSFB as the expression of junctional proteins and formation of tight junctions, a high electrical resistance and minimal levels of macromolecular flux when grown on transwell filters. Importantly, when challenged with the zoonotic pathogen Streptococcus suis or the human pathogenic bacterium Neisseria meningitidis the HIBCPP show polar bacterial invasion only from the physiologically relevant basolateral side. Meningococcal invasion is attenuated by the presence of a capsule and translocated N. meningitidis form microcolonies on the apical side of HIBCPP opposite of sites of entry. As a functionally relevant human model of the BCSFB the HIBCPP offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens.     

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.     

Atorvastatin partially prevents an inflammatory barrier breakdown of cultured human brain endothelial cells at a pharmacologically relevant concentration

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (i.e. statins) are currently under clinical investigation as a prophylactic immunomodulatory treatment for neurological diseases where an inflammatory disruption of the blood-brain barrier plays a pathogenic role. Here, we investigated whether atorvastatin pre-treatment modulates inflammatory-induced barrier dysfunction of cultured human brain microvascular endothelial cells (HBMEC). Pre-treatment of immortalized HBMEC with atorvastatin (50 nmol/L to 1 micromol/L) dose-dependently prevented an inflammatory up-regulation of monocyte chemoattractant protein-1/CCL2 but not of interleukin-8/CXCL8 and intercellular adhesion molecule-1 expression by tumor necrosis factor-alpha or interleukin-1beta. It antagonized an inflammatory up-regulation of claudin-3 expression while zonula occludens-1 and occludin protein levels remained unaltered. Like immortalized HBMEC, primary HBMEC also showed a reduction of claudin-3 and of inducible CCL2 expression following atorvastatin pre-treatment. On a functional level, atorvastatin pre-treatment of HBMEC strongly and dose-dependently reduced adhesion of activated T lymphocytes to pre-activated primary endothelium. Atorvastatin effects could partially be abolished by parallel mevalonate treatment. These anti-inflammatory effects of atorvastatin were observed already at a pharmacologically relevant concentration of 50 nmol/L. Our results obtained with human brain endothelial cells demonstrate how statins may partially prevent an inflammatory-mediated blood-brain barrier breakdown in humans.     

Current concepts on Escherichia coli K1 translocation of the blood-brain barrier

The mortality and morbidity associated with neonatal gram-negative meningitis have remained significant despite advances in antimicrobial chemotherapy. Escherichia coli K1 is the most common gram-negative organism causing neonatal meningitis. Our incomplete knowledge of the pathogenesis of this disease is one of the main reasons for this high mortality and morbidity. We have previously established both in vitro and in vivo models of the blood-brain barrier (BBB) using human brain microvascular endothelial cells (HBMEC) and hematogenous meningitis in neonatal rats, respectively. With these in vitro and in vivo models, we have shown that successful crossing of the BBB by circulating E. coli requires a high-degree of bacteremia, E. coli binding to and invasion of HBMEC, and E. coli traversal of the BBB as live bacteria. Our previous studies using TnphoA, signature-tagged mutagenesis and differential fluorescence induction identified several E. coli K1 determinants such as OmpA, Ibe proteins, AslA, TraJ and CNF1 contributing to invasion of HBMEC in vitro and traversal of the blood-brain barrier in vivo. We have shown that some of these determinants interact with specific receptors on HBMEC, suggesting E. coli translocation of the BBB is the result of specific pathogen-host cell interactions. Recent studies using functional genomics techniques have identified additional E. coli K1 factors that contribute to the high degree of bacteremia and HBMEC binding/invasion/transcytosis. In this review, we summarize the current knowledge on the mechanisms underlying the successful E. coli translocation of the BBB.     

Cryptococcus neoformans Activates RhoGTPase Proteins Followed by Protein Kinase C,Focal Adhesion Kinase,and Ezrin to Promote Traversal across the Blood-Brain Barrier

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis. Previous studies have demonstrated that Cryptococcus binding and invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for transmigration across the blood-brain barrier. However, the molecular mechanism involved in the cryptococcal blood-brain barrier traversal is poorly understood. In this study we examined the signaling events in HBMEC during interaction with C. neoformans. Analysis with inhibitors revealed that cryptococcal association, invasion, and transmigration require host actin cytoskeleton rearrangement. Rho pulldown assays revealed that Cryptococcus induces activation of three members of RhoGTPases, e.g. RhoA, Rac1, and Cdc42, and their activations are required for cryptococcal transmigration across the HBMEC monolayer. Western blot analysis showed that Cryptococcus also induces phosphorylation of focal adhesion kinase (FAK), ezrin, and protein kinase C α (PKCα), all of which are involved in the rearrangement of host actin cytoskeleton. Down-regulation of FAK, ezrin, or PKCα by shRNA knockdown, dominant-negative transfection, or inhibitors significantly reduces cryptococcal ability to traverse the HBMEC monolayer, indicating their positive role in cryptococcal transmigration. In addition, activation of RhoGTPases is the upstream event for phosphorylation of FAK, ezrin, and PKCα during C. neoformans-HBMEC interaction. Taken together, our findings demonstrate that C. neoformans activates RhoGTPases and subsequently FAK, ezrin, and PKCα to promote their traversal across the HBMEC monolayer, which is the critical step for cryptococcal brain infection and development of meningitis.     

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.     

Transforming growth factor-beta increases Escherichia coli K1 adherence,invasion, and transcytosis in human brain microvascular endothelial cells

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.     

The K1 capsule modulates trafficking of E. coli-containing vacuoles and enhances intracellular bacterial survival in human brain microvascular endothelial cells

Escherichia coli K1 has been shown to invade human brain microvascular endothelial cells (HBMEC) in vitro and translocate the blood-brain barrier in vivo, but it is unclear how E. coli K1 traverses HBMEC. We have previously shown that internalized E. coli K1 is localized within membrane-bound vacuole in HBMEC. The present study was carried out to understand intracellular trafficking of E. coli K1 containing vacuoles (ECVs) in HBMEC. ECVs initially acquired two early endosomal marker proteins, EEA1 and transferrin receptor. Rab7 and Lamp-1, markers for late endosome and late endosome/lysosome, respectively, were subsequently recruited on the ECVs, which was confirmed with flow cytometry analysis of ECVs. However, ECVs did not obtain cathepsin D, a lysosomal enzyme, even after 120 min incubation, suggesting that E. coli K1 avoids lysosomal fusion. In contrast, isogenic K1 capsule-deletion mutant obtained early and late endosomal markers on vacuolar membranes and allowed lysosomal fusion with subsequent degradation inside vacuoles. This observation was consistent with the decreased intracellular survival of K1 capsule-deletion mutant, even though the binding and internalization rates of the mutant were higher than those of the parent E. coli K1 strain. This is the first demonstration that E. coli K1, via the K1 capsule on the bacterial surface, modulates the maturation process of ECVs and prevents fusion with lysosomes, which is an event necessary for traversal of the blood-brain barrier as live bacteria.     

Pro-inflammatory cytokine and chemokine release by human brain microvascular endothelial cells stimulated by Streptococcus suis serotype 2

Streptococcus suis serotype 2 is a world-wide agent of diseases among pigs including meningitis, septicemia and arthritis. This microorganism is also recognized as an important zoonotic agent. The pathogenesis of the meningitis caused by S. suis is poorly understood. We have previously shown that S. suis is able to adhere to human brain microvascular endothelial cells (BMEC), but not to human umbilical vein endothelial cells (HUVEC). The objective of this work was to study the ability of S. suis serotype 2 to induce the release of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1); IL-6 and the chemokines IL-8 and monocyte chemotactic protein-1 (MCP-1) by human BMEC and HUVEC, using a sandwich enzyme-linked immunosorbent assay. S. suis was able to stimulate the production of IL-6, IL-8 and MCP-1 by BMEC but not HUVEC, in a time- and concentration-dependent manner. Bacterial cell wall components were largely responsible for such stimulation. The human and pig origin of strains does not seem to affect the intensity of the response; indeed, a very heterogeneous pattern of cytokine and chemokine production was observed for the different strains tested in this study. In situ production of cytokines and chemokines by BMEC may be the result of specific adhesion of S. suis to this cell type, with several consequences such as increased recruitment of leukocytes and an increase in the blood-brain barrier permeability.     

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-α), 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.     

Cannabinoids inhibit HIV-1 Gp120-mediated insults in brain microvascular endothelial cells

HIV-1 infection has significant effect on the immune system as well as on the nervous system. Breakdown of the blood-brain barrier (BBB) is frequently observed in patients with HIV-associated dementia (HAD) despite lack of productive infection of human brain microvascular endothelial cells (HBMEC). Cellular products and viral proteins secreted by HIV-1 infected cells, such as the HIV-1 Gp120 envelope glycoprotein, play important roles in BBB impairment and HIV-associated dementia development. HBMEC are a major component of the BBB. Using cocultures of HBMEC and human astrocytes as a model system for human BBB as well as in vivo model, we show for the first time that cannabinoid agonists inhibited HIV-1 Gp120-induced calcium influx mediated by substance P and significantly decreased the permeability of HBMEC as well as prevented tight junction protein down-regulation of ZO-1, claudin-5, and JAM-1 in HBMEC. Furthermore, cannabinoid agonists inhibited the transmigration of human monocytes across the BBB and blocked the BBB permeability in vivo. These results demonstrate that cannabinoid agonists are able to restore the integrity of HBMEC and the BBB following insults by HIV-1 Gp120. These studies may lead to better strategies for treatment modalities targeted to the BBB following HIV-1 infection of the brain based on cannabinoid pharmacotherapies.     

Crystal structure of cytotoxin protein suilysin from Streptococcus suis

Cholesterol-dependent cytolysins (CDC) are pore forming toxins. A prototype of the CDC family members is perfringolysin O (PFO), which directly binds to cholesterol rich cell membrane and lyses the cell. However, as an exception of this general observation, Streptococcus intermedius intermedilysin (ILY) requires human CD59 as its receptor in addition to cholesterol when exhibiting hemolytic activity. It was attempted to explain this functional difference based on a conformational variation in the C-terminal domain of the two toxin proteins, particularly a highly conserved undecapeptide termed tryptophan rich motif. Here, we present the crystal structure of suilysin, a CDC toxin from the swine infectious pathogen Streptococcus suis. Like PFO, suilysin does not require a host receptor for hemolytic activity; yet in the suilysin crystal it shares a similar conformation in the tryptophan rich motif with ILY. This observation suggests that current views of structure-function relationship of CDC proteins in membrane association are still far from complete.     

Poliovirus entry into human brain microvascular cells requires receptor-induced activation of SHP-2

Viruses use specific receptor molecules to bind selectively to target cells. Receptors have often been considered as mere docking sites, but they may also possess intrinsic signaling capacities that serve to prime the cell for entry and infection. Poliovirus (PV) initiates infection by binding to the PV receptor (PVR) and causes paralytic poliomyelitis by replicating within motor neurons of the brain and spinal cord. We have examined the process by which PV enters cultured human brain microvascular endothelial cells (HBMEC), an in vitro model of the blood-brain barrier. We found that PV enters HBMEC by dynamin-dependent caveolar endocytosis, and that entry depends on intracellular signals triggered by virus attachment to PVR. Tyrosine kinase and RhoA GTPase activation initiated by PVR ligation were both essential. Virus attachment also induced tyrosine phosphorylation of PVR; this permitted the association of PVR with SHP-2, a protein tyrosine phosphatase whose activation was required for entry and infection. The results indicate that receptor-induced signals promote virus entry and suggest a role for tyrosine phosphatases in viral pathogenesis.     

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.