Sphingosine 1-Phosphate Activation of EGFR As a Novel Target for Meningitic Escherichia coli Penetration of the Blood-Brain Barrier.

Central nervous system (CNS) infection continues to be an important cause of mortality and morbidity, necessitating new approaches for investigating its pathogenesis, prevention and therapy. Escherichia coli is the most common Gram-negative bacillary organism causing meningitis, which develops following penetration of the blood-brain barrier (BBB). By chemical library screening, we identified epidermal growth factor receptor (EGFR) as a contributor to E. coli invasion of the BBB in vitro. Here, we obtained the direct evidence that CNS-infecting E. coli exploited sphingosine 1-phosphate (S1P) for EGFR activation in penetration of the BBB in vitro and in vivo. We found that S1P was upstream of EGFR and participated in EGFR activation through S1P receptor as well as through S1P-mediated up-regulation of EGFR-related ligand HB-EGF, and blockade of S1P function through targeting sphingosine kinase and S1P receptor inhibited EGFR activation, and also E. coli invasion of the BBB. We further found that both S1P and EGFR activations occurred in response to the same E. coli proteins (OmpA, FimH, NlpI), and that S1P and EGFR promoted E. coli invasion of the BBB by activating the downstream c-Src. These findings indicate that S1P and EGFR represent the novel host targets for meningitic E. coli penetration of the BBB, and counteracting such targets provide a novel approach for controlling E. coli meningitis in the era of increasing resistance to conventional antibiotics.

Cysteinyl leukotrienes as novel host factors facilitating Cryptococcus neoformans penetration into the brain.

Cryptococcus neoformas infection of the central nervous system (CNS) continues to be an important cause of mortality and morbidity, and a major contributing factor is our incomplete knowledge of the pathogenesis of this disease. Here, we provide the first direct evidence that C. neoformans exploits host cysteinyl leukotrienes (LTs), formed via LT biosynthetic pathways involving cytosolic phospholipase A2 α (cPLA2 α) and 5-lipoxygenase (5-LO) and acting via cysteinyl leukotriene type 1 receptor (CysLT1), for penetration of the blood-brain barrier. Gene deletion of cPLA2 α and 5-LO and pharmacological inhibition of cPLA2 α, 5-LO and CysLT1 were effective in preventing C. neoformans penetration of the blood-brain barrier in vitro and in vivo. A CysLT1 antagonist enhanced the efficacy of an anti-fungal agent in therapy of C. neoformans CNS infection in mice. These findings demonstrate that host cysteinyl LTs, dependent on the actions of cPLA2 α and 5-LO, promote C. neoformans penetration of the blood-brain barrier and represent novel targets for elucidating the pathogenesis and therapeutic development of C. neoformans CNS infection.

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.

IbeA and OmpA of Escherichia coli K1 exploit Rac1 activation for invasion of human brain microvascular endothelial cells.

Meningitis-causing Escherichia coli K1 internalization of the blood-brain barrier is required for penetration into the brain, but the host-microbial interactions involved in E. coli entry of the blood-brain barrier remain incompletely understood. We show here that a meningitis-causing E. coli K1 strain RS218 activates Rac1 (GTP-Rac1) of human brain microvascular endothelial cells (HBMEC) in a time-dependent manner. Both activation and bacterial invasion were significantly inhibited in the presence of a Rac1 inhibitor. We further showed that the guanine nucleotide exchange factor Vav2, not ß-Pix, was involved in E. coli K1-mediated Rac1 activation. Since activated STAT3 is known to bind GTP-Rac1, the relationship between STAT3 and Rac1 was examined in E. coli K1 invasion of HBMEC. Downregulation of STAT3 resulted in significantly decreased E. coli invasion compared to control HBMEC, as well as a corresponding decrease in GTP-Rac1, suggesting that Rac1 activation in response to E. coli is under the control of STAT3. More importantly, two E. coli determinants contributing to HBMEC invasion, IbeA and OmpA, were shown to affect both Rac1 activation and their association with STAT3. These findings demonstrate for the first time that specific E. coli determinants regulate a novel mechanism of STAT3 cross talk with Rac1 in E. coli K1 invasion of HBMEC.

Extracellular loops of the Eschericia coli outer membrane protein A contribute to the pathogenesis of meningitis.

Neonatal meningitis by Eschericia coli RS218 occurs due to bacteremia and its transmigration across the blood-brain barrier. Although the outer membrane protein A (OmpA), a molecule with extracellular loops has been shown to contribute to the above phenomenon, we do not know the exact the role of these individual loops. Using bacterial strains whose individual loops have been removed, we demonstrated that whereas Loops1 and 2 contribute to 70%-80% bacterial survival in serum, bacterial entry into human brain microvascular endothelial cells (HBMEC) is governed by Loops1, 2, and 3. Cellular invasion was shown to require activation of host cytosolic phospholipase A2 (cPLA2α) by Loops1 and 2 but not 3. This suggests 2 distinct pathways for bacterial entry into host cells. Loop 4 played no role in either serum survival, cellular entry, or cPLA2α signaling. These findings demonstrate for the first time the different contributions of extracellular loops of OmpA to the pathogenesis of E. coli meningitis.

Host cytosolic phospholipase A2α contributes to group B Streptococcus penetration of the blood-brain barrier.

Group B Streptococcus (GBS) is the most common bacterium causing neonatal meningitis, and neonatal GBS meningitis continues to be an important cause of mortality and morbidity. Here we provide the first direct evidence that host cytosolic phospholipase A2α (cPLA2α) contributes to type III GBS invasion of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier and penetration into the brain, the key step required for the development of GBS meningitis. This was shown by our demonstration that pharmacological inhibition and gene deletion of cPLA2α significantly decreased GBS invasion of the HBMEC monolayer and penetration into the brain. cPLA2α releases arachidonic acid from membrane phospholipids, and we showed that the contribution of cPLA2α to GBS invasion of HBMEC involved lipoxygenated metabolites of arachidonic acid, cysteinyl leukotrienes (LTs). In addition, type III GBS invasion of the HBMEC monolayer involves protein kinase Cα (PKCα), as shown by time-dependent PKCα activation in response to GBS as well as decreased GBS invasion in HBMEC expressing dominant-negative PKCα. PKCα activation in response to GBS, however, was abolished by inhibition of cPLA2α and cysteinyl LTs, suggesting that cPLA2α and cysteinyl LTs contribute to type III GBS invasion of the HBMEC monolayer via PKCα. These findings demonstrate that specific host factors involving cPLA2α and cysteinyl LTs contribute to type III GBS penetration of the blood-brain barrier and their contribution involves PKCα.

Arachidonic acid metabolism regulates Escherichia coli penetration of the blood-brain barrier.

Escherichia coli K1 meningitis occurs following penetration of the blood-brain barrier, but the underlying mechanisms involved in E. coli penetration of the blood-brain barrier remain incompletely understood. We have previously shown that host cytosolic phospholipase A(2)α (cPLA(2)α) contributes to E. coli invasion of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, but the underlying mechanisms remain unclear. cPLA(2)α selectively liberates arachidonic acid from membrane phospholipids. Here, we provide the first direct evidence that host 5-lipoxygenase and lipoxygenase products of arachidonic acid, cysteinyl leukotrienes (LTs), contribute to E. coli K1 invasion of HBMEC and penetration into the brain, and their contributions involve protein kinase C alpha (PKCα). These findings demonstrate that arachidonic acid metabolism regulates E. coli penetration of the blood-brain barrier, and studies are needed to further elucidate the mechanisms involved with metabolic products of arachidonic acid for their contribution to E. coli invasion of the blood-brain barrier.

NlpI contributes to Escherichia coli K1 strain RS218 interaction with human brain microvascular endothelial cells.

Escherichia coli K1 is the most common Gram-negative bacillary organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMECs) is a prerequisite for its traversal of the blood-brain barrier (BBB) and penetration into the brain. In the present study, we identified NlpI as a novel bacterial determinant contributing to E. coli K1 interaction with HBMECs. The deletion of nlpI did not affect the expression of the known bacterial determinants involved in E. coli K1-HBMEC interaction, such as type 1 fimbriae, flagella, and OmpA, and the contribution of NlpI to HBMECs binding and invasion was independent of those bacterial determinants. Previous reports have shown that the nlpI mutant of E. coli K-12 exhibits growth defect at 42 degrees C at low osmolarity, and its thermosensitive phenotype can be suppressed by a mutation on the spr gene. The nlpI mutant of strain RS218 exhibited similar thermosensitive phenotype, but additional spr mutation did not restore the ability of the nlpI mutant to interact with HBMECs. These findings suggest the decreased ability of the nlpI mutant to interact with HBMECs is not associated with the thermosensitive phenotype. NlpI was determined as an outer membrane-anchored protein in E. coli, and the nlpI mutant was defective in cytosolic phospholipase A(2)alpha (cPLA(2)alpha) phosphorylation compared to the parent strain. These findings illustrate the first demonstration of NlpI's contribution to E. coli K1 binding to and invasion of HBMECs, and its contribution is likely to involve cPLA(2)alpha.

Acquisition of factor H by a novel surface protein on group B Streptococcus promotes complement degradation.

Binding of the host complement regulator, factor H (FH), by some pathogenic microbes constitutes an important virulence mechanism, whereby complement is broken down to help microbes survive in the host. Although it has been hypothesized for the past two decades that GBS type III binds FH via sialic acid present on its capsule, neither the binding of FH to GBS has been demonstrated nor the mechanism of interaction identified. We observed that FH bound to both wild-type and capsule or sialic acid-deficient GBS that were used as negative controls. Wild-type and acapsular GBS were incubated with serum or pure FH degraded almost 90% of C3b, suggesting that the GBS-bound FH maintained cofactor activity. In addition, dot-blot analysis showed approximately 5-10% of C5 and C9 formation, as compared to an Escherichia coli control, suggesting breakdown at the C3b level. Protease treatment of the bacteria completely abolished binding of FH. Using overlay assays and mass spectroscopic analysis, we identified the FH receptor as the streptococcal histidine triad (SHT) surface protein. The ability of binding FH to SHT was further confirmed by using recombinant SHT. This report describes the identification of the SHT as an FH-binding protein on the surface of GBS type III, revealing a novel mechanism by which the bacterium acquires FH to evade complement opsonization.

Escherichia coli interaction with human brain microvascular endothelial cells induces signal transducer and activator of transcription 3 association with the C-terminal domain of Ec-gp96, the outer membrane protein A receptor for invasion.

Our inability to develop new therapeutic strategies to prevent meningitis due to Escherichia coli K1 is attributed to our incomplete understanding of the pathophysiology of the disease. Previously, we demonstrated that outer membrane protein A of E. coli interacts with a gp96 homologue, Ec-gp96, on human brain microvascular endothelial cells (HBMEC) for invasion. However, signalling events mediated by Ec-gp96 that allow internalization of E. coli are incompletely understood. Here, we demonstrate that signal transducer and activator of transcription 3 (Stat3) activation and its interaction with Ec-gp96 were critical for E. coli invasion. The activated Stat3 was colocalized with Ec-gp96 at the actin condensation sites, and overexpressing a dominant negative (DN) form of Stat3 in HBMEC significantly abrogated the invasion. Furthermore, overexpression of Ec-gp96Delta200, the C-terminal 214-amino-acid truncated Ec-gp96, prevented the invasion of E. coli in HBMEC. In contrast, lack of ATP binding by gp96 did not affect the invasion. Overexpression of DN forms of either phosphatidyl inositol-3 kinase (PI3-kinase) subunit p85 or protein kinase C-alpha (PKC-alpha) had no effect on the activation of Stat3 and its association with Ec-gp96, whereas overexpression of DN-Stat3 abolished the activation of both PI3-kinase and PKC-alpha. Together, our findings identified a novel interaction of Stat3 with Ec-gp96, upstream of PI3-kinase and PKC-alpha activation that is required for the invasion of E. coli into HBMEC.

Effects of complement regulators bound to Escherichia coli K1 and Group B Streptococcus on the interaction with host cells.

Escherichia coli K1 and Group B Streptococcus (GBS) are the most common bacteria that cause meningitis during the neonatal period. Complement, the first line of defence in the host, acts on these bacteria to opsonize with various components of complement for subsequent presentation to phagocytes. To counteract these opsonization effects, E. coli and GBS bind to the complement regulators C4 binding protein and Factor H, respectively. Nonetheless, the deposition of complement components on these two bacteria from neonatal serum and their effect on the host cell interaction is unclear. Here we demonstrated that the deposition of complement proteins from adult serum prevented the invasion of E. coli into human brain microvascular endothelial cells, whereas the invasion of GBS was enhanced. In contrast, treatment with cord serum had no effect on the invasion of both these bacteria. We also examined the effect of the deposited complement proteins on phagocytosis using THP-1 cells and THP-1 cells differentiated into macrophages. Escherichia coli treated with adult serum neither attached nor entered these cells, whereas GBS was phagocytosed and survived efficiently. We further demonstrate that the inhibitory effect of complement proteins is the result of the bound complement inhibitors C4b-binding protein, in the case of E. coli, and Factor H, in the case of GBS. Taken together, these results suggest that E. coli and GBS utilize contrasting mechanisms of complement-mediated interactions with their target cells for successful establishment of disease.

Plasminogen activator inhibitor-1 deficiency has renal benefits but some adverse systemic consequences in diabetic mice.

BACKGROUND: Elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) are observed in patients with obesity, hypertension and diabetes, and several observations suggest that PAI-1 mediates diabetic vascular complications. Although increased intrarenal expression of PAI-1 is also a feature of diabetic nephropathy, evidence that PAI-1 plays a primary pathogenetic role in the renal pathology is lacking. METHODS: This study was designed to investigate the renal effects of genetic PAI-1 deficiency in db/db mice with obesity, hyperinsulinemia and hyperglycemia. For comparison the effects of PAI-1 deficiency were also examined in a cohort of mice with insulin-deficient streptozotocin (STZ)-induced diabetes. The findings are reported for 4 study groups at 8 months of age: PAI-1+/+ controls, PAI-1+/+ diabetics, PAI-1-/- controls and PAI-1-/- diabetics. RESULTS: PAI-1 deficiency had an unexpected negative impact on the db/db mice. Overall 33% of the diabetic mice died prematurely, and 63% of the db/db PAI-1-/- males had an obese body habitus but were runts. The final analyses were limited to the female db/db mice. Several nephropathy parameters were improved in the db/db PAI-1-/- group compared to the db/db PAI-1+/+ group including: albumin-to-creatinine ratios (57 +/- 45 vs. 145 +/- 71 microg/mg x10), change in glomerular extracellular matrix (ECM) area (decrease of 10% compared to controls vs. an increase of 31%) and increased total kidney collagen (47% increased vs. 96% in the PAI-1+/+ diabetics). The serum glucose levels were 15-25% lower in the PAI-1-/- nondiabetic control groups and remained lower in the db/dbPAI-1-/- mice. The STZ study was performed in males. None of the mice developed a runted phenotype or died prematurely. After diabetes of 6 months' duration changes in glomerular ECM area (-15 vs. +64%) and total kidney collagen (+8 vs. +40%) were lower in the PAI-1-/- mice compared to the PAI-1+/+ mice. The serum cholesterol levels were significantly lower in the PAI-1-/- mice, both controls (47 +/- 3 vs. 53 +/- 10 mg/dl) and diabetics (48 +/- 3 vs. 74 +/- 9 mg/dl). CONCLUSION: These data suggest a direct role for PAI-1 in renal matrix expansion and metabolic control in diabetes, but they also highlight important adverse outcomes that include male runting and premature death in mice with diabetes due to an inactive leptin receptor.

Exogenous bone morphogenetic protein-7 fails to attenuate renal fibrosis in rats with overload proteinuria.

BACKGROUND: Bone morphogenetic protein-7 (BMP-7) plays a critical role in renal development, accelerates recovery from acute renal injury, and more recently it has been shown to delay progressive renal disease. The present study was designed to investigate the effect of BMP-7 on interstitial fibrosis in the rat protein-overloaded model. METHODS: Renal disease was induced in 26 rats by daily intraperitoneal injections of bovine serum albumin (BSA); controls (n = 28) were injected with saline. Half of the rats in each group were treated with human recombinant BMP-7 (300 microg/kg i.p. 3 times weekly) and half with placebo. Animals were killed after 3 or 6 weeks. RESULTS: Compared to the saline control groups, the BSA groups had evidence of chronic renal disease: significantly increased urinary protein excretion rates; total kidney collagen content, and increased fibronectin and collagen III interstitial areas. By 6 weeks the BSA + BMP-7 group compared to the BSA + placebo group had a nonsignificant decrease in blood urea nitrogen (40 +/- 13 vs. 46 +/- 11 mg/dl), total kidney collagen (10.8 +/- 2.1 vs. 12.2 +/- 3.5 microg/kidney), fibronectin interstitial area (23 +/- 4 vs. 25 +/- 8%) and collagen III interstitial area (22 +/- 6 vs. 28 +/- 7%). Despite these results, renal gene expression profiles actually predicted worse fibrosis in the BSA + BMP-7 group with significantly higher total kidney mRNA levels for alpha(1)(III) procollagen (2.8 +/- 0.5 vs. 1.6 +/- 0.6, p < 0.05) and fibronectin at 6 weeks (1.9 +/- 0.3 vs. 1.2 +/- 0.5, p < 0.05). Renal BMP-7 mRNA levels at 6 weeks were significantly increased in the BSA + placebo group compared to the saline + placebo group with no difference between the BSA + BMP-7 and the BSA + placebo groups. Both cortical and medullary tubules expressed BMP-7 protein but BMP-7 was only detected in the tubular lumina and urine of proteinuric animals. CONCLUSIONS: In rats with protein-overload proteinuria, renal tubules continue to express BMP-7 but some of the endogenous protein is secreted into the urinary space. Administration of exogenous recombinant BMP-7 had no effect on proteinuria but was associated with a nonsignificant trend towards less interstitial fibrosis at 6 weeks despite significantly higher kidney extracellular matrix gene mRNA levels. These findings suggest that BMP-7 treatment may have anti-fibrotic effects through enhancement of matrix turnover, although overall these effects are modest in proteinuric states in the absence of significant tubular epithelial cell apoptosis and epithelial-mesenchymal transition.

Logarithmic phase Escherichia coli K1 efficiently avoids serum killing by promoting C4bp-mediated C3b and C4b degradation.

Meningitis caused by Escherichia coli K1 is a serious illness in neonates with neurological sequelae in up to 50% of survivors. A high degree of bacteremia is required for E. coli K1 to cross the blood-brain barrier, which suggests that the bacterium must evade the host defence mechanisms and survive in the bloodstream. We previously showed that outer membrane protein A (OmpA) of E. coli binds C4b-binding protein (C4bp), an inhibitor of complement activation via the classical pathway. Nevertheless, the exact mechanism by which E. coli K1 survives in serum remains elusive. Here, we demonstrate that log phase (LP) OmpA+ E. coli K1 avoids serum bactericidal activity more effectively than postexponential phase bacteria. OmpA- E. coli cannot survive in serum grown to either phase. The increased serum resistance of LP OmpA+ E. coli is the result of increased binding of C4bp, with a concomitant decrease in the deposition of C3b and the downstream complement proteins responsible for the formation of the membrane attack complex. C4bp bound to E. coli K1 acts as a cofactor to factor I in the cleavage of both C3b and C4b, which shuts down the ensuing complement cascade. Accordingly, a peptide corresponding to the complement control protein domain 3 of C4bp sequence, was able to compete with C4bp binding to OmpA and cause increased deposition of C3b. Thus, binding of C4bp appears to be responsible for survival of E. coli K1 in human serum.