Interleukin-22 Contributes to Blood-Brain Barrier Disruption via STAT3/VEGFA Activation in Escherichia coli Meningitis.

Escherichia coli continues to be the predominant Gram-negative pathogen causing neonatal meningitis worldwide. Inflammatory mediators have been implicated in the pathogenesis of meningitis and are key therapeutic targets. The role of interleukin-22 (IL-22) in various diseases is diverse, with both protective and pathogenic effects. However, little is understood about the mechanisms underlying the damaging effects of IL-22 on the blood-brain barrier (BBB) in E. coli meningitis. We observed that meningitic E. coli infection induced IL-22 expression in the serum and brain of mice. The tight junction proteins (TJPs) components ZO-1, Occludin, and Claudin-5 were degraded in the mouse brain and human brain microvascular endothelial cells (hBMEC) following IL-22 administration. Moreover, the meningitic E. coli-caused increase in BBB permeability in wild-type mice was restored by knocking out IL-22. Mechanistically, IL-22 activated the STAT3-VEGFA signaling cascade in E. coli meningitis, thus eliciting the degradation of TJPs to induce BBB disruption. Our data indicated that IL-22 is an essential host accomplice during E. coli-caused BBB disruption and could be targeted for the therapy of bacterial meningitis.

TGFß1-induced hedgehog signaling suppresses the immune response of brain microvascular endothelial cells elicited by meningitic Escherichia coli.

BACKGROUND: Meningitic Escherichia coli (E. coli) is the major etiological agent of bacterial meningitis, a life-threatening infectious disease with severe neurological sequelae and high mortality. The major cause of central nervous system (CNS) damage and sequelae is the bacterial-induced inflammatory storm, where the immune response of the blood-brain barrier (BBB) is crucial. METHODS: Western blot, real-time PCR, enzyme-linked immunosorbent assay, immunofluorescence, and dual-luciferase reporter assay were used to investigate the suppressor role of transforming growth factor beta 1 (TGFß1) in the immune response of brain microvascular endothelial cells elicited by meningitic E. coli. RESULT: In this work, we showed that exogenous TGFß1 and induced noncanonical Hedgehog (HH) signaling suppressed the endothelial immune response to meningitic E. coli infection via upregulation of intracellular miR-155. Consequently, the increased miR-155 suppressed ERK1/2 activation by negatively regulating KRAS, thereby decreasing IL-6, MIP-2, and E-selectin expression. In addition, the exogenous HH signaling agonist SAG demonstrated promising protection against meningitic E. coli-induced neuroinflammation. CONCLUSION: Our work revealed the effect of TGFß1 antagonism on E. coli-induced BBB immune response and suggested that activation of HH signaling may be a potential protective strategy for future bacterial meningitis therapy. Video Abstract.

Egr-1 is a key regulator of the blood-brain barrier damage induced by meningitic Escherichia coli.

Bacterial meningitis remains a leading cause of infection-related mortality worldwide. Although Escherichia coli (E. coli) is the most common etiology of neonatal meningitis, the underlying mechanisms governing bacterial blood-brain barrier (BBB) disruption during infection remain elusive. We observed that infection of human brain microvascular endothelial cells with meningitic E. coli triggers the activation of early growth response 1 (Egr-1), a host transcriptional activator. Through integrated chromatin immunoprecipitation sequencing and transcriptome analysis, we identified Egr-1 as a crucial regulator for maintaining BBB integrity. Mechanistically, Egr-1 induced cytoskeletal changes and downregulated tight junction protein expression by directly targeting VEGFA, PDGFB, and ANGPTL4, resulting in increased BBB permeability. Meanwhile, Egr-1 also served as a master regulator in the initiation of neuroinflammatory response during meningitic E. coli infection. Our findings support an Egr-1-dependent mechanism of BBB disruption by meningitic E. coli, highlighting a promising therapeutic target for bacterial meningitis.

Nucleotide receptors mediate protection against neonatal sepsis and meningitis caused by alpha-hemolysin expressing Escherichia coli K1.

Neonatal meningitis-associated Escherichia coli (NMEC) is among the leading causes of bacterial meningitis and sepsis in newborn infants. Several virulence factors have been identified as common among NMEC, and have been shown to play an important role in the development of bacteremia and/or meningitis. However, there is significant variability in virulence factor expression between NMEC isolates, and relatively little research has been done to assess the impact of variable virulence factor expression on immune cell activation and the outcome of infection. Here, we investigated the role of NMEC strain-dependent P2X receptor (P2XR) signaling on the outcome of infection in neonatal mice. We found that alpha-hemolysin (HlyA)-expressing NMEC (HlyA+ ) induced robust P2XR-dependent macrophage cell death in vitro, while HlyA- NMEC did not. P2XR-dependent cell death was inflammasome independent, suggesting an uncoupling of P2XR and inflammasome activation in the context of NMEC infection. In vivo inhibition of P2XRs was associated with increased mortality in neonatal mice infected with HlyA+ NMEC, but had no effect on the survival of neonatal mice infected with HlyA- NMEC. Furthermore, we found that P2XR-dependent protection against HlyA+ NMEC in vivo required macrophages, but not neutrophils or NLRP3. Taken together, these data suggest that HlyA+ NMEC activates P2XRs which in turn confers macrophage-dependent protection against infection in neonates. In addition, our findings indicate that strain-dependent virulence factor expression should be taken into account when studying the immune response to NMEC.

miR-155 and miR-146a collectively regulate meningitic Escherichia coli infection-mediated neuroinflammatory responses.

BACKGROUND: Escherichia coli is the most common Gram-negative bacterium causing meningitis, and E. coli meningitis is associated with high mortality and morbidity throughout the world. Our previous study showed that E. coli can colonize the brain and cause neuroinflammation. Increasing evidence supports the involvement of miRNAs as key regulators of neuroinflammation. However, it is not clear whether these molecules participate in the regulation of meningitic E. coli-mediated neuroinflammation. METHODS: The levels of miR-155 and miR-146a, as well as their precursors, in E. coli-infected astrocytes were measured using quantitative real-time PCR (qPCR). Overexpression and knockdown studies of miR-155 and miR-146a were performed to observe the effects on bacterial loads, cytokines, chemokines, and NF-κB signaling pathways. Bioinformatics methods were utilized to predict the target genes, and these target genes were validated using qPCR, Western blotting, and luciferase reporter system. In vivo knockdown of miR-155 and miR-146a was carried out to observe the effects on bacterial loads, inflammatory genes, astrocyte activation, microglia activation, and survival in a mouse model. RESULTS: The levels of miR-155, miR-146a, and their precursors were significantly increased in astrocytes during E. coli infection. miR-155 and miR-146a were induced by the NF-κB-p65 signaling pathway upon infection. Overexpressing and inhibiting miR-155 and miR-146a in astrocytes did not affect the bacterial loads. Further, the in vitro overexpression of miR-155 and miR-146a suppressed the E. coli-induced inflammatory response, whereas the inhibition of miR-155 and miR-146a enhanced it. Mechanistically, miR-155 inhibited TAB2, and miR-146a targeted IRAK1 and TRAF6; therefore, they functioned collaboratively to modulate TLR-mediated NF-κB signaling. In addition, both miR-155 and miR-146a could regulate the EGFR-NF-κB signaling pathway. Finally, the in vivo suppression of E. coli-induced miR-155 and miR-146a further promoted the production of inflammatory cytokines, aggravated astrocyte and microglia activation, and decreased mouse survival time, without affecting the bacterial loads in the blood and brain. CONCLUSIONS: E. coli infection induced miR-155 and miR-146a, which collectively regulated bacteria-triggered neuroinflammatory responses through negative feedback regulation involving the TLR-mediated NF-κB and EGFR-NF-κB signaling pathways, thus protecting the central nervous system from further neuroinflammatory damage.

Prophylactic Palmitoylethanolamide Prolongs Survival and Decreases Detrimental Inflammation in Aged Mice With Bacterial Meningitis.

Easy-to-achieve interventions to promote healthy longevity are desired to diminish the incidence and severity of infections, as well as associated disability upon recovery. The dietary supplement palmitoylethanolamide (PEA) exerts anti-inflammatory and neuroprotective properties. Here, we investigated the effect of prophylactic PEA on the early immune response, clinical course, and survival of old mice after intracerebral E. coli K1 infection. Nineteen-month-old wild type mice were treated intraperitoneally with two doses of either 0.1 mg PEA/kg in 250 µl vehicle solution (n = 19) or with 250 µl vehicle solution only as controls (n = 19), 12 h and 30 min prior to intracerebral E. coli K1 infection. The intraperitoneal route was chosen to reduce distress in mice and to ensure exact dosing. Survival time, bacterial loads in cerebellum, blood, spleen, liver, and microglia counts and activation scores in the brain were evaluated. We measured the levels of IL-1ß, IL-6, MIP-1α, and CXCL1 in cerebellum and spleen, as well as of bioactive lipids in serum in PEA- and vehicle-treated animals 24 h after infection. In the absence of antibiotic therapy, the median survival time of PEA-pre-treated infected mice was prolonged by 18 h compared to mice of the vehicle-pre-treated infected group (P = 0.031). PEA prophylaxis delayed the onset of clinical symptoms (P = 0.037). This protective effect was associated with lower bacterial loads in the spleen, liver, and blood compared to those of vehicle-injected animals (P ≤ 0.037). PEA-pre-treated animals showed diminished levels of pro-inflammatory cytokines and chemokines in spleen 24 h after infection, as well as reduced serum concentrations of arachidonic acid and of one of its metabolites, 20-hydroxyeicosatetraenoic acid. In the brain, prophylactic PEA tended to reduce bacterial titers and attenuated microglial activation in aged infected animals (P = 0.042). Our findings suggest that prophylactic PEA can counteract infection associated detrimental responses in old animals. Accordingly, PEA treatment slowed the onset of infection symptoms and prolonged the survival of old infected mice. In a clinical setting, prophylactic administration of PEA might extend the potential therapeutic window where antibiotic therapy can be initiated to rescue elderly patients.

New insights into meningitic Escherichia coli infection of brain microvascular endothelial cells from quantitative proteomics analysis.

BACKGROUND: Bacterial meningitis remains a big threat to the integrity of the central nervous system (CNS), despite the advancements in antimicrobial reagents. Escherichia coli is a bacterial pathogen that can disrupt the CNS function, especially in neonates. E. coli meningitis occurs after bacteria invade the brain microvascular endothelial cells (BMECs) that form a direct and essential barrier restricting the entry of circulating microbes and toxins to the brain. Previous studies have reported on several cellular proteins that function during meningitic E. coli infections; however, more comprehensive investigations to elucidate the potential targets involved in E. coli meningitis are essential to better understand this disease and discover new treatments for it. METHODS: The isobaric tags for relative and absolute quantification (iTRAQ) approach coupled with LC-MS/MS were applied to compare and characterize the different proteomic profiles of BMECs in response to meningitic or non-meningitic E. coli strains. KEGG and gene ontology annotations, ingenuity pathways analysis, and functional experiments were combined to identify the key host molecules involved in the meningitic E. coli-induced tight junction breakdown and neuroinflammatory responses. RESULTS: A total of 13 cellular proteins were found to be differentially expressed by meningitic E. coli strains PCN033 and RS218, including one that was also affected by HB101, a non-meningitic E. coli strain. Through bioinformatics analysis, we identified the macrophage migration inhibitory factor (MIF), granzyme A, NF-κB signaling, and mitogen-activated protein kinase (MAPK) pathways as being biologically involved in the meningitic E. coli-induced tight junction breakdown and neuroinflammation. Functionally, we showed that MIF facilitated meningitic E. coli-induced production of cytokines and chemokines and also helped to disrupt the blood-brain barrier by decreasing the expression of tight junction proteins like ZO-1, occludin. Moreover, we demonstrated the significant activation of NF-κB and MAPK signaling in BMECs in response to meningitic E. coli strains, which dominantly determined the generation of the proinflammatory cytokines including IL-6, IL-8, TNF-α, and IL-1ß. CONCLUSIONS: Our work identified 12 host cellular targets that are affected by meningitic E. coli strains and revealed MIF to be an important contributor to meningitic E. coli-induced cytokine production and tight junction disruption, and also the NF-κB and MAPK signaling pathways that are mainly involved in the infection-induced cytokines production. Characterization of these distinct proteins and pathways in BMECs will facilitate further elucidation of meningitis-causing mechanisms in humans and animals, thereby enabling the development of novel preventative and therapeutic strategies against infection with meningitic E. coli.

Mesenchymal stem cells transplantation attenuates brain injury and enhances bacterial clearance in Escherichia coli meningitis in newborn rats.

OBJECTIVE: Neonatal meningitis caused by Escherichia coli results in significant mortality and neurological disabilities, with few effective treatments. Recently, we demonstrated that human umbilical cord blood-derived mesenchymal stem cell (hUCB-MSC) transplantation attenuated E. coli-induced severe pneumonia, primarily by reducing inflammation and enhancing bacterial clearance. This study aimed to determine whether intraventricular transplantation of hUCB-MSCs attenuated the brain injury in E. coli meningitis in newborn rats. METHODS: Meningitis without concomitant bacteremia was induced by intraventricular injection of 5 × 102 colony forming units of K1 (-) E. coli in rats at postnatal day (P)11, and hUCB-MSCs (1 × 105) were transplanted intraventricularly 6 h after induction of meningitis. Antibiotics was started 24 h after modeling. RESULT: Meningitis modeling induced robust proliferation of E. coli in the cerebrospinal fluid and increased mortality in rat pups, and MSC transplantation significantly reduced this bacterial growth and the mortality rate. Impaired sensorimotor function in the meningitis rats was ameliorated by MSCs injection. MSCs transplantation also attenuated meningitis caused brain injury including cerebral ventricular dilatation, brain cell death, reactive gliosis, and inflammatory response. CONCLUSION: Intraventricular transplantation of hUCB-MSCs significantly improved survival and attenuated the brain injury via anti-inflammatory and antibacterial effects in experimental neonatal E. coli meningitis.

Caspr1 is a host receptor for meningitis-causing Escherichia coli.

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis, but the pathogenesis of E. coli meningitis remains elusive. E. coli penetration of the blood-brain barrier (BBB) is the critical step for development of meningitis. Here, we identify Caspr1, a single-pass transmembrane protein, as a host receptor for E. coli virulence factor IbeA to facilitate BBB penetration. Genetic ablation of endothelial Caspr1 and blocking IbeA-Caspr1 interaction effectively prevent E. coli penetration into the brain during meningitis in rodents. IbeA interacts with extracellular domain of Caspr1 to activate focal adhesion kinase signaling causing E. coli internalization into the brain endothelial cells of BBB. E. coli can invade hippocampal neurons causing apoptosis dependent on IbeA-Caspr1 interaction. Our results indicate that E. coli exploits Caspr1 as a host receptor for penetration of BBB resulting in meningitis, and that Caspr1 might be a useful target for prevention or therapy of E. coli meningitis.

Temporal changes of oxidative stress markers in Escherichia coli K1-induced experimental meningitis in a neonatal rat model.

Despite advances in antimicrobial therapy and advanced critical care neonatal bacterial meningitis has a mortality rate of over 10% and induces neurological sequelae in 20-50% of cases. Escherichia coli K1 (E. coli K1) is the most common gram-negative organism causing neonatal meningitis and is the second most common cause behind group B streptococcus. We previously reported that an E. coli K1 experimental meningitis infection in neonatal rats resulted in habituation and aversive memory impairment and a significant increase in cytokine levels in adulthood. In this present study, we investigated the oxidative stress profile including malondialdehyde (MDA) levels, carbonyl protein formation, myeloperoxidase activity (MPO) activity, superoxide dismutase (SOD) activity and catalase (CAT) activity 6, 12, 24, 48, 72 and 96h after E. coli K1 experimental meningitis infection. In addition, sulfhydryl groups, nitrite and nitrate levels and activity of the mitochondrial respiratory chain enzymes were also measured in the frontal cortex and hippocampus of neonatal rats. The results from this study demonstrated a significant increase in MDA, protein carbonyls and MPO activity and a simultaneous decrease in SOD activity in the hippocampus of the neonatal meningitis survivors but the same was not observed in frontal cortex. In addition, we also observed a significant increase in complex IV activity in the hippocampus and frontal cortex of meningitis survivor rats. Thus, the results from this study reaffirmed the possible role of oxidative stress, nitric oxide and its related compounds in the complex pathophysiology of E. coli K1-induced bacterial meningitis.

IQGAP1 Mediates Hcp1-Promoted Escherichia coli Meningitis by Stimulating the MAPK Pathway.

Escherichia coli-induced meningitis remains a life-threatening disease despite recent advances in the field of antibiotics-based therapeutics, necessitating continued research on its pathogenesis. The current study aims to elucidate the mechanism through which hemolysin-coregulated protein 1 (Hcp1) induces the apoptosis of human brain microvascular endothelial cells (HBMEC). Co-immunoprecipitation coupled with mass spectrometric (MS) characterization led to the identification of IQ motif containing GTPase activating protein 1 (IQGAP1) as a downstream target of Hcp1. IQGAP1 was found to be up-regulated by Hcp1 treatment and mediate the stimulation of HBMEC apoptosis. It was shown that Hcp1 could compete against Smurf1 for binding to IQGAP1, thereby rescuing the latter from ubiquitin-dependent degradation. Subsequent study suggested that IQGAP1 could stimulate the MAPK signaling pathway by promoting the phosphorylation of ERK1/2, an effect that was blocked by U0126, an MAPK inhibitor. Furthermore, U0126 also demonstrated therapeutic potential against E. coli meningitis in a mouse model. Taken together, our results suggested the feasibility of targeting the MAPK pathway as a putative therapeutic strategy against bacterial meningitis.

Differential transcription profiles of long non-coding RNAs in primary human brain microvascular endothelial cells in response to meningitic Escherichia coli.

Accumulating studies have indicated the influence of long non-coding RNAs (lncRNAs) on various biological processes as well as disease development and progression. However, the lncRNAs involved in bacterial meningitis and their regulatory effects are largely unknown. By RNA-sequencing, the transcriptional profiles of host lncRNAs in primary human brain microvascular endothelial cells (hBMECs) in response to meningitic Escherichia coli were demonstrated. Here, 25,257 lncRNAs were identified, including 24,645 annotated lncRNAs and 612 newly found ones. A total of 895 lncRNAs exhibited significant differences upon infection, among which 382 were upregulated and 513 were downregulated (≥2-fold, p < 0.05). Via bioinformatic analysis, the features of these lncRNAs, their possible functions, and the potential regulatory relationships between lncRNAs and mRNAs were predicted. Moreover, we compared the transcriptional specificity of these differential lncRNAs among hBMECs, human astrocyte cell U251, and human umbilical vein endothelial cells, and demonstrated the novel regulatory effects of proinflammatory cytokines on these differential lncRNAs. To our knowledge, this is the first time the transcriptional profiles of host lncRNAs involved in E. coli-induced meningitis have been reported, which shall provide novel insight into the regulatory mechanisms behind bacterial meningitis involving lncRNAs, and contribute to better prevention and therapy of CNS infection.

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.

Neuropathogenic Escherichia coli K1 does not exhibit proteolytic activities to exert its pathogenicity.

BACKGROUND: Proteases are well-known virulence factors that promote survival, pathogenesis and immune evasion of many pathogens. Several lines of evidence suggest that the blood-brain barrier permeability is a prerequisite in microbial invasion of the central nervous system. Because proteases are frequently associated with vascular permeability by targeting junctional proteins, here it is hypothesized that neuropathogenic Escherichia coli K1 exhibit proteolytic activities to exert its pathogenicity. METHODS: Zymographic assays were performed using collagen and gelatin as substrates. The lysates of whole E. coli K1 strain E44, or E. coli K-12 strain HB101 were tested for proteolytic activities. The conditioned media were prepared by incubating bacteria in RPMI-1640 in the presence or absence of serum. The cell-free supernatants were collected and tested for proteases in zymography as mentioned above. Additionally, proteolytic degradation of host immune factors was determined by co-incubating conditioned media with albumin/immunoglobulins using protease assays. RESULTS: When collagen or gelatin were used as substrates in zymographic assays, neither whole bacteria nor conditioned media exhibited proteolytic activities. The conditioned media of neuropathogenic E. coli K1 strain E44, or E. coli K-12 strain HB101 did not affect degradation of albumin and immunoglobulins using protease assays. CONCLUSIONS: Neither zymographic assays nor protease assays detected proteolytic activities in either the whole bacteria or conditioned media of E. coli K1 strain E44 and E. coli K-12 strain HB101. These findings suggest that host cell monolayer disruptions and immune evasion strategies are likely independent of proteolytic activities of neuropathogenic E. coli K1.

Attenuation of biopterin synthesis prevents Escherichia coli K1 invasion of brain endothelial cells and the development of meningitis in newborn mice.

Elevated levels of pterins and nitric oxide (NO) are observed in patients with septic shock and bacterial meningitis. We demonstrate that Escherichia coli K1 infection of human brain microvascular endothelial cells (HBMECs) induces the expression of guanosine triphosphate cyclohydrolase (GCH1), the rate-limiting enzyme in pterin synthesis, thereby elevating levels of biopterin. DAHP (2,4-diamino hydroxyl pyrimidine), a specific inhibitor of GCH1, prevented biopterin and NO production and invasion of E. coli K1 in HBMECs. GCH1 interaction with Ecgp96, the receptor for outer membrane protein A of E. coli K1, also increases on infection, and suppression of Ecgp96 expression prevents GCH1 activation and biopterin synthesis. Pretreatment of newborn mice with DAHP prevented the production of biopterin and the development of meningitis. These results suggest a novel role for biopterin synthesis in the pathogenesis of E. coli K1 meningitis.

Involvement of IbeA in meningitic Escherichia coli K1-induced polymorphonuclear leukocyte transmigration across brain endothelial cells.

Transmigration of neutrophil [polymorphonuclear neutrophil (PMN)] across the blood-brain barrier (BBB) is a critical event in the pathogenesis of bacterial meningitis. We have shown that IbeA is able to induce meningitic Escherichia coli invasion of brain microvascular endothelial cells (BMECs), which constitutes the BBB. In this report, we provide evidence that IbeA and its receptor, vimentin, play a key role in E. coli-induced PMN transmigration across BMEC. In vitro and in vivo studies indicated that the ibeA-deletion mutant ZD1 was significantly less active in stimulating PMN transmigration than the parent strain E44. ZD1 was fully complemented by the ibeA gene and its product. E. coli-induced PMN transmigration was markedly inhibited by withaferin A, a dual inhibitor of vimentin and proteasome. These cellular effects were significantly stimulated and blocked by overexpression of vimentin and its head domain deletion mutant in human BMEC, respectively. Our studies further demonstrated that IbeA-induced PMN migration was blocked by bortezomib, a proteasomal inhibitor and correlated with upregulation of endothelial ICAM-1 and CD44 expression through proteasomal regulation of NFκB activity. Taken together, our data suggested that IbeA and vimentin contribute to E. coli K1-stimulated PMN transendothelial migration that is correlated with upregulation of adhesion molecule expression at the BBB.

Deciphering the roles of outer membrane protein A extracellular loops in the pathogenesis of Escherichia coli K1 meningitis.

Outer membrane protein A (OmpA) has been implicated as an important virulence factor in several gram-negative bacterial infections such as Escherichia coli K1, a leading cause of neonatal meningitis associated with significant mortality and morbidity. In this study, we generated E. coli K1 mutants that express OmpA in which three or four amino acids from various extracellular loops were changed to alanines, and we examined their ability to survive in several immune cells. We observed that loop regions 1 and 2 play an important role in the survival of E. coli K1 inside neutrophils and dendritic cells, and loop regions 1 and 3 are needed for survival in macrophages. Concomitantly, E. coli K1 mutants expressing loop 1 and 2 mutations were unable to cause meningitis in a newborn mouse model. Of note, mutations in loop 4 of OmpA enhance the severity of the pathogenesis by allowing the pathogen to survive better in circulation and to produce high bacteremia levels. These results demonstrate, for the first time, the roles played by different regions of extracellular loops of OmpA of E. coli K1 in the pathogenesis of meningitis and may help in designing effective preventive strategies against this deadly disease.

Fcγ receptor I alpha chain (CD64) expression in macrophages is critical for the onset of meningitis by Escherichia coli K1.

Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa(-/-) mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa(-/-) macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa(-/-) mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1.

Effects of dopamine infusion on cerebral blood flow, brain cell membrane function and energy metabolism in experimental Escherichia coli meningitis in the newborn piglet.

In the present study, we tested whether maintenance of adequate cerebral perfusion pressure (CPP) by pharmacologically preventing systemic hypotension with dopamine infusion would prevent cerebral ischemia and attenuate energy depletion and neuronal injury even though intracranial pressure remains elevated in a newborn piglet meningitis model. Cerebral blood flow, measured at the end of the experiment using fluorescent microspheres, was significantly increased by dopamine infusion. The decreased cerebral cortical cell membrane Na+, K+ -ATPase activity and increased lipid peroxidation products, indicative of meningitis-induced brain damage, were significantly attenuated by dopamine infusion. Dopamine also significantly attenuated the meningitis-induced reduction in both brain ATP and phosphocreatine levels and the increase in brain lactate level. In summary, maintenance of adequate CPP with dopamine prevented cerebral ischemia, reduced cerebral energy depletion, and attenuated brain injury in neonatal bacterial meningitis.

Effects of MK-801 (dizocilpine) on brain cell membrane function and energy metabolism in experimental Escherichia coli meningitis in the newborn piglet.

We evaluated the efficacy of non-competitive N-methyl-D-aspartate receptor antagonist MK-801 (dizocilpine) as an adjuvant therapy in experimental neonal bacterial meningitis. Meningitis was induced by injecting 10(6) colony forming units of Escherichia coli into the cisterna magna. MK-801 3 mg/kg was given as a bolus intravenous injection, 30 min before the induction of meningitis. MK-801 did not down-modulate the inflammatory parameters, such as increased intracranial pressure, cerebrospinal fluid (CSF) leukocytosis, increased lactate and TNF-alpha levels in the CSF, and hypoglycorrhachia observed in the meningitis group. MK-801 did not significantly attenuate the elevated glutamate concentration in the CSF. However, MK-801 showed some neuroprotective effects as evidenced by significant attenuation of cerebral lipid peroxidation products (conjugated dienes) and increase of brain high-energy phosphate compounds (ATP and PCr). Improvement in cerebral cortical cell membrane Na+, K+ -ATPase activity did not reach a statistical significance. These results suggest that MK-801 was effective in ameliorating brain injury in neonatal bacterial meningitis, although it failed to attenuate the inflammatory responses.