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.

Taste vs. pain: A sensory feast in bacterial meningitis.

During meningitis, sensory neurons detect bacterial toxins and metabolites. Early activation of pain receptors suppresses host defense in the meninges while, later, taste receptors amplify inflammation in the spinal cord. Is a neuroimmune axis a clue to new treatments?

An Integrative Approach to Investigate the Mode of Action of (-)-Dendroparishiol in Bacterial Meningitis: Computer-Aided Estimation of Biological Activity and Network Pharmacology.

Bacterial meningitis remains one of the most prevalent infectious diseases worldwide. Although advances in medical care have improved mortality and morbidity, neurological complications remain high. Therefore, aside from antibiotics, therapeutic adjuvants targeting neuroinflammation are essential to combat the long-term neuronal sequelae of bacterial meningitis. In the present study, we propose (-)-dendroparishiol as a potential add-on therapy to improve neuroinflammation associated with bacterial meningitis. The biological activity of (-)-dendroparishiol was first predicted by computational analysis and further confirmed in vitro using a cell-based assay with LPS-induced BV-2 microglial cells. Biological pathways involved with (-)-dendroparishiol were identified by applying network pharmacology. Computational predictions of biological activity indicated possible attenuation of several inflammatory processes by (-)-dendroparishiol. In LPS-induced BV-2 microglial cells, (-)-dendroparishiol significantly reduced the expression of inflammatory mediators: iNOS, NO, COX-2, IL-6, and TNF-α. Molecular docking results demonstrated the potential iNOS and COX-2 inhibitory activity of (-)-dendroparishiol. Network pharmacological analysis indicated the plausible role of (-)-dendroparishiol in biological processes involved in oxidative stress and neuroinflammation with enrichment in neuroinflammatory pathways. Overall, this study provides scientific evidence for the potential application of (-)-dendroparishiol in the management of bacterial meningitis-associated neuroinflammation.

Blood-Brain Barrier Integrity Damage in Bacterial Meningitis: The Underlying Link, Mechanisms, and Therapeutic Targets.

Despite advances in supportive care and antimicrobial treatment, bacterial meningitis remains the most serious infection of the central nervous system (CNS) that poses a serious risk to life. This clinical dilemma is largely due to our insufficient knowledge of the pathology behind this disease. By controlling the entry of molecules into the CNS microenvironment, the blood-brain barrier (BBB), a highly selective cellular monolayer that is specific to the CNS's microvasculature, regulates communication between the CNS and the rest of the body. A defining feature of the pathogenesis of bacterial meningitis is the increase in BBB permeability. So far, several contributing factors for BBB disruption have been reported, including direct cellular damage brought on by bacterial virulence factors, as well as host-specific proteins or inflammatory pathways being activated. Recent studies have demonstrated that targeting pathological factors contributing to enhanced BBB permeability is an effective therapeutic complement to antimicrobial therapy for treating bacterial meningitis. Hence, understanding how these meningitis-causing pathogens affect the BBB permeability will provide novel perspectives for investigating bacterial meningitis's pathogenesis, prevention, and therapies. Here, we summarized the recent research progress on meningitis-causing pathogens disrupting the barrier function of BBB. This review provides handy information on BBB disruption by meningitis-causing pathogens, and helps design future research as well as develop potential combination therapies.

The VraSR two-component signal transduction system contributes to the damage of blood-brain barrier during Streptococcus suis meningitis.

Streptococcus suis (S. suis) is an important zoonotic pathogen that can cause high morbidity and mortality in both humans and swine. As the most important life-threatening infection of the central nervous system (CNS), meningitis is an important syndrome of S. suis infection. The vancomycin resistance associated sensor/regulator (VraSR) is a critical two-component signal transduction system that affects the ability of S. suis to resist the host innate immune system and promotes its ability to adhere to brain microvascular endothelial cells (BMECs). Prior work also found mice infected with ΔvraSR had no obvious neurological symptoms, unlike mice infected with wild-type SC19. Whether and how VraSR participates in the development of S. suis meningitis remains unknown. Here, we found ΔvraSR-infected mice did not show obvious meningitis, compared with wild-type SC19-infected mice. Moreover, the proinflammatory cytokines and chemokines in serum and brains of ΔvraSR-infected mice, including IL-6, TNF-α, MCP-1 and IFN-γ, were significantly lower than wild-type infected group. Besides, blood-brain barrier (BBB) permeability also confirmed that the mutant had lower ability to disrupt BBB. Furthermore, in vivo and in vitro experiments showed that SC19 could increase BBB permeability by downregulating tight junction (TJ) proteins such as ZO-1, ß-Catenin, Occludin, and Clauidn-5, compared with mutant ΔvraSR. These findings provide new insight into the influence of S. suis VraSR on BBB disruption during the pathogenic process of streptococcal meningitis, thereby offering potential targets for future preventative and therapeutic strategies against this disease.

Targeting the BspC-vimentin interaction to develop anti-virulence therapies during Group B streptococcal meningitis.

Bacterial infections are a major cause of morbidity and mortality worldwide and the rise of antibiotic resistance necessitates development of alternative treatments. Pathogen adhesins that bind to host cells initiate disease pathogenesis and represent potential therapeutic targets. We have shown previously that the BspC adhesin in Group B Streptococcus (GBS), the leading cause of bacterial neonatal meningitis, interacts with host vimentin to promote attachment to brain endothelium and disease development. Here we determined that the BspC variable (V-) domain contains the vimentin binding site and promotes GBS adherence to brain endothelium. Site directed mutagenesis identified a binding pocket necessary for GBS host cell interaction and development of meningitis. Using a virtual structure-based drug screen we identified compounds that targeted the V-domain binding pocket, which blocked GBS adherence and entry into the brain in vivo. These data indicate the utility of targeting the pathogen-host interface to develop anti-virulence therapeutics.

Long non-coding RNA lncC11orf54-1 modulates neuroinflammatory responses by activating NF-κB signaling during meningitic Escherichia coli infection.

Escherichia coli is the most common gram-negative pathogenic bacterium causing meningitis. It penetrates the blood-brain barrier (BBB) and activates nuclear factor kappa B (NF-κB) signaling, which are vital events leading to the development of meningitis. Long non-coding RNAs (lncRNAs) have been implicated in regulating neuroinflammatory signaling, and our previous study showed that E. coli can induce differential expression of lncRNAs, including lncC11orf54-1, in human brain microvascular endothelial cells (hBMECs). The hBMECs constitute the structural and functional basis for the BBB, however, it is unclear whether lncRNAs are involved in the regulation of inflammatory responses of hBMECs during meningitic E. coli infection. In this study, we characterized an abundantly expressed lncRNA, lncC11orf54-1, which was degraded by translocated coilin to produce mgU2-19 and mgU2-30 in hBMECs during E. coli infection. Functionally, lncC11orf54-1-originated non-coding RNA mgU2-30 interacted with interleukin-1 receptor-associated kinase 1 (IRAK1) to induce its oligomerization and autophosphorylation, thus promoting the activation of NF-κB signaling and facilitating the production of pro-inflammatory cytokines. In summary, our study uncovers the involvement of lncC11orf54-1 in IRAK1-NF-κB signaling, and it functions as a positive regulator of inflammatory responses in meningitic E. coli-induced neuroinflammation, which may be a valuable therapeutic and diagnostic target for bacterial meningitis.

miR-141-3p inhibits the activation of astrocytes and the release of inflammatory cytokines in bacterial meningitis through down-regulating HMGB1.

BACKGROUND: Bacterial meningitis (BM) is a serious infectious disease of the central nervous system that often occurs in children and adolescents. Many studies have suggested that microRNAs (miRNAs) are involved in BM. This study aimed to address the effects of miR-141-3p on astrocyte activation and inflammatory response in BM through HMGB1. METHODS: The 3-week-old rats were injected with Streptococcus pneumoniae (SP) into the lateral ventricle to establish a BM model. Loeffler scoring method was used to evaluate the recovery of neurological function. Brain pathological damage was observed by hematoxylin and eosin (H&E) staining. Primary astrocytes were isolated from brain tissues of BM or non-infected SD rats. The levels of TNF-α, IL-1ß, and IL-6 in brain tissues and astrocyte culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). The targeting relationship between miR-141-3p and HMGB1 was tested using dual-luciferase reporter assay. The expression of miR-141-3p, HMGB1, and the astrocytic marker glial fibrillary acidic protein (GFAP) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blotting. Methylation-specific PCR (MSP) analysis was performed to measure the methylation status of miR-141 promoter. RESULTS: The results showed that lower Loeffler scores were exhibited in rats with BM. The subarachnoid space of brain tissues of BM rats was widened, and obvious inflammatory cells were observed. miR-141-3p expression was reduced in BM rats and SP-treated astrocytes. Additionally, we found that overexpression of miR-141-3p led to the downregulation of HMGB1, GFAP, and inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in astrocytes. Furthermore, the results of dual-luciferase reporter assay confirmed that miR-141-3p directly targeted HMGB1. Overexpression of miR-141-3p inhibited the levels of GFAP, TNF-α, IL-1ß, and IL-6 in astrocytes, which was eliminated by the up-regulation of HMGB1. The results of MSP analysis indicated that miR-141 promoter was highly methylated in brain tissues and astrocytes. DNMT1 was involved in the methylation of miR-141 promoter in BM. CONCLUSION: The present study verified that miR-141-3p affected inflammatory response by suppressing HMGB1 in SP-induced astrocytes and BM rat model.

Implication of long non-coding RNA NEAT1 in the pathogenesis of bacterial meningitis-induced blood-brain barrier damage.

PURPOSE: Blood-brain barrier (BBB) damage is closely related to various neurological disorders, including bacterial meningitis (BM). Determining a reliable strategy to prevent BBB damage in the context of infection would be highly desirable. In the present study, we investigated the implications of the long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) in moderating BBB damage. METHODS: In vitro BBB models were developed by co-culturing hCMEC/D3 cells with glioma cells, whereupon the glioma-exposed endothelial cells (GECs) were treated with a series of mimics, inhibitors, overexpression plasmids, and shRNAs for evaluating whether NEAT1, microRNA-135a (miR-135a) and hypoxia-inducible factor 1α (HIF1α) mediated BBB integrity and permeability. Furthermore, the in vivo biological function of NEAT1 was validated in a mouse model of BBB damage. RESULTS: NEAT1 and HIF1α were determined to be up-regulated, while miR-135a was under-expressed in GECs. As demonstrated by chromatin immunoprecipitation and dual-luciferase reporter assays, NEAT1 could bind to miR-135a, and HIF1α was confirmed as a target of miR-135a. Either overexpression of NEAT1 or depletion of miR-135a impaired the integrity and augmented the permeability of BBB. However, HIF1α silencing could reverse the BBB damage induced by NEAT1 overexpression or by inhibition of miR-135a. In vivo experiments substantiated that knockdown of NEAT1 could alleviate BBB damage in living mice. CONCLUSIONS: Hence, NEAT1 knockdown prevents BBB disruption and exerts promise as a potential target for BM treatment.

Combined testing of cerebrospinal fluid IL-12 (p40) and serum C-reactive protein as a possible discriminator of acute bacterial neuroinfections.

INTRODUCTION: Central nervous system infections (CNS) are life-threatening diseases, with meningitis being the most common. Viral infections are usually self-limiting diseases but bacterial pathogens are associated with higher mortality rates and persistent neurological sequelae. We aimed to study the role of IL-6, IL-8, IL-10, IL-12(p40), TNF-α cytokines, classical cerebrospinal fluid (CSF) parameters, and serum C-reactive protein levels (CRP) for discriminating bacterial from viral central nervous system infections. MATERIAL AND METHODS: This prospective study included 80 patients with clinical signs and abnormal cerebrospinal fluid laboratory findings typical for neuroinfection admitted to St. George University Hospital-Plovdiv. Routine methods such as direct microscopy, culturing and identification were used for microbiological analysis as well as latex-agglutination test and multiplex PCR. Cytokines' concentrations were measured by ELISA. CRP and CSF parameters were collected from the patients' medical records. RESULTS: We observed the highest discriminatory power among cytokines for cerebrospinal IL-12(p40) (AUC = 0.925; p = 0.000). CSF protein levels were the best predictor for bacterial neuroinfection (AUC = 0.973; p = 0.000). The AUC for the serum CRP as a stand-alone biomarker was estimated to be 0.943. The discriminatory power can be increased up to 0.995 (p = 0.000) when combining cerebrospinal fluid IL-12(p40) and serum CRP, with an optimal cut-off value of 144 (Sensitivity 100%; Specificity 90.9%). CONCLUSION: The combined testing of CSF IL-12(p40) and serum CRP is associated with the highest diagnostic accuracy.

CC17 group B Streptococcus exploits integrins for neonatal meningitis development.

Group B Streptococcus (GBS) is the major cause of human neonatal infections. A single clone, designated CC17-GBS, accounts for more than 80% of meningitis cases, the most severe form of the infection. However, the events allowing blood-borne GBS to penetrate the brain remain largely elusive. In this study, we identified the host transmembrane receptors α5ß1 and αvß3 integrins as the ligands of Srr2, a major CC17-GBS-specific adhesin. Two motifs located in the binding region of Srr2 were responsible for the interaction between CC17-GBS and these integrins. We demonstrated in a blood-brain-barrier cellular model that both integrins contributed to the adhesion and internalization of CC17-GBS. Strikingly, both integrins were overexpressed during the postnatal period in the brain vessels of the blood-brain barrier and blood-cerebrospinal fluid barrier and contributed to juvenile susceptibility to CC17 meningitis. Finally, blocking these integrins decreased the ability of CC17-GBS to cross into the CNS of juvenile mice in an in vivo model of meningitis. Our study demonstrated that CC17-GBS exploits integrins in order to cross the brain vessels, leading to meningitis. Importantly, it provides host molecular insights into neonate's susceptibility to CC17-GBS meningitis, thereby opening new perspectives for therapeutic and prevention strategies of GBS-elicited meningitis.

Individual responsiveness of macrophage migration inhibitory factor predicts long-term cognitive impairment after bacterial meningitis.

BACKGROUND: Patients with pneumococcal meningitis are at risk for death and neurological sequelae including cognitive impairment. Functional genetic polymorphisms of macrophage migration inhibitory factor (MIF) alleles have shown to predict mortality of pneumococcal meningitis. METHODS: We investigated whether MIF concentrations during the acute phase of disease were predictive for death in a nationwide prospective cohort study. Subsequently, we studied whether individual ex vivo MIF response years after meningitis was associated with the development of cognitive impairment. RESULTS: We found that in the acute illness of pneumococcal meningitis, higher plasma MIF concentrations were predictive for mortality (p = 0.009). Cognitive impairment, examined 1-5 years after meningitis, was present in 11 of 79 patients after pneumococcal meningitis (14%), as compared to 1 of 63 (2%) in controls, and was consistently associated with individual variability in MIF production by peripheral blood mononuclear cells after ex vivo stimulation with various infectious stimuli. CONCLUSIONS: Our study confirms the role of MIF in poor disease outcome of pneumococcal meningitis. Inter-individual differences in MIF production were associated with long-term cognitive impairment years after pneumococcal meningitis. The present study provides evidence that MIF mediates long-term cognitive impairment in bacterial meningitis survivors and suggests a potential role for MIF as a target of immune-modulating adjunctive therapy.

Identification of Zinc-Dependent Mechanisms Used by Group B Streptococcus To Overcome Calprotectin-Mediated Stress.

Nutritional immunity is an elegant host mechanism used to starve invading pathogens of necessary nutrient metals. Calprotectin, a metal-binding protein, is produced abundantly by neutrophils and is found in high concentrations within inflammatory sites during infection. Group B Streptococcus (GBS) colonizes the gastrointestinal and female reproductive tracts and is commonly associated with severe invasive infections in newborns such as pneumonia, sepsis, and meningitis. Although GBS infections induce robust neutrophil recruitment and inflammation, the dynamics of GBS and calprotectin interactions remain unknown. Here, we demonstrate that disease and colonizing isolate strains exhibit susceptibility to metal starvation by calprotectin. We constructed a mariner transposon (Krmit) mutant library in GBS and identified 258 genes that contribute to surviving calprotectin stress. Nearly 20% of all underrepresented mutants following treatment with calprotectin are predicted metal transporters, including known zinc systems. As calprotectin binds zinc with picomolar affinity, we investigated the contribution of GBS zinc uptake to overcoming calprotectin-imposed starvation. Quantitative reverse transcriptase PCR (qRT-PCR) revealed a significant upregulation of genes encoding zinc-binding proteins, adcA, adcAII, and lmb, following calprotectin exposure, while growth in calprotectin revealed a significant defect for a global zinc acquisition mutant (ΔadcAΔadcAIIΔlmb) compared to growth of the GBS wild-type (WT) strain. Furthermore, mice challenged with the ΔadcAΔadcAIIΔlmb mutant exhibited decreased mortality and significantly reduced bacterial burden in the brain compared to mice infected with WT GBS; this difference was abrogated in calprotectin knockout mice. Collectively, these data suggest that GBS zinc transport machinery is important for combatting zinc chelation by calprotectin and establishing invasive disease.IMPORTANCE Group B Streptococcus (GBS) asymptomatically colonizes the female reproductive tract but is a common causative agent of meningitis. GBS meningitis is characterized by extensive infiltration of neutrophils carrying high concentrations of calprotectin, a metal chelator. To persist within inflammatory sites and cause invasive disease, GBS must circumvent host starvation attempts. Here, we identified global requirements for GBS survival during calprotectin challenge, including known and putative systems involved in metal ion transport. We characterized the role of zinc import in tolerating calprotectin stress in vitro and in a mouse model of infection. We observed that a global zinc uptake mutant was less virulent than the parental GBS strain and found calprotectin knockout mice to be equally susceptible to infection by wild-type (WT) and mutant strains. These findings suggest that calprotectin production at the site of infection results in a zinc-limited environment and reveals the importance of GBS metal homeostasis to invasive disease.

Poldip2 mediates blood-brain barrier disruption and cerebral edema by inducing AQP4 polarity loss in mouse bacterial meningitis model.

BACKGROUND: Specific highly polarized aquaporin-4 (AQP4) expression is reported to play a crucial role in blood-brain barrier (BBB) integrity and brain water transport balance. The upregulation of polymerase δ-interacting protein 2 (Poldip2) was involved in aggravating BBB disruption following ischemic stroke. This study aimed to investigate whether Poldip2-mediated BBB disruption and cerebral edema formation in mouse bacterial meningitis (BM) model occur via induction of AQP4 polarity loss. METHODS AND RESULTS: Mouse BM model was induced by injecting mice with group B hemolytic streptococci via posterior cistern. Recombinant human Poldip2 (rh-Poldip2) was administered intranasally at 1 hour after BM induction. Small interfering ribonucleic acid (siRNA) targeting Poldip2 was administered by intracerebroventricular (i.c.v) injection at 48 hours before BM induction. A specific inhibitor of matrix metalloproteinases (MMPs), UK383367, was administered intravenously at 0.5 hour before BM induction. Western blotting, immunofluorescence staining, quantitative real-time PCR, neurobehavioral test, brain water content test, Evans blue (EB) permeability assay, transmission electron microscopy (TEM), and gelatin zymography were carried out. The results showed that Poldip2 was upregulated and AQP4 polarity was lost in mouse BM model. Both Poldip2 siRNA and UK383367 improved neurobehavioral outcomes, alleviated brain edema, preserved the integrity of BBB, and relieved the loss of AQP4 polarity in BM model. Rh-Poldip2 upregulated the expression of MMPs and glial fibrillary acidic protein (GFAP) and downregulated the expression of ß-dystroglycan (ß-DG), zonula occludens-1 (ZO-1), occludin, and claudin-5; whereas Poldip2 siRNA downregulated the expression of MMPs and GFAP, and upregulated ß-DG, ZO-1, occludin, and claudin-5. Similarly, UK383367 downregulated the expression of GFAP and upregulated the expression of ß-DG, ZO-1, occludin, and claudin-5. CONCLUSION: Poldip2 inhibition alleviated brain edema and preserved the integrity of BBB partially by relieving the loss of AQP4 polarity via MMPs/ß-DG pathway.

Purinergic signaling in infectious diseases of the central nervous system.

The incidence of infectious diseases affecting the central nervous system (CNS) has been increasing over the last several years. Among the reasons for the expansion of these diseases and the appearance of new neuropathogens are globalization, global warming, and the increased proximity between humans and wild animals due to human activities such as deforestation. Neurotropism affecting normal brain function is shared by organisms such as viruses, bacteria, fungi, and parasites. Neuroinfections caused by these agents activate immune responses, inducing neuroinflammation, excitotoxicity, and neurodegeneration. Purinergic signaling is an evolutionarily conserved signaling pathway associated with these neuropathologies. During neuroinfections, host cells release ATP as an extracellular danger signal with pro-inflammatory activities. ATP is metabolized to its derivatives by ectonucleotidases such as CD39 and CD73; ATP and its metabolites modulate neuronal and immune mechanisms through P1 and P2 purinergic receptors that are involved in pathophysiological mechanisms of neuroinfections. In this review we discuss the beneficial or deleterious effects of various components of the purinergic signaling pathway in infectious diseases that affect the CNS, including human immunodeficiency virus (HIV-1) infection, herpes simplex virus type 1 (HSV-1) infection, bacterial meningitis, sepsis, cryptococcosis, toxoplasmosis, and malaria. We also provide a description of this signaling pathway in emerging viral infections with neurological implications such as Zika and SARS-CoV-2.

Bacteriophage K1F targets Escherichia coli K1 in cerebral endothelial cells and influences the barrier function.

Bacterial neonatal meningitis results in high mortality and morbidity rates for those affected. Although improvements in diagnosis and treatment have led to a decline in mortality rates, morbidity rates have remained relatively unchanged. Bacterial resistance to antibiotics in this clinical setting further underlines the need for developing other technologies, such as phage therapy. We exploited an in vitro phage therapy model for studying bacterial neonatal meningitis based on Escherichia coli (E. coli) EV36, bacteriophage (phage) K1F and human cerebral microvascular endothelial cells (hCMECs). We show that phage K1F is phagocytosed and degraded by constitutive- and PAMP-dependent LC3-assisted phagocytosis and does not induce expression of inflammatory cytokines TNFα, IL-6, IL-8 or IFNß. Additionally, we observed that phage K1F temporarily decreases the barrier resistance of hCMEC cultures, a property that influences the barrier permeability, which could facilitate the transition of immune cells across the endothelial vessel in vivo. Collectively, we demonstrate that phage K1F can infect intracellular E. coli EV36 within hCMECs without themselves eliciting an inflammatory or defensive response. This study illustrates the potential of phage therapy targeting infections such as bacterial neonatal meningitis and is an important step for the continued development of phage therapy targeting antibiotic-resistant bacterial infections generally.

Derivation of a metabolic signature associated with bacterial meningitis in infants.

BACKGROUND: Diagnosis of bacterial meningitis (BM) is challenging in newborn infants. Presently, biomarkers of BM have limited diagnostic accuracy. Analysis of cerebrospinal fluid (CSF) metabolites may be a useful diagnostic tool in BM. METHODS: In a nested case-control study, we examined >400 metabolites in CSF of uninfected infants and infants with culture-confirmed BM using gas and liquid chromatography mass spectrometry. Preterm and full-term infants in a Level III or IV Neonatal Intensive Care Unit were prospectively enrolled when evaluated for serious bacterial infection. RESULTS: Over 200 CSF metabolites significantly differed in uninfected infants and infants with BM. Using machine learning, we found that as few as 6 metabolites distinguished infants with BM from uninfected infants in this pilot cohort. Further analysis demonstrated three metabolites associated with Group B Streptococcal meningitis. CONCLUSIONS: We report the first comprehensive metabolic analysis of CSF in infants with BM. In our pilot cohort, we derived a metabolic signature that predicted the presence or absence of BM, irrespective of gestational age, postnatal age, sex, race and ethnicity, presence of neurosurgical hardware, white blood cell count in CSF, and red blood cell contamination in CSF. Metabolic analysis may aid diagnosis of BM and facilitate clinical decision-making in infants. IMPACT: In a pilot cohort, metabolites in cerebrospinal fluid distinguished infants with bacterial meningitis from uninfected infants.We report the first comprehensive metabolic analysis of cerebrospinal fluid in infants with bacterial meningitis.Our findings may be used to improve diagnosis of bacterial meningitis and to offer mechanistic insights into the pathophysiology of bacterial meningitis in infants.

Virulence Factors of Meningitis-Causing Bacteria: Enabling Brain Entry across the Blood-Brain Barrier.

Infections of the central nervous system (CNS) are still a major cause of morbidity and mortality worldwide. Traversal of the barriers protecting the brain by pathogens is a prerequisite for the development of meningitis. Bacteria have developed a variety of different strategies to cross these barriers and reach the CNS. To this end, they use a variety of different virulence factors that enable them to attach to and traverse these barriers. These virulence factors mediate adhesion to and invasion into host cells, intracellular survival, induction of host cell signaling and inflammatory response, and affect barrier function. While some of these mechanisms differ, others are shared by multiple pathogens. Further understanding of these processes, with special emphasis on the difference between the blood-brain barrier and the blood-cerebrospinal fluid barrier, as well as virulence factors used by the pathogens, is still needed.

The Group B Streptococcal surface antigen I/II protein, BspC, interacts with host vimentin to promote adherence to brain endothelium and inflammation during the pathogenesis of meningitis.

Streptococcus agalactiae (Group B Streptococcus, GBS) normally colonizes healthy adults but can cause invasive disease, such as meningitis, in the newborn. To gain access to the central nervous system, GBS must interact with and penetrate brain or meningeal blood vessels; however, the exact mechanisms are still being elucidated. Here, we investigate the contribution of BspC, an antigen I/II family adhesin, to the pathogenesis of GBS meningitis. Disruption of the bspC gene reduced GBS adherence to human cerebral microvascular endothelial cells (hCMEC), while heterologous expression of BspC in non-adherent Lactococcus lactis conferred bacterial attachment. In a murine model of hematogenous meningitis, mice infected with ΔbspC mutants exhibited lower mortality as well as decreased brain bacterial counts and inflammatory infiltrate compared to mice infected with WT GBS strains. Further, BspC was both necessary and sufficient to induce neutrophil chemokine expression. We determined that BspC interacts with the host cytoskeleton component vimentin and confirmed this interaction using a bacterial two-hybrid assay, microscale thermophoresis, immunofluorescent staining, and imaging flow cytometry. Vimentin null mice were protected from WT GBS infection and also exhibited less inflammatory cytokine production in brain tissue. These results suggest that BspC and the vimentin interaction is critical for the pathogenesis of GBS meningitis.