Central Nervous System-Infecting Pathogens Escherichia coli and Cryptococcus neoformans Exploit the Host Pdlim2 for Intracellular Traversal and Exocytosis in the Blood-Brain Barrier.

Microbial penetration of the blood-brain barrier, a prerequisite for the development of central nervous system (CNS) infection, involves microbial invasion, intracellular traversal, and exocytosis. Microbial invasion of the blood-brain barrier has been investigated, but the molecular basis for microbial traversal and exit from the blood-brain barrier remains unknown. We performed transcriptome analysis of human brain microvascular endothelial cells (HBMEC) infected with Escherichia coli and Cryptococcus neoformans, representative bacterial and fungal pathogens common in CNS infections. Among the targets upregulated in response to E. coli and C. neoformans infection, PDLIM2 was knocked down by small hairpin RNA (shRNA) in HBMEC for further investigation. We demonstrated that Pdlim2 specifically regulated microbial traversal and exit from HBMEC by assessing microbial invasion, transcytosis, intracellular multiplication, and egression. Additionally, the defective exocytosis of internalized E. coli cells from the PDLIM2 shRNA knockdown cells was restored by treatment with a calcium ionophore (ionomycin). Moreover, we performed proximity-dependent biotin labeling with the biotin ligase BioID2 and identified 210 potential Pdlim2 interactors. Among the nine Pdlim2 interactors enriched in response to both E. coli and C. neoformans infection, we selected MPRIP and showed that HBMEC with knockdown of MPRIP mimicked the phenotype of PDLIM2 knockdown cells. These results suggest that the CNS-infecting microbes hijack Pdlim2 and Mprip for intracellular traversal and exocytosis in the blood-brain barrier.

A prospective cohort study on the intersectionality of obesity, chronic disease, social factors, and incident risk of COVID-19 in US low-income minority middle-age mothers.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) has disproportionally affected communities of color. We aimed to determine what factors are associated with COVID-19 testing and test positivity in an underrepresented, understudied, and underreported (U3) population of mothers. METHODS: This study included 2996 middle-aged mothers of the Boston Birth Cohort (a sample of predominantly urban, low-income, Black and Hispanic mothers) who were enrolled shortly after they gave birth and followed onward at the Boston Medical Center. COVID-19 testing and test positivity were defined by the SARS-CoV-2 nucleic acid test. Two-probit Heckman selection models were performed to identify factors associated with test positivity while accounting for potential selection associated with COVID testing. RESULTS: The mean (SD) age of study mothers was 41.9 (±7.7) years. In the sample, 1741 (58.1%) and 667 (22.3%) mothers were self-identified as Black and Hispanic, respectively. A total of 396 mothers had COVID-19 testing and of those, 95 mothers tested positive from March 2020 to February 2021. Among a multitude of factors examined, factors associated with the probability of being tested were obesity (RR = 1.27; 95% confidence interval (CI): 1.08-1.49); and presence of preexisting chronic medical conditions including hypertension, asthma, stroke, and other comorbidities (coronary heart disease, chronic kidney disease, and sickle cell disease) with a corresponding RR = 1.40 (95% CI: 1.23-1.60); 1.29 (95% CI: 1.11-1.50); 1.44 (95% CI: 1.23-1.68); and 1.37 (95% CI: 1.12-1.67), respectively. Factors associated with higher incident risk of a positive COVID-19 test were body mass index, birthplace outside of the USA, and being without a college-level education. CONCLUSIONS: This study demonstrated the intersectionality of obesity and social factors in modulating incident risk of COVID-19 in this sample of US Black and Hispanic middle-aged mothers. Methodologically, our findings underscore the importance of accounting for potential selection bias in COVID-19 testing in order to obtain unbiased estimates of COVID-19 infection.

Targeting E. coli invasion of the blood-brain barrier for investigating the pathogenesis and therapeutic development of E. coli meningitis.

Escherichia coli is the most common Gram-negative bacillary organism causing neonatal meningitis. Escherichia coli meningitis remains an important cause of mortality and morbidity, but the pathogenesis of E. coli penetration of the blood-brain barrier remains incompletely understood. Escherichia coli entry into the brain occurs in the meningeal and cortex capillaries, not in the choroid plexus, and exploits epidermal growth factor receptor (EGFR) and cysteinyl leukotrienes (CysLTs) for invasion of the blood-brain barrier. The present study examined whether EGFR and CysLTs are inter-related in their contribution to E. coli invasion of the blood-brain barrier and whether counteracting EGFR and CysLTs is a beneficial adjunct to antibiotic therapy of E. coli meningitis. We showed that (a) meningitis isolates of E. coli exploit EGFR and CysLTs for invasion of the blood-brain barrier, (b) the contribution of EGFR is upstream of that of CysLTs, and (c) counteracting EGFR and CysLTs as an adjunctive therapy improved the outcome (survival, neuronal injury and memory impairment) of animals with E. coli meningitis. These findings suggest that investigation of host factors contributing to E. coli invasion of the blood-brain barrier will help in enhancing the pathogenesis and development of new therapeutic targets for E. coli meningitis in the era of increasing resistance to conventional antibiotics.

Heterogeneity in pneumolysin expression governs the fate of Streptococcus pneumoniae during blood-brain barrier trafficking.

Outcome of host-pathogen encounter is determined by the complex interplay between protective bacterial and host defense strategies. This complexity further amplifies with the existence of cell-to-cell phenotypic heterogeneity in pathogens which remains largely unexplored. In this study, we illustrated that heterogeneous expression of pneumolysin (Ply), a pore-forming toxin of the meningeal pathogen, S. pneumoniae (SPN) gives rise to stochastically different bacterial subpopulations with variable fate during passage across blood-brain barrier (BBB). We demonstrate that Ply mediated damage to pneumococcus containing vacuolar (PCV) membrane leads to recruitment of cytosolic "eat-me" signals, galectin-8 and ubiquitin, targeting SPN for autophagic clearance. However, a majority of high Ply producing subset extensively damages autophagosomes leading to pneumococcal escape into cytosol and efficient clearance by host ubiquitination machinery. Interestingly, a low Ply producing subset halts autophagosomal maturation and evades all intracellular defense mechanisms, promoting its prolonged survival and successful transcytosis across BBB, both in vitro and in vivo. Ply therefore acts as both, sword and shield implying that its smart regulation ensures optimal disease manifestation. Our elucidation of heterogeneity in Ply expression leading to disparate infection outcomes attempts to resolve the dubious role of Ply in pneumococcal pathogenesis.

Effect of catechins and high-temperature-processed green tea extract on scavenging reactive oxygen species and preventing Aß1-42 fibrils' formation in brain microvascular endothelium.

The present study investigated the effect of high-temperature-processed green tea extract (HTP_GTE) and its bioactive components on the reduction of reactive oxygen species (ROS) and amyloid-beta (Aß) protein in human microvascular endothelial cells. Compared to Aß1-42-only treatment, pretreatment of HTP_GTE was revealed to effectively inhibit ROS generation (P<0.05). HTP_GTE and catechins not only inhibit Aß1-42 fibril formation but also destabilize preformed Aß1-42 fibrils. The presence of HTP_GTE, Aß1-42 fibril formation was significantly inhibited in a dose-dependent manner at 12.5-100 µg/ml of HTP_GTE, showing 86-56%, respectively. Treatment of various concentrations of HTP_GTE and catechins steadily destabilized the preformed Aß1-42 fibrils for 24 h in a dose-dependent manner. It was observed that the gallated groups such as epigallocatechin gallate, epicatechin gallate, gallocatechin gallate, and catechin gallate more effectively disturbed Aß1-42 fibril formation and destabilized the preformed Aß1-42 fibrils than the non-gallated group. Taken together, these findings supported that sterilized green tea could be promising natural anti-amyloidogenic agents associated with therapeutic approaches in Alzheimer's disease by scavenging ROS generation and Aß fibril in the brain tissue.

Meningitic Escherichia coli-induced upregulation of PDGF-B and ICAM-1 aggravates blood-brain barrier disruption and neuroinflammatory response.

BACKGROUND: Blood-brain barrier (BBB) disruption and neuroinflammation are considered key mechanisms of pathogenic Escherichia coli invasion of the brain. However, the specific molecules involved in meningitic E. coli-induced BBB breakdown and neuroinflammatory response remain unclear. Our previous RNA-sequencing data from human brain microvascular endothelial cells (hBMECs) revealed two important host factors: platelet-derived growth factor-B (PDGF-B) and intercellular adhesion molecule-1 (ICAM-1), which were significantly upregulated in hBMECs after meningitic E. coli infection. Whether and how PDGF-B and ICAM-1 contribute to the development of E. coli meningitis are still unclear. METHODS: The western blot, real-time PCR, enzyme-linked immunosorbent assay, immunohistochemistry, and immunofluorescence were applied to verify the significant induction of PDGF-B and ICAM-1 by meningitic E. coli in vivo and in vitro. Evan's blue assay and electric cell-substrate impedance sensing assay were combined to identify the effects of PDGF-B on BBB permeability. The CRISPR/Cas9 technology, cell-cell adhesion assay, and electrochemiluminescence assay were used to investigate the role of ICAM-1 in neuroinflammation subversion. RESULTS: We verified the significant induction of PDGF-B and ICAM-1 by meningitic E. coli in mouse as well as monolayer hBMECs models. Functionally, we showed that the increase of PDGF-B may directly enhance the BBB permeability by decreasing the expression of tight junction proteins, and the upregulation of ICAM-1 contributed to neutrophils or monocytes recruitment as well as neuroinflammation subversion in response to meningitic E. coli infection. CONCLUSIONS: Our findings demonstrated the roles of PDGF-B and ICAM-1 in mediating bacterial-induced BBB damage as well as neuroinflammation, providing new concepts and potential targets for future prevention and treatment of bacterial meningitis.

Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury.

Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors, we provide new insights into the pathogenesis of EHEC O157 infections. Our data have implications for considering O157 OMVs as vaccine candidates.

IL-1ß promotes transendothelial migration of PBMCs by upregulation of the FN/α5ß1 signalling pathway in immortalised human brain microvascular endothelial cells.

Neuroinflammation is often associated with pathological changes in the function of the blood-brain barrier (BBB) caused by disassembly of tight and adherens junctions that under physiological conditions are important for the maintenance of the BBB integrity. Consequently, in inflammation the BBB becomes dysfunctional, facilitating leukocyte traversal of the barrier and accumulation of immune cells within the brain. The extracellular matrix (ECM) also contributes to BBB integrity but the significance of the main ECM receptors, the ß1 integrins also expressed on endothelial cells, is less well understood. To evaluate whether ß1 integrin function is affected during inflammation and impacts barrier function, we used a transformed human brain microvascular endothelial cell (THBMEC)-based Interleukin 1ß (IL-1ß)-induced inflammatory in vitro BBB model. We demonstrate that IL-1ß increases cell-matrix adhesion and induces a redistribution of active ß1 integrins to the basal surface. In particular, binding of α5ß1 integrin to its ligand fibronectin is enhanced and α5ß1 integrin-dependent signalling is upregulated. Additionally, localisation of the tight junction protein claudin-5 is altered. Blockade of the α5ß1 integrin reduces the IL-1ß-induced transendothelial migration of peripheral blood mononuclear cells (PBMCs). These data imply that IL-1ß-induced inflammation not only destabilizes tight junctions but also increases α5ß1 integrin-dependent cell-matrix adhesion to fibronectin.

High-Throughput Chip Assay for Investigating Escherichia coli Interaction with the Blood-Brain Barrier Using Microbial and Human Proteome Microarrays (Dual-Microarray Technology).

Bacterial meningitis in neonates and infants is an acute lethal disease and occurs in response to microbial exploitation of the blood-brain barrier (BBB), resulting in the intracranial inflammation. Several pathogens, such as Escherichia coli ( E. coli), can cause this devastating disease; however, the underlying molecular mechanisms by which these pathogens exploit the BBB remain incompletely understood. To identify important players on both the pathogen and host sides that govern the E. coli-BBB cell interactions, we took advantage of the E. coli and human proteome microarrays (i.e., HuProt) as an unbiased, proteome-wide tool for identification of important players on both sides. Using the E. coli proteome microarrays, we developed a unique high throughput chip-based cell probing assay to probe with fluorescent live human brain microvascular endothelial cells (HBMEC, which constitute the BBB). We identified several transmembrane proteins, which effectively bound to live HBMEC. We focused on YojI protein for further study. By probing the HuProt arrays with YojI, interferon-alpha receptor (IFNAR2) was identified as one of its binding proteins. The importance of YojI and IFNAR2 involved in E. coli-HBMEC interactions was characterized using the YojI knockout bacteria and IFNAR2-knock down HBMEC and further confirmed by E. coli binding assay in HBMEC. This study represents a new paradigm (dual-microarray technology) that enables rapid, unbiased discovery of both pathogen and host players that are involved in pathogen-host interactions for human infectious diseases in a high throughput manner.

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.

Glial Cell-Elicited Activation of Brain Microvasculature in Response to Brucella abortus Infection Requires ASC Inflammasome-Dependent IL-1ß Production.

Blood-brain barrier activation and/or dysfunction are a common feature of human neurobrucellosis, but the underlying pathogenic mechanisms are largely unknown. In this article, we describe an immune mechanism for inflammatory activation of human brain microvascular endothelial cells (HBMEC) in response to infection with Brucella abortus Infection of HBMEC with B. abortus induced the secretion of IL-6, IL-8, and MCP-1, and the upregulation of CD54 (ICAM-1), consistent with a state of activation. Culture supernatants (CS) from glial cells (astrocytes and microglia) infected with B. abortus also induced activation of HBMEC, but to a greater extent. Although B. abortus-infected glial cells secreted IL-1ß and TNF-α, activation of HBMEC was dependent on IL-1ß because CS from B. abortus-infected astrocytes and microglia deficient in caspase-1 and apoptosis-associated speck-like protein containing a CARD failed to induce HBMEC activation. Consistently, treatment of CS with neutralizing anti-IL-1ß inhibited HBMEC activation. Both absent in melanoma 2 and Nod-like receptor containing a pyrin domain 3 are partially required for caspase-1 activation and IL-1ß secretion, suggesting that multiple apoptosis-associated speck-like protein containing CARD-dependent inflammasomes contribute to IL-1ß-induced activation of the brain microvasculature. Inflammasome-mediated IL-1ß secretion in glial cells depends on TLR2 and MyD88 adapter-like/TIRAP. Finally, neutrophil and monocyte migration across HBMEC monolayers was increased by CS from Brucella-infected glial cells in an IL-1ß-dependent fashion, and the infiltration of neutrophils into the brain parenchyma upon intracranial injection of B. abortus was diminished in the absence of Nod-like receptor containing a pyrin domain 3 and absent in melanoma 2. Our results indicate that innate immunity of the CNS set in motion by B. abortus contributes to the activation of the blood-brain barrier in neurobrucellosis and IL-1ß mediates this phenomenon.

Erythropoietin-mediated activation of aquaporin-4 channel for the treatment of experimental hydrocephalus.

OBJECTIVE: In this study, we investigate a neuroprotective agent, erythropoietin (EPO), in animal hydrocephalus model and its potential reversal effects on hydrocephalus by altering the expression of aquaporin-4 (AQP4). METHODS: Obstructive hydrocephalus was induced in 2-week-old rat pups by injecting kaolin (50 µl, 10 mg/ml in saline) into the cisterna magna, while the control pups received only saline. Kaolin-injected pups were divided into two groups on the fifth day after kaolin injection; one group received intra-peritoneal (i.p.) EPO (1 µg/pup) for 5 consecutive days, while other group received i.p. saline for 5 days. The effects of EPO on hydrocephalus were investigated by studying cerebral ventricle size and structural ependymal changes. We examined also the EPO effects on AQP4 expression and microRNA expression. RESULTS: EPO treatment significantly reduced dilation of the cerebral ventricle and denudation of ependymal line in hydrocephalic pups comparing with the control group. Increased expression of AQP4 in periventricular ependymal lining and cultured astrocytes and increased vascular formation were noted after EPO treatment. Additionally, we identified miR-668 as an endogenous regulator of AQP4 in response to EPO. Anti-miR-668 dampened EPO-induced activation of AQP4 expression. CONCLUSIONS: Together, our results show that EPO-mediated upregulation of AQP4 significantly reduces dilation of the cerebral ventricles in obstructive hydrocephalus pups and may lead to potential therapeutic options for hydrocephalus.

Cytotoxic Necrotizing Factor-1 (CNF1) does not promote E. coli infection in a murine model of ascending pyelonephritis.

BACKGROUND: Urinary tract infections (UTI) are among the most common and costly infections in both hospitalized and ambulatory patients. Uropathogenic E. coli (UPEC) represent the majority of UTI isolates and are a diverse group of bacteria that utilize a variety of virulence factors to establish infection of the genitourinary tract. The virulence factor cytotoxic necrotizing factor-1 (CNF1) is frequently expressed in clinical UPEC isolates. To date, there have been conflicting reports on the role of CNF1 in the pathogenesis of E. coli urinary tract infections. RESULTS: We examined the importance of CNF1 in a murine ascending kidney infection/ pyelonephritis model by performing comparative studies between a clinical UPEC isolate strain and a CNF1-deletion mutant. We found no alterations in bacterial burden with the loss of CNF1, whereas loss of the virulence factor fimH decreased bacterial burdens. In addition, we found no evidence that CNF1 contributed to the recruitment of inflammatory infiltrates in the kidney or bladder in vivo. CONCLUSIONS: While further examination of CNF-1 may reveal a role in UTI pathogenesis, our data casts doubt on the role of CNF-1 in the pathogenesis of UPEC UTI. As with other infections, different models and approaches are needed to elucidate the contribution of CNF1 to E. coli UTI.

Impact of commercial cigarette smoke condensate on brain tissue co-cultured with astrocytes and blood-brain barrier endothelial cells.

The purpose of the current study was to investigate the effect of two commercial cigarette smoke condensates (CCSC) on oxidative stress and cell cytotoxicity in human brain (T98G) or astrocytes (U-373 MG) in the presence of human brain microvascular endothelial cells (HBMEC). Cell viability of mono-culture of T98G or U-373 MG was markedly decreased in a concentration-dependent manner, and T98G was more susceptible than U-373 MG to CCSC exposure. Cytotoxicity was less prominent when T98G was co-cultured with HBMEC than when T98G was co-cultured with U-373 MG. Significant reduction in trans-epithelial electric resistance (TEER), a biomarker of cellular integrity was noted in HBMEC co-cultured with T98G (HBMEC-T98G co-culture) and U-373 MG co-cultured with T98G (U-373 MG-T98G co-culture) after 24 or 48 hr CCSC exposure, respectively. TEER value of U-373 MG co-cultured with T98G (79-84%) was higher than HBMEC co-cultured with T98G (62-63%) within 120-hr incubation with CCSC. Reactive oxygen species (ROS) generated by CCSC in mono-culture of T98G and U-373 MG reached highest levels at 4 and 16 mg/ml, respectively. ROS production by T98G fell when co-cultured with HBMEC or U-373MG. These findings suggest that adverse consequences of CCSC treatment on brain cells may be protected by blood-brain barrier or astrocytes, but with chronic exposure toxicity may be worsened due to destruction of cellular integrity.

The effect of tobacco smoke exposure on the generation of reactive oxygen species and cellular membrane damage using co-culture model of blood brain barrier with astrocytes.

Brain tissue is known to be vulnerable to the exposure by tobacco smoke. Tobacco smoke can induce generation of reactive oxygen species (ROS), causing inflammatory activity and blood-brain barrier (BBB) impairment. The aim of the present study was to investigate the effect of tobacco smoke on cell cytotoxicity, generation of ROS, and cellular membrane damage in astrocytes and BBB using a co-culture system. Cell viability of U373MG cells was reduced in a dose-dependent manner, ranging from 96.7% to 40.3% by tobacco smoke condensate (TSC). Cell viability of U373MG co-cultured with human brain microvascular endothelial cells (HBMECs) was 104.9% at the IC50 value of TSC. Trans-epithelial electric resistance values drastically decreased 80% following 12-h incubation. The value was maintained until 48 h and then increased at 72-h incubation (85%). It then decreased to 75% at 120 h. Generation of ROS increased in a dose-dependent manner, ranging from 102.7% to 107.9%, when various concentrations of TSC (4-16 mg/mL) were administered to the U373MG monoculture. When TSC was added into U373MG co-cultured with HBMECs, production of ROS ranged from 101.7% to 102.6%, slightly increasing over 12 h. Maximum exposure-generated ROS of 104.8% was reached at 24 h. Cell cytotoxicity and oxidative stress levels in the U373MG co-culture model system with HBMECs were lower than U373MG monoculture. HBMECs effectively acted as a barrier to protect the astrocytes (U373MG) from toxicity of TSC.

Multiple Functions of Glutamate Uptake via Meningococcal GltT-GltM L-Glutamate ABC Transporter in Neisseria meningitidis Internalization into Human Brain Microvascular Endothelial Cells.

We previously reported that Neisseria meningitidis internalization into human brain microvasocular endothelial cells (HBMEC) was triggered by the influx of extracellular L-glutamate via the GltT-GltM L-glutamate ABC transporter, but the underlying mechanism remained unclear. We found that the ΔgltT ΔgltM invasion defect in assay medium (AM) was alleviated in AM without 10% fetal bovine serum (FBS) [AM(-S)]. The alleviation disappeared again in AM(-S) supplemented with 500 µM glutamate. Glutamate uptake by the ΔgltT ΔgltM mutant was less efficient than that by the wild-type strain, but only upon HBMEC infection. We also observed that both GltT-GltM-dependent invasion and accumulation of ezrin, a key membrane-cytoskeleton linker, were more pronounced when N. meningitidis formed larger colonies on HBMEC under physiological glutamate conditions. These results suggested that GltT-GltM-dependent meningococcal internalization into HBMEC might be induced by the reduced environmental glutamate concentration upon infection. Furthermore, we found that the amount of glutathione within the ΔgltT ΔgltM mutant was much lower than that within the wild-type N. meningitidis strain only upon HBMEC infection and was correlated with intracellular survival. Considering that the L-glutamate obtained via GltT-GltM is utilized as a nutrient in host cells, l-glutamate uptake via GltT-GltM plays multiple roles in N. meningitidis internalization into HBMEC.

The West Nile virus-like flavivirus Koutango is highly virulent in mice due to delayed viral clearance and the induction of a poor neutralizing antibody response.

UNLABELLED: The mosquito-borne West Nile virus (WNV) is responsible for outbreaks of viral encephalitis in humans, horses, and birds, with particularly virulent strains causing recent outbreaks of disease in eastern Europe, the Middle East, North America, and Australia. Previous studies have phylogenetically separated WNV strains into two main genetic lineages (I and II) containing virulent strains associated with neurological disease. Several WNV-like strains clustering outside these lineages have been identified and form an additional five proposed lineages. However, little is known about whether these strains have the potential to induce disease. In a comparative analysis with the highly virulent lineage I American strain (WNVNY99), the low-pathogenicity lineage II strain (B956), a benign Australian strain, Kunjin (WNVKUN), the African WNV-like Koutango virus (WNVKOU), and a WNV-like isolate from Sarawak, Malaysia (WNVSarawak), were assessed for neuroinvasive properties in a murine model and for their replication kinetics in vitro. While WNVNY99 replicated to the highest levels in vitro, in vivo mouse challenge revealed that WNVKOU was more virulent, with a shorter time to onset of neurological disease and higher morbidity. Histological analysis of WNVKOU- and WNVNY99-infected brain and spinal cords demonstrated more prominent meningoencephalitis and the presence of viral antigen in WNVKOU-infected mice. Enhanced virulence of WNVKOU also was associated with poor viral clearance in the periphery (sera and spleen), a skewed innate immune response, and poor neutralizing antibody development. These data demonstrate, for the first time, potent neuroinvasive and neurovirulent properties of a WNV-like virus outside lineages I and II. IMPORTANCE: In this study, we characterized the in vitro and in vivo properties of previously uncharacterized West Nile virus strains and West Nile-like viruses. We identified a West Nile-like virus, Koutango virus (WNVKOU), that was more virulent than a known virulent lineage I virus, WNVNY99. The enhanced virulence of WNVKOU was associated with poor viral clearance and the induction of a poor neutralizing antibody response. These findings provide new insights into the pathogenesis of West Nile virus.

Enterohemorrhagic Escherichia coli hemolysin employs outer membrane vesicles to target mitochondria and cause endothelial and epithelial apoptosis.

Enterohemorrhagic Escherichia coli (EHEC) strains cause diarrhea and hemolytic uremic syndrome resulting from toxin-mediated microvascular endothelial injury. EHEC hemolysin (EHEC-Hly), a member of the RTX (repeats-in-toxin) family, is an EHEC virulence factor of increasingly recognized importance. The toxin exists as free EHEC-Hly and as EHEC-Hly associated with outer membrane vesicles (OMVs) released by EHEC during growth. Whereas the free toxin is lytic towards human endothelium, the biological effects of the OMV-associated EHEC-Hly on microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, are unknown. Using microscopic, biochemical, flow cytometry and functional analyses of human brain microvascular endothelial cells (HBMEC) and Caco-2 cells we demonstrate that OMV-associated EHEC-Hly does not lyse the target cells but triggers their apoptosis. The OMV-associated toxin is internalized by HBMEC and Caco-2 cells via dynamin-dependent endocytosis of OMVs and trafficked with OMVs into endo-lysosomal compartments. Upon endosome acidification and subsequent pH drop, EHEC-Hly is separated from OMVs, escapes from the lysosomes, most probably via its pore-forming activity, and targets mitochondria. This results in decrease of the mitochondrial transmembrane potential and translocation of cytochrome c to the cytosol, indicating EHEC-Hly-mediated permeabilization of the mitochondrial membranes. Subsequent activation of caspase-9 and caspase-3 leads to apoptotic cell death as evidenced by DNA fragmentation and chromatin condensation in the intoxicated cells. The ability of OMV-associated EHEC-Hly to trigger the mitochondrial apoptotic pathway in human microvascular endothelial and intestinal epithelial cells indicates a novel mechanism of EHEC-Hly involvement in the pathogenesis of EHEC diseases. The OMV-mediated intracellular delivery represents a newly recognized mechanism for a bacterial toxin to enter host cells in order to target mitochondria.

Nutrition and the microbiome 2015.

Carlo Agostoni, MD, and Kwang Sik Kim, MD, are the Guest Editors for this annual review issue on Nutrition and the Microbiome. Dr Agostoni is a Professor of Pediatrics at the Department of Clinical Sciences and Community Health, University of Milan, within the Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan. Dr Kim is Professor of Pediatrics and Molecular Microbiology and Immunology, Johns Hopkins University School of Medicine and Bloomberg School of Public Health; and Director, Division of Pediatric Infectious Diseases, Johns Hopkins Children's Center, Baltimore, MD.