The phosphoproteome of choroid plexus epithelial cells following infection with Neisseria meningitidis.

The Gram-negative bacterium Neisseria meningitidis, which causes meningitis in humans, has been demonstrated to manipulate or alter host signalling pathways during infection of the central nervous system (CNS). However, these complex signalling networks are not completely understood. We investigate the phosphoproteome of an in vitro model of the blood-cerebrospinal fluid barrier (BCSFB) based on human epithelial choroid plexus (CP) papilloma (HIBCPP) cells during infection with the N. meningitidis serogroup B strain MC58 in presence and absence of the bacterial capsule. Interestingly, our data demonstrates a stronger impact on the phosphoproteome of the cells by the capsule-deficient mutant of MC58. Using enrichment analyses, potential pathways, molecular processes, biological processes, cellular components and kinases were determined to be regulated as a consequence of N. meningitidis infection of the BCSFB. Our data highlight a variety of protein regulations that are altered during infection of CP epithelial cells with N. meningitidis, with the regulation of several pathways and molecular events only being detected after infection with the capsule-deficient mutant. Mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD038560.

Capsule-dependent impact of MAPK signalling on host cell invasion and immune response during infection of the choroid plexus epithelium by Neisseria meningitidis.

BACKGROUND: The Gram-negative bacterium Neisseria meningitidis (Nm) can cause meningitis in humans, but the host signalling pathways manipulated by Nm during central nervous system (CNS) entry are not completely understood. METHODS: We investigate the role of the mitogen-activated protein kinases (MAPK) Erk1/2 and p38 in an in vitro model of the blood-cerebrospinal fluid barrier (BCSFB) based on human epithelial choroid plexus (CP) papilloma (HIBCPP) cells during infection with Nm serogroup B (NmB) and serogroup C (NmC) strains. A transcriptome analysis of HIBCPP cells following infection with Nm by massive analysis of cDNA ends (MACE) was done to further characterize the cellular response to infection of the barrier. RESULTS: Interestingly, whereas NmB and NmC wild type strains required active Erk1/2 and p38 pathways for infection, invasion by capsule-deficient mutants was independent of Erk1/2 and, in case of the NmB strain, of p38 activity. The transcriptome analysis of HIBCPP cells following infection with Nm demonstrated specific regulation of genes involved in the immune response dependent on Erk1/2 signalling. Gene ontology (GO) analysis confirmed loss of MAPK signalling after Erk1/2 inhibition and revealed an additional reduction of cellular responses including NFκB and JAK-STAT signalling. Interestingly, GO terms related to TNF signalling and production of IL6 were lost specifically following Erk1/2 inhibition during infection with wild type Nm, which correlated with the reduced infection rates by the wild type in absence of Erk1/2 signalling. CONCLUSION: Our data point towards a role of MAPK signalling during infection of the CP epithelium by Nm, which is strongly influenced by capsule expression, and affects infection rates as well as the host cell response.

Invasion of the choroid plexus epithelium by Neisseria meningitidis is differently mediated by Arp2/3 signaling and possibly by dynamin dependent on the presence of the capsule.

Neisseria meningitis (Nm) is a human-specific bacterial pathogen that can cause sepsis and meningitis. To cause meningitis Nm must enter the central nervous system (CNS) across one of the barriers between the blood and the brain. We have previously shown that a capsule-depleted Serogroup B strain of Nm displays enhanced invasion into human choroid plexus (CP) epithelial papilloma (HIBCPP) cells, which represent an in vitro model of the blood-cerebrospinal fluid barrier (BCSFB). Still, the processes involved during CNS invasion by Nm, especially the role of host cell actin cytoskeleton remodeling, are not investigated in detail. Here, we demonstrate that invasion into CP epithelial cells by encapsulated and capsule-depleted Nm is mediated by distinct host cell pathways. Whereas a Serogroup B wild-type strain enters HIBCPP cells by a possibly dynamin-independent, but actin related protein 2/3 (Arp2/3)-dependent mechanism, invasion by a capsule-depleted mutant is reduced by the dynamin inhibitor dynasore and Arp2/3-independent. Both wild-type and mutant bacteria require Src kinase activity for entry into HIBCPP cells. Our data show that Nm can employ different mechanisms for invasion into the CP epithelium dependent on the presence of a capsule.

Interactions and Signal Transduction Pathways Involved during Central Nervous System Entry by Neisseria meningitidis across the Blood-Brain Barriers.

The Gram-negative diplococcus Neisseria meningitidis, also called meningococcus, exclusively infects humans and can cause meningitis, a severe disease that can lead to the death of the afflicted individuals. To cause meningitis, the bacteria have to enter the central nervous system (CNS) by crossing one of the barriers protecting the CNS from entry by pathogens. These barriers are represented by the blood-brain barrier separating the blood from the brain parenchyma and the blood-cerebrospinal fluid (CSF) barriers at the choroid plexus and the meninges. During the course of meningococcal disease resulting in meningitis, the bacteria undergo several interactions with host cells, including the pharyngeal epithelium and the cells constituting the barriers between the blood and the CSF. These interactions are required to initiate signal transduction pathways that are involved during the crossing of the meningococci into the blood stream and CNS entry, as well as in the host cell response to infection. In this review we summarize the interactions and pathways involved in these processes, whose understanding could help to better understand the pathogenesis of meningococcal meningitis.

Non-Typeable Haemophilus influenzae Invade Choroid Plexus Epithelial Cells in a Polar Fashion.

Non-typeable Haemophilus influenzae (NTHI) is a pathogen of the human respiratory tract causing the majority of invasive H. influenzae infections. Severe invasive infections such as septicemia and meningitis occur rarely, but the lack of a protecting vaccine and the increasing antibiotic resistance of NTHI impede treatment and emphasize its relevance as a potential meningitis causing pathogen. Meningitis results from pathogens crossing blood-brain barriers and invading the immune privileged central nervous system (CNS). In this study, we addressed the potential of NTHI to enter the brain by invading cells of the choroid plexus (CP) prior to meningeal inflammation to enlighten NTHI pathophysiological mechanisms. A cell culture model of human CP epithelial cells, which form the blood-cerebrospinal fluid barrier (BCSFB) in vivo, was used to analyze adhesion and invasion by immunofluorescence and electron microscopy. NTHI invade CP cells in vitro in a polar fashion from the blood-facing side. Furthermore, NTHI invasion rates are increased compared to encapsulated HiB and HiF strains. Fimbriae occurrence attenuated adhesion and invasion. Thus, our findings underline the role of the BCSFB as a potential entry port for NTHI into the brain and provide strong evidence for a function of the CP during NTHI invasion into the CNS during the course of meningitis.

The Lyme disease bacterium, Borrelia burgdorferi, stimulates an inflammatory response in human choroid plexus epithelial cells.

The main functions of the choroid plexus (CP) are the production of cerebral spinal fluid (CSF), the formation of the blood-CSF barrier, and regulation of immune response. This barrier allows for the exchange of specific nutrients, waste, and peripheral immune cells between the blood stream and CSF. Borrelia burgdorferi (Bb), the causative bacteria of Lyme disease, is associated with neurological complications including meningitis-indeed, Bb has been isolated from the CSF of patients. While it is accepted that B. burgdorferi can enter the central nervous system (CNS) of patients, it is unknown how the bacteria crosses this barrier and how the pathogenesis of the disease leads to the observed symptoms in patients. We hypothesize that during infection Borrelia burgdorferi will induce an immune response conducive to the chemotaxis of immune cells and subsequently lead to a pro-inflammatory state with the CNS parenchyma. Primary human choroid plexus epithelial cells were grown in culture and infected with B. burgdorferi strain B31 MI-16 for 48 hours. RNA was isolated and used for RNA sequencing and RT-qPCR validation. Secreted proteins in the supernatant were analyzed via ELISA. Transcriptome analysis based on RNA sequencing determined a total of 160 upregulated genes and 98 downregulated genes. Pathway and biological process analysis determined a significant upregulation in immune and inflammatory genes specifically in chemokine and interferon related pathways. Further analysis revealed downregulation in genes related to cell to cell junctions including tight and adherens junctions. These results were validated via RT-qPCR. Protein analysis of secreted factors showed an increase in inflammatory chemokines, corresponding to our transcriptome analysis. These data further demonstrate the role of the CP in the modulation of the immune response in a disease state and give insight into the mechanisms by which Borrelia burgdorferi may disseminate into, and act upon, the CNS. Future experiments aim to detail the impact of B. burgdorferi on the blood-CSF-barrier (BCSFB) integrity and inflammatory response within animal models.

Virulence factor-dependent basolateral invasion of choroid plexus epithelial cells by pathogenic Escherichia coli in vitro.

Escherichia coli is the most common Gram-negative causative agent of neonatal meningitis and E. coli meningitis is associated with high morbidity and mortality. Previous research has been carried out with regard to the blood-brain barrier and thereby unveiled an assortment of virulence factors involved in E. coli meningitis. Little, however, is known about the role of the blood-cerebrospinal fluid (CSF) barrier (BCSFB), in spite of several studies suggesting that the choroid plexus (CP) is a possible entry point for E. coli into the CSF spaces. Here, we used a human CP papilloma (HIBCPP) cell line that was previously established as valid model for the study of the BCSFB. We show that E. coli invades HIBCPP cells in a polar fashion preferentially from the physiologically relevant basolateral side. Moreover, we demonstrate that deletion of outer membrane protein A, ibeA or neuDB genes results in decreased cell infection, while absence of fimH enhances invasion, although causing reduced adhesion to the apical side of HIBCPP cells. Our findings suggest that the BCSFB might constitute an entry point for E. coli into the central nervous system, and HIBCPP cells are a valuable tool for investigating E. coli entry of the BCSFB.

The choroid plexus epithelium as a novel player in the stomach-brain axis during Helicobacter infection.

Several studies suggest a link between shifts in gut microbiota and neurological disorders. Recently, we reported a high prevalence of Helicobacter suis (H. suis) in patients with Parkinson's disease. Here, we evaluated the effect of gastric H. suis infection on the brain in mice. One month of infection with H. suis resulted in increased brain inflammation, reflected in activation of microglia and cognitive decline. Additionally, we detected choroid plexus inflammation and disruption of the epithelial blood-cerebrospinal fluid (CSF) barrier upon H. suis infection, while the endothelial blood-brain barrier (BBB) remained functional. These changes were accompanied by leakage of the gastrointestinal barrier and low-grade systemic inflammation, suggesting that H. suis-evoked gastrointestinal permeability and subsequent peripheral inflammation induces changes in brain homeostasis via changes in blood-CSF barrier integrity. In conclusion, this study shows for the first time that H. suis infection induces inflammation in the brain associated with cognitive decline and that the choroid plexus is a novel player in the stomach-brain axis.

Mitogen-activated protein kinases are required for effective infection of human choroid plexus epithelial cells by Listeria monocytogenes.

Listeria monocytogenes, a Gram-positive bacterium, can cause meningitis after invading the human central nervous system. The blood-cerebrospinal fluid barrier (BCSFB), located at the epithelium of the choroid plexus, is a possible entry site for L. monocytogenes into the brain, and in vitro L. monocytogenes invades human choroid plexus epithelial papilloma (HIBCPP) cells. Although host cell signal transduction subsequent to infection by L. monocytogenes has been investigated, the role of mitogen-activated protein kinases (MAPK) is not clarified yet. We show that infection with L. monocytogenes causes activation of the MAPKs Erk1/2 and p38 preferentially when bacteria are added to the physiologically more relevant basolateral side of HIBCPP cells. Deletion of the listerial virulence factors Internalin (InlA) and InlB reduces MAPK activation. Whereas inhibition of either Erk1/2 or p38 signaling significantly attenuates infection of HIBCPP cells with L. monocytogenes, simultaneous inhibition of both MAPK pathways shows an additive effect, and Erk1/2 and p38 are involved in regulation of cytokine and chemokine expression following infection. Blocking of endocytosis with the synthetic dynamin inhibitor dynasore strongly abrogates infection of HIBCPP cells with L. monocytogenes. Concurrent inhibition of MAPK signaling further reduces infection, suggesting MAPKs mediate infection with L. monocytogenes during inhibition of dynamin-mediated endocytosis.

Non-invasive model of neuropathogenic Escherichia coli infection in the neonatal rat.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Atypical clinical and radiological presentation of cryptococcal choroid plexitis in an immunocompetent woman.

Central nervous system cryptococcal infections usually manifests as meningitis, meningoencephalitis, encephalitis or ventriculitis. Cryptococcal choroid plexus inflammation is a particularly rare entity most often presenting with signs and symptoms of intracranial hypertension, hydrocephalus or meningitis due to a delayed diagnosis. Herein we reported the case of a 63-year-old immunocompetent woman with a history of temporal lobe epilepsy and behavioral disorders. Magnetic resonance imaging (MRI) and fluorodeoxyglucose positron emission tomographic (FDG-PET) images revealed atypical cryptococcal choroid plexitis with surrounding bitemporal edema without features of meningitis, intraparenchymal cryptococcoma or hydrocephalus. The patient underwent serial MRI and FDG-PET images performed before and after antifungal therapy that caused a marked clinical improvement. Our case also suggests a potential role of FDG-PET in the monitoring antifungal therapeutic efficacy.

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

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

In vitro transcriptome analysis of porcine choroid plexus epithelial cells in response to Streptococcus suis: release of pro-inflammatory cytokines and chemokines.

The Gram-positive zoonotic bacterium Streptococcus suis (S. suis) is responsible for a wide range of diseases including meningitis in pigs and humans. The blood-cerebrospinal fluid (CSF) barrier is constituted by the epithelial cells of the choroid plexus, which execute barrier function also after bacteria have entered the central nervous system (CNS). We show that the bacterial capsule, a major virulence factor, strongly attenuates adhesion of S. suis to the apical side of porcine choroid plexus epithelial cells (PCPEC). Oligonucleotide microarray analysis and quantitative PCR surprisingly demonstrated that adherent wild-type and capsule-deficient S. suis influenced expression of a pronounced similar pattern of genes in PCPEC. Investigation of purified capsular material provided no evidence for a significant role of the capsule. Enriched among the regulated genes were those involved in "inflammatory response", "defense response" and "cytokine activity". These comprised several cytokines and chemokines including the interleukins 6 and 8, which could be detected on protein level. We show that after infection with S. suis the choroid plexus contributes to the immune response by actively producing cytokines and chemokines. Other virulence factors than the bacterial capsule may be relevant in inducing a strong inflammatory response in the CNS during S. suis meningitis.

The distribution of E-cadherin expression in listeric rhombencephalitis of ruminants indicates its involvement in Listeria monocytogenes neuroinvasion.

AIM: To investigate the expression of E-cadherin, a major host cell receptor for Listeria monocytogenes (LM) internalin A, in the ruminant nervous system and its putative role in brainstem invasion and intracerebral spread of LM in the natural disease. METHODS: Immunohistochemistry and double immunofluorescence was performed on brains, cranial nerves and ganglia of ruminants with and without natural LM rhombencephalitis using antibodies against E-cadherin, protein gene product 9.5, myelin-associated glycoprotein and LM. RESULTS: In the ruminant brain, E-cadherin is expressed in choroid plexus epithelium, meningothelium and restricted neuropil areas of the medulla, but not in the endothelium. In cranial nerves and ganglia, E-cadherin is expressed in satellite cells and myelinating Schwann cells. Expression does not differ between ruminants with or without listeriosis and does not overlap with the presence of microabscesses in the medulla. LM is observed in phagocytes, axons, Schwann cells, satellite cells and ganglionic neurones. CONCLUSION: Our results support the view that the specific ligand-receptor interaction between LM and host E-cadherin is involved in the neuropathogenesis of ruminant listeriosis. They suggest that oral epithelium and Schwann cells expressing E-cadherin provide a port of entry for free bacteria offering a site of primary intracellular replication, from where the bacterium may invade the axonal compartment by cell-to-cell spread. As E-cadherin expression in the ruminant central nervous system is weak, only very locally restricted and not related to the presence of microabscesses, it is likely that further intracerebral spread is independent of E-cadherin and relies primarily on axonal spread.

Central nervous system marginal zone B-cell lymphoma associated with Chlamydophila psittaci infection.

Extranodal marginal zone B-cell lymphomas are linked to bacterial infections that vary according to the anatomical site. The occurrence of these lymphomas in the central nervous system is a very rare event, and the identification of specific bacteria in this setting has not been previously addressed. Herein, we report for the first time a case of primary central nervous system marginal zone B-cell lymphoma involving the choroid plexus associated with Chlamydophila psittaci infection. No concomitant ocular involvement was detected. C psittaci was identified with 3 independent methods, and through immunohistochemistry, it was visualized in the cytoplasm of monocytes/macrophages present within lymphomatous tissues. This observation points toward the opportunity to investigate the prevalence of C psittaci infection in central nervous system lymphomas, particularly in those with low-grade histologic features.

Cryptococcal choroid plexitis: rare imaging findings of central nervous system cryptococcal infection in an immunocompetent individual.

Central nervous system (CNS) cryptococcosis is a common opportunistic fungal infection in immunocompromised patients, and the imaging findings differ from those in immunocompetent patients. Here, we present the imaging findings in an immunocompetent woman of a rare case of central nervous system cryptococcal choroid plexitis with trapped temporal horns, enlarged enhancing bilateral choroid plexuses and multiple intraventricular choroid plexus cysts.

Polar bacterial invasion and translocation of Streptococcus suis across the blood-cerebrospinal fluid barrier in vitro.

Previous experimental studies in a standard Transwell culture system have shown Streptococcus suis ability to compromise barrier function of porcine choroid plexus epithelial cells (PCPEC). The development of an 'inverted' Transwell filter system of PCPEC enables us now for the first time to investigate bacterial invasion and translocation from the physiologically relevant basolateral (blood) to the apical (cerebrospinal fluid) side. Most importantly, we observed specific invasion and translocation of S. suis across the PCPEC exclusively from the basolateral side. During this process, bacterial viability and the presence of a capsule as well as cytoskeletal regulation of PCPEC seemed to play an important role. No loss of barrier function was observed. Bacterial translocation could be significantly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002, but not by its inactive analogue Ly303511 or dexamethasone. Apotome imaging as well as electron microscopy revealed intracellular bacteria often in cell vacuoles. Thus, possibly regulated by the presence of a capsule, S. suis induces signals that depend on the lipid kinase phosphatidylinositol 3-kinase pathway, which paves the way for cellular uptake during the bacterial transcellular translocation process. Taken together, our data underline the relevance of the blood-cerebrospinal fluid barrier as a gate for bacterial entry into the central nervous system.

A novel cell wall-anchored peptidoglycan hydrolase (autolysin), IspC, essential for Listeria monocytogenes virulence: genetic and proteomic analysis.

We have recently concluded that a Listeria monocytogenes 86 kDa immunogenic surface protein, IspC, is a cell wall-anchored peptidoglycan hydrolase (autolysin), capable of degrading the cell wall peptidoglycan of the bacterium itself. To determine if this enzyme has any biological functions and/or plays a role in virulence, we in-frame-deleted the ispC gene from the L. monocytogenes chromosome. This DeltaispC mutant exhibited complete abrogation of expression of IspC and displayed no defects in in vitro growth, colony and microscopic morphologies, or biochemical characteristics. Lack of IspC led to attenuated virulence in mice, evidenced by a significant reduction in bacterial counts in livers and brains and no mortality compared with the wild-type. Furthermore, the data from assays using various eukaryotic cells for adhesion, invasion, actin tail formation, plaque formation and intracellular growth indicated that the mutant was severely attenuated in virulence in a cell culture model in a cell type-dependent manner. The findings that (i) the mutant was impaired for adhesion to certain eukaryotic cells, and (ii) both purified IspC and its C-terminal cell wall-binding domain were capable of binding sheep choroid plexus (SCP) epithelial cells and Vero cells, supported the role of IspC as an adhesin in virulence. The DeltaispC mutant exhibited a marked defect in adhesion to and invasion of SCP cells but not human brain microvascular endothelial cells (HBMEC), suggesting that IspC is necessary for crossing the blood-cerebrospinal fluid barrier. Proteomic and immunological analysis showed a reduced surface expression of some known or putative virulence factors (e.g. ActA, InlC2 and a flagellin homologue, FlaA) due to IspC deficiency. Altogether, this study demonstrates that IspC, expressed as a minor autolysin in vitro, is not important for cell division or separation but is essential for full virulence of L. monocytogenes in vivo.

Choroid plexitis in a case of systemic nocardiosis.

Although rare, primary choroid plexitis can occur as an early presentation of a central nervous system (CNS) infection most commonly with cryptococcosis, tuberculosis, and nocardiosis. In the appropriate clinical setting, an enlarged, intensely enhancing choroid plexus should raise suspicion for choroid plexitis. It is important to recognize this entity early as aggressive diagnostic and therapeutic intervention may be necessary. We review the existing literature and present a case of infectious choroid plexitis in a patient with systemic nocardiosis; computed tomography and magnetic resonance imaging demonstrated the characteristic findings of choroid plexitis, which later developed into a parenchymal abscess.