Toxoplasma-Induced Hypermigration of Primary Cortical Microglia Implicates GABAergic Signaling.
ABSTRACT: Toxoplasma gondii is a widespread obligate intracellular parasite that causes chronic infection and life-threatening acute infection in the central nervous system. Previous work identified Toxoplasma-infected microglia and astrocytes during reactivated infections in mice, indicating an implication of glial cells in acute toxoplasmic encephalitis. However, the mechanisms leading to the spread of Toxoplasma in the brain parenchyma remain unknown. Here, we report that, shortly after invasion by T. gondii tachyzoites, parasitized microglia, but not parasitized astrocytes, undergo rapid morphological changes and exhibit dramatically enhanced migration in 2-dimensional and 3-dimensional matrix confinements. Interestingly, primary microglia secreted the neurotransmitter ?-aminobutyric acid (GABA) in the supernatant as a consequence of T. gondii infection but not upon stimulation with LPS or heat-inactivated T. gondii. Further, microglia transcriptionally expressed components of the GABAergic machinery, including GABA-A receptor subunits, regulatory molecules and voltage-dependent calcium channels (VDCCs). Further, their transcriptional expression was modulated by challenge with T. gondii. Transcriptional analysis indicated that GABA was synthesized via both, the conventional pathway (glutamate decarboxylases GAD65 and GAD67) and a more recently characterized alternative pathway (aldehyde dehydrogenases ALDH2 and ALDH1a1). Pharmacological inhibitors targeting GABA synthesis, GABA-A receptors, GABA-A regulators and VDCC signaling inhibited Toxoplasma-induced hypermotility of microglia. Altogether, we show that primary microglia express a GABAergic machinery and that T. gondii induces hypermigration of microglia in a GABA-dependent fashion. We hypothesize that migratory activation of parasitized microglia by Toxoplasma may promote parasite dissemination in the brain parenchyma.
Project description:Disseminated toxoplasmosis in the central nervous system (CNS) is often accompanied by a lethal outcome. Studies with murine models of infection have focused on the role of systemic immunity in control of toxoplasmic encephalitis, while knowledge remains limited on the contributions of resident cells with immune functions in the CNS. In this study, the role of glial cells was addressed in the setting of recrudescent Toxoplasma infection in mice. Activated astrocytes and microglia were observed in the close vicinity of foci with replicating parasites in situ in the brain parenchyma. Toxoplasma gondii tachyzoites were allowed to infect primary microglia and astrocytes in vitro. Microglia were permissive to parasite replication, and infected microglia readily transmigrated across transwell membranes and cell monolayers. Thus, infected microglia, but not astrocytes, exhibited a hypermotility phenotype reminiscent of that recently described for infected dendritic cells. In contrast to gamma interferon-activated microglia, Toxoplasma-infected microglia did not upregulate major histocompatibility complex (MHC) class II molecules and the costimulatory molecule CD86. Yet Toxoplasma-infected microglia and astrocytes exhibited increased sensitivity to T cell-mediated killing, leading to rapid parasite transfer to effector T cells in vitro. We hypothesize that glial cells and T cells, besides their role in triggering antiparasite immunity, may also act as "Trojan horses," paradoxically facilitating dissemination of Toxoplasma within the CNS. To our knowledge, this constitutes the first report of migratory activation of a resident CNS cell by an intracellular parasite.
Project description:During acute infection in human and animal hosts, the obligate intracellular protozoan Toxoplasma gondii infects a variety of cell types, including leukocytes. Poised to respond to invading pathogens, dendritic cells (DC) may also be exploited by T. gondii for spread in the infected host. Here, we report that human and mouse myeloid DC possess functional ?-aminobutyric acid (GABA) receptors and the machinery for GABA biosynthesis and secretion. Shortly after T. gondii infection (genotypes I, II and III), DC responded with enhanced GABA secretion in vitro. We demonstrate that GABA activates GABA(A) receptor-mediated currents in T. gondii-infected DC, which exhibit a hypermigratory phenotype. Inhibition of GABA synthesis, transportation or GABA(A) receptor blockade in T. gondii-infected DC resulted in impaired transmigration capacity, motility and chemotactic response to CCL19 in vitro. Moreover, exogenous GABA or supernatant from infected DC restored the migration of infected DC in vitro. In a mouse model of toxoplasmosis, adoptive transfer of infected DC pre-treated with GABAergic inhibitors reduced parasite dissemination and parasite loads in target organs, e.g. the central nervous system. Altogether, we provide evidence that GABAergic signaling modulates the migratory properties of DC and that T. gondii likely makes use of this pathway for dissemination. The findings unveil that GABA, the principal inhibitory neurotransmitter in the brain, has activation functions in the immune system that may be hijacked by intracellular pathogens.
Project description:The obligate intracellular parasite Toxoplasma gondii exploits cells of the immune system to disseminate. Upon T. gondii-infection, ?-aminobutyric acid (GABA)/GABAA receptor signaling triggers a hypermigratory phenotype in dendritic cells (DCs) by unknown signal transduction pathways. Here, we demonstrate that calcium (Ca2+) signaling in DCs is indispensable for T. gondii-induced DC hypermotility and transmigration in vitro. We report that activation of GABAA receptors by GABA induces transient Ca2+ entry in DCs. Murine bone marrow-derived DCs preferentially expressed the L-type voltage-dependent Ca2+ channel (VDCC) subtype Cav1.3. Silencing of Cav1.3 by short hairpin RNA or selective pharmacological antagonism of VDCCs abolished the Toxoplasma-induced hypermigratory phenotype. In a mouse model of toxoplasmosis, VDCC inhibition of adoptively transferred Toxoplasma-infected DCs delayed the appearance of cell-associated parasites in the blood circulation and reduced parasite dissemination to target organs. The present data establish that T. gondii-induced hypermigration of DCs requires signaling via VDCCs and that Ca2+ acts as a second messenger to GABAergic signaling via the VDCC Cav1.3. The findings define a novel motility-related signaling axis in DCs and unveil that interneurons and DCs share common GABAergic motogenic pathways. T. gondii employs GABAergic non-canonical pathways to induce host cell migration and facilitate dissemination.
Project description:The balance between controlling infection and limiting inflammation is particularly precarious in the brain because of its unique vulnerability to the toxic effects of inflammation. Astrocytes have been implicated as key regulators of neuroinflammation in CNS infections, including infection with Toxoplasma gondii, a protozoan parasite that naturally establishes a chronic CNS infection in mice and humans. In CNS toxoplasmosis, astrocytes are critical to controlling parasite growth. They secrete proinflammatory cytokines and physically encircle parasites. However, the molecular mechanisms used by astrocytes to limit neuroinflammation during toxoplasmic encephalitis have not yet been identified. TGF-? signaling in astrocytes is of particular interest because TGF-? is universally upregulated during CNS infection and serves master regulatory and primarily anti-inflammatory functions. We report in this study that TGF-? signaling is activated in astrocytes during toxoplasmic encephalitis and that inhibition of astrocytic TGF-? signaling increases immune cell infiltration, uncouples proinflammatory cytokine and chemokine production from CNS parasite burden, and increases neuronal injury. Remarkably, we show that the effects of inhibiting astrocytic TGF-? signaling are independent of parasite burden and the ability of GFAP(+) astrocytes to physically encircle parasites.
Project description:UNLABELLED:During infections with the protozoan parasite Toxoplasma gondii, gamma-aminobutyric acid (GABA) is utilized as a carbon source for parasite metabolism and also to facilitate parasite dissemination by stimulating dendritic-cell motility. The best-recognized function for GABA, however, is its role in the nervous system as an inhibitory neurotransmitter that regulates the flow and timing of excitatory neurotransmission. When this pathway is altered, seizures develop. Human toxoplasmosis patients suffer from seizures, suggesting that Toxoplasma interferes with GABA signaling in the brain. Here, we show that while excitatory glutamatergic presynaptic proteins appeared normal, infection with type II ME49 Toxoplasma tissue cysts led to global changes in the distribution of glutamic acid decarboxylase 67 (GAD67), a key enzyme that catalyzes GABA synthesis in the brain. Alterations in GAD67 staining were not due to decreased expression but rather to a change from GAD67 clustering at presynaptic termini to a more diffuse localization throughout the neuropil. Consistent with a loss of GAD67 from the synaptic terminals, Toxoplasma-infected mice develop spontaneous seizures and are more susceptible to drugs that induce seizures by antagonizing GABA receptors. Interestingly, GABAergic protein mislocalization and the response to seizure-inducing drugs were observed in mice infected with type II ME49 but not type III CEP strain parasites, indicating a role for a polymorphic parasite factor(s) in regulating GABAergic synapses. Taken together, these data support a model in which seizures and other neurological complications seen in Toxoplasma-infected individuals are due, at least in part, to changes in GABAergic signaling. IMPORTANCE:Infections of the central nervous system can cause seizures. While inflammation in the brain has been proposed to initiate the onset of the seizures, relatively little is known about how inflammation impacts the structure and function of the neurons. Here we used a parasite called Toxoplasma gondii that infects the brain and showed that seizures arise due to a defect in signaling of GABA, which is the neurotransmitter primarily responsible for preventing the onset of seizures.
Project description:Toxoplasma gondii is capable of persisting in the brain, although it is efficiently eliminated by cellular immune responses in most other sites. While Toll-like receptor 2 (TLR2) reportedly plays important roles in protective immunity against the parasite, the relationship between neurological disorders induced by T. gondii infection and TLR2 function in the brain remains controversial with many unknowns. In this study, primary cultured astrocytes, microglia, neurons, and peritoneal macrophages obtained from wild-type and TLR2-deficient mice were exposed to T. gondii tachyzoites. To characterize TLR2-dependent functional pathways activated in response to T. gondii infection, gene expression of different cell types was profiled by RNA sequencing.During T. gondii infection, a total of 611, 777, 385, and 1105 genes were upregulated in astrocytes, microglia, neurons, and macrophages, respectively, while 163, 1207, 158, and 1274 genes were downregulated, respectively, in a TLR2-dependent manner. Overrepresented Gene Ontology (GO) terms for TLR2-dependently upregulated genes were associated with immune and stress responses in astrocytes, immune responses and developmental processes in microglia, metabolic processes and immune responses in neurons, and metabolic processes and gene expression in macrophages. Overrepresented GO terms for downregulated genes included ion transport and behavior in astrocytes, cell cycle and cell division in microglia, metabolic processes in neurons, and response to stimulus, signaling and cell motility in macrophages.To our knowledge, this is the first transcriptomic study of TLR2 function across different cell types during T. gondii infection. Results of RNA-sequencing demonstrated roles for TLR2 varied by cell type during T. gondii infection. Our findings facilitate understanding of the detailed relationship between TLR2 and T. gondii infection, and elucidate mechanisms underlying neurological changes during infection.
Project description:The obligate intracellular parasite Toxoplasma gondii exploits cells of the immune system to disseminate. Upon infection, parasitized dendritic cells (DCs) and microglia exhibit a hypermigratory phenotype in vitro that has been associated with enhancing parasite dissemination in vivo in mice. One unresolved question is how parasites commandeer parasitized cells to achieve systemic dissemination by a 'Trojan-horse' mechanism. By chromatography and mass spectrometry analyses, we identified an orthologue of the 14-3-3 protein family, T. gondii 14-3-3 (Tg14-3-3), as mediator of DC hypermotility. We demonstrate that parasite-derived polypeptide fractions enriched for Tg14-3-3 or recombinant Tg14-3-3 are sufficient to induce the hypermotile phenotype when introduced by protein transfection into murine DCs, human DCs or microglia. Further, gene transfer of Tg14-3-3 by lentiviral transduction induced hypermotility in primary human DCs. In parasites expressing Tg14-3-3 in a ligand-regulatable fashion, overexpression of Tg14-3-3 was correlated with induction of hypermotility in parasitized DCs. Localization studies in infected DCs identified Tg14-3-3 within the parasitophorous vacuolar space and a rapid recruitment of host cell 14-3-3 to the parasitophorous vacuole membrane. The present work identifies a determinant role for Tg14-3-3 in the induction of the migratory activation of immune cells by T. gondii. Collectively, the findings reveal Tg14-3-3 as a novel target for an intracellular pathogen that acts by hijacking the host cell's migratory properties to disseminate.
Project description:BACKGROUND:More than 100 different pathogens can cause encephalitis. Testing of all the neurological pathogens by conventional methods can be difficult. Metagenomic next-generation sequencing (NGS) could identify the infectious agents in a target-independent manner. The role of this novel method in clinical diagnostic microbiology still needs to be evaluated. In present study, we used metagenomic NGS to search for an infectious etiology in a human immunodeficiency virus (HIV)-infected patient with lethally diffuse brain lesions. Sequences mapping to Toxoplasma gondii were unexpectedly detected. CASE PRESENTATION:A 31-year-old HIV-infected patient presented to hospital in a critical ill condition with a Glasgow coma scale score of 3. Brain magnetic resonance imaging showed diffuse brain abnormalities with contrast enhancement. Metagenomic NGS was performed on DNA extract from 300 μL patient's cerebrospinal fluid (CSF) with the BGISEQ-50 platform. The sequencing detection identified 65,357 sequence reads uniquely aligned to the Toxoplasma gondii genome. Presence of Toxoplasma gondii genome in CSF was further verified by Toxoplasma gondii-specific polymerase chain reaction and Sanger sequencing. Altogether, those results confirmed the diagnosis of toxoplasmic encephalitis. CONCLUSIONS:This study suggests that metagenomic NGS may be a useful diagnostic tool for toxoplasmic encephalitis. As metagenomic NGS is able to identify all pathogens in a single run, it may be a promising strategy to explore the clinical causative pathogens in central nervous system infections with atypical features.
Project description:Toxoplasmic encephalitis in patients with AIDS is a life-threatening disease mostly due to reactivation of Toxoplasma gondii cysts in the brain. The main objective of this study was to evaluate the performance of real-time PCR assay in peripheral blood samples for the diagnosis of toxoplasmic encephalitis in AIDS patients in the French West Indies and Guiana.Adult patients with HIV and suspicion of toxoplasmic encephalitis with start of specific antitoxoplasmic therapy were included in this study during 40 months. The real-time PCR assay targeting the 529 bp repeat region of T. gondii was performed in two different centers for all blood samples. A Neighbor-Joining tree was reconstructed from microsatellite data to examine the relationships between strains from human cases of toxoplasmosis in South America and the Caribbean. A total of 44 cases were validated by a committee of experts, including 36 cases with toxoplasmic encephalitis. The specificity of the PCR assay in blood samples was 100% but the sensitivity was only 25% with moderate agreement between the two centers. Altered level of consciousness and being born in the French West Indies and Guiana were the only two variables that were associated with significantly decreased risk of false negative results with the PCR assay.Our results showed that PCR sensitivity in blood samples increased with severity of toxoplasmic encephalitis in AIDS patients. Geographic origin of patients was likely to influence PCR sensitivity but there was little evidence that it was caused by differences in T. gondii strains.ClinicalTrials.gov NCT00803621.
Project description:Several treatment failures have been reported for the treatment of toxoplasmic encephalitis, chorioretinitis, and congenital toxoplasmosis. Recently we found three Toxoplasma gondii strains naturally resistant to sulfadiazine and we developed in vitro two sulfadiazine resistant strains, RH-R(SDZ) and ME-49-R(SDZ), by gradual pressure. In Plasmodium, common mechanisms of drug resistance involve, among others, mutations and/or amplification within genes encoding the therapeutic targets dhps and dhfr and/or the ABC transporter genes family. To identify genotypic and/or phenotypic markers of resistance in T. gondii, we sequenced and analyzed the expression levels of therapeutic targets dhps and dhfr, three ABC genes, two Pgp, TgABC.B1 and TgABC.B2, and one MRP, TgABC.C1, on sensitive strains compared to sulfadiazine resistant strains. Neither polymorphism nor overexpression was identified. Contrary to Plasmodium, in which mutations and/or overexpression within gene targets and ABC transporters are involved in antimalarial resistance, T. gondii sulfadiazine resistance is not related to these toxoplasmic genes studied.