RESUMO
Host cytosolic proteins are endocytosed by Toxoplasma gondii and degraded in its lysosome-like compartment, the vacuolar compartment (VAC), but the dynamics and route of endocytic trafficking remain undefined. Conserved endocytic components and plant-like features suggest T. gondii endocytic trafficking involves transit through early and late endosome-like compartments (ELCs) and potentially the trans-Golgi network (TGN) as in plants. However, exocytic trafficking to regulated secretory organelles, micronemes and rhoptries, also proceeds through ELCs and requires classical endocytic components, including a dynamin-related protein, DrpB. Here, we show that host cytosolic proteins are endocytosed within 7 minutes post-invasion, trafficked through ELCs en route to the VAC, and degraded within 30 minutes. We could not definitively interpret if ingested protein is trafficked through the TGN. We also found that parasites ingest material from the host cytosol throughout the parasite cell cycle. Ingested host proteins colocalize with immature microneme proteins, proM2AP and proMIC5, in transit to the micronemes, but not with the immature rhoptry protein proRON4, indicating that endocytic trafficking of ingested protein intersects with exocytic trafficking of microneme proteins. Finally, we show that conditional expression of a DrpB dominant negative mutant increases T. gondii ingestion of host-derived proteins, suggesting that DrpB is not required for parasite endocytosis.
Assuntos
Endocitose , Exocitose , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Endossomos/metabolismo , Complexo de Golgi/metabolismoRESUMO
Anti-NMDA receptor (NMDAR) autoantibodies have been postulated to play a role in the pathogenesis of NMDAR hypofunction, which contributes to the etiology of psychotic symptoms. Toxoplasma gondii is a pathogen implicated in psychiatric disorders and associated with elevation of NMDAR autoantibodies. However, it remains unclear whether parasite infection is the cause of NMDAR autoantibodies. By using mouse models, we found that NMDAR autoantibody generation had a strong temporal association with tissue cyst formation, as determined by MAG1 antibody seroreactivity (r = 0.96; P < 0.0001), which is a serologic marker for the cyst burden. The presence of MAG1 antibody response, but not T. gondii IgG response, was required for NMDAR autoantibody production. The pathogenic relevance of NMDAR autoantibodies to behavioral abnormalities (blunted response to amphetamine-triggered activity and decreased locomotor activity and exploration) and reduced expression of synaptic proteins (the GLUN2B subtype of NMDAR and PSD-95) has been demonstrated in infected mice. Our study suggests that NMDAR autoantibodies are specifically induced by persistent T. gondii infection and are most likely triggered by tissue cysts. NMDAR autoantibody seroreactivity may be a novel pathological hallmark of chronic toxoplasmosis, which raises questions about NMDAR hypofunction and neurodegeneration in the infected brain.
Assuntos
Autoanticorpos/imunologia , Encéfalo/patologia , Receptores de N-Metil-D-Aspartato/imunologia , Toxoplasma/fisiologia , Toxoplasmose/parasitologia , Toxoplasmose/psicologia , Animais , Comportamento Animal , Encéfalo/imunologia , Encéfalo/parasitologia , Encéfalo/fisiopatologia , Doença Crônica , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Atividade Motora , Neuropatologia , Toxoplasmose/imunologia , Toxoplasmose/patologiaRESUMO
Chloroviruses (family Phycodnaviridae) are large DNA viruses known to infect certain eukaryotic green algae and have not been previously shown to infect humans or to be part of the human virome. We unexpectedly found sequences homologous to the chlorovirus Acanthocystis turfacea chlorella virus 1 (ATCV-1) in a metagenomic analysis of DNA extracted from human oropharyngeal samples. These samples were obtained by throat swabs of adults without a psychiatric disorder or serious physical illness who were participating in a study that included measures of cognitive functioning. The presence of ATCV-1 DNA was confirmed by quantitative PCR with ATCV-1 DNA being documented in oropharyngeal samples obtained from 40 (43.5%) of 92 individuals. The presence of ATCV-1 DNA was not associated with demographic variables but was associated with a modest but statistically significant decrease in the performance on cognitive assessments of visual processing and visual motor speed. We further explored the effects of ATCV-1 in a mouse model. The inoculation of ATCV-1 into the intestinal tract of 9-11-wk-old mice resulted in a subsequent decrease in performance in several cognitive domains, including ones involving recognition memory and sensory-motor gating. ATCV-1 exposure in mice also resulted in the altered expression of genes within the hippocampus. These genes comprised pathways related to synaptic plasticity, learning, memory formation, and the immune response to viral exposure.
Assuntos
Comportamento Animal , Chlorella/virologia , Cognição , Laringe/virologia , Memória , Mariposas/virologia , Phycodnaviridae , Animais , Feminino , Humanos , Masculino , CamundongosRESUMO
Elucidating the molecular basis of complex human psychiatric disorders is challenging due to the multitude of factors that underpin these disorders. Genetic and chromosomal changes are two factors that have been suggested to be involved in psychiatric disorders. Indeed, numerous risk loci have been identified in autism spectrum disorders, schizophrenia, and related psychiatric disorders. Here, we introduce genetic animal models that disturb excitatory-inhibitory balance in the brain and animal models mirroring human chromosomal abnormalities, both of which may be implicated in autism spectrum disorder pathophysiology. In addition, we discuss recent unique translational research using rodent models, such as Cntnap2 knockout mouse, Mecp2 mutant mouse, Pick1 knockout mouse, and neonatal ventral hippocampal lesion rat. By using these models, several types of drugs are administered during the developmental period to see the effect on psychotic symptoms and neural activities in adults. The accumulating evidence from recent animal studies provides an informative intervention strategy as a translational research.
Assuntos
Transtornos Cromossômicos/genética , Modelos Animais de Doenças , Transtornos Mentais/genética , Animais , Intervenção Médica Precoce/métodosRESUMO
BACKGROUND: Toxoplasma gondii is a pathogen implicated in psychiatric disorders. As elevated antibodies to T. gondii are also present in non-symptomatic individuals, we hypothesized that the age during first exposure to the pathogen may affect symptom manifestation. We tested this hypothesis by evaluating neurobehavioral abnormalities and the immune response in mice following adolescent or adult T. gondii infection. METHODS: Mice were infected with T. gondii at postnatal day 33 (adolescent/juvenile) or 61 (adult). At 8weeks post-infection (wpi), pre-pulse inhibition of the acoustic startle (PPI) in mice administered MK-801 (0.1 and 0.3mg/kg) and amphetamine (5 and 10mg/kg) was assessed. Peripheral (anti-T. gondii, C1q-associated IgG and anti-GLUN2 antibodies) and central (C1q and Iba1) markers of the immune response were also evaluated. In addition, regional brain expression of N-methyl-d-aspartate receptor (NMDAR) subunits (GLUN1 and GLUN2A), glutamatergic (vGLUT1, PSD95) and GABAergic (GAD67) markers, and monoamines (DA, NE, 5-HT) and their metabolites were measured. RESULTS: Juvenile and adult infected mice exhibited opposite effects of MK-801 on PPI, with decreased PPI in juveniles and increased PPI in adults. There was a significantly greater elevation of GLUN2 autoantibodies in juvenile-compared to adult-infected mice. In addition, age-dependent differences were found in regional expression of NMDAR subunits and markers of glutamatergic, GABAergic, and monoaminergic systems. Activated microglia and C1q elevations were found in both juvenile- and adult-T. gondii infected mice. CONCLUSIONS: Our study demonstrates that the age at first exposure to T. gondii is an important factor in shaping distinct behavioral and neurobiological abnormalities. Elevation in GLUN2 autoantibodies or complement protein C1q may be a potential underlying mechanism. A better understanding of these age-related differences may lead to more efficient treatments of behavioral disorders associated with T. gondii infection.
Assuntos
Autoanticorpos/imunologia , Encéfalo/patologia , Encéfalo/parasitologia , Transtornos Mentais/patologia , Receptores de N-Metil-D-Aspartato/imunologia , Toxoplasma , Envelhecimento , Animais , Imunoglobulina G/metabolismo , Masculino , Camundongos Endogâmicos BALB C , ToxoplasmoseRESUMO
Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Disease pathogenesis derives, at least in part, from the long polyglutamine tract encoded by mutant HTT. Therefore, considerable effort has been dedicated to the development of therapeutic strategies that significantly reduce the expression of the mutant HTT protein. Antisense oligonucleotides (ASOs) targeted to the CAG repeat region of HTT transcripts have been of particular interest due to their potential capacity to discriminate between normal and mutant HTT transcripts. Here, we focus on phosphorodiamidate morpholino oligomers (PMOs), ASOs that are especially stable, highly soluble and non-toxic. We designed three PMOs to selectively target expanded CAG repeat tracts (CTG22, CTG25 and CTG28), and two PMOs to selectively target sequences flanking the HTT CAG repeat (HTTex1a and HTTex1b). In HD patient-derived fibroblasts with expanded alleles containing 44, 77 or 109 CAG repeats, HTTex1a and HTTex1b were effective in suppressing the expression of mutant and non-mutant transcripts. CTGn PMOs also suppressed HTT expression, with the extent of suppression and the specificity for mutant transcripts dependent on the length of the targeted CAG repeat and on the CTG repeat length and concentration of the PMO. PMO CTG25 reduced HTT-induced cytotoxicity in vitro and suppressed mutant HTT expression in vivo in the N171-82Q transgenic mouse model. Finally, CTG28 reduced mutant HTT expression and improved the phenotype of Hdh(Q7/Q150) knock-in HD mice. These data demonstrate the potential of PMOs as an approach to suppressing the expression of mutant HTT.
Assuntos
Morfolinos/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Animais , Sequência de Bases , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Camundongos , Camundongos Transgênicos , Morfolinos/química , Mutação , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Oligonucleotídeos Antissenso/química , RNA Mensageiro/metabolismo , Expansão das Repetições de TrinucleotídeosRESUMO
Exposure to the neurotropic parasite, Toxoplasma gondii, causes significant brain and behavioral anomalies in humans and other mammals. Understanding the cellular mechanisms of T. gondii-generated brain pathologies would aid the advancement of novel strategies to reduce disease. Complement factor C1q is part of a classic immune pathway that functions peripherally to tag and remove infectious agents and cellular debris from circulation. In the developing and adult brain, C1q modifies neuronal architecture through synapse marking and pruning. T. gondii exposure and complement activation have both been implicated in the development of complex brain disorders such as schizophrenia. Thus, it seems logical that mechanistically, the physiological pathways associated with these two factors are connected. We employed a rodent model of chronic infection to investigate the extent to which cyst presence in the brain triggers activation of cerebral C1q. Compared to uninfected mice, cortical C1q was highly expressed at both the RNA and protein levels in infected animals bearing a high cyst burden. In these mice, C1q protein localized to cytoplasm, adjacent to GFAP-labeled astrocytes, near degenerating cysts, and in punctate patterns along processes. In summary, our results demonstrated an upregulation of cerebral C1q in response to latent T. gondii infection. Our data preliminarily suggest that this complement activity may aid in the clearance of this parasite from the CNS and in so doing, have consequences for the connectivity of neighboring cells and synapses.
Assuntos
Córtex Cerebral/imunologia , Córtex Cerebral/parasitologia , Complemento C1q/metabolismo , Toxoplasmose/imunologia , Animais , Doença Crônica , Cistos/imunologia , Feminino , CamundongosRESUMO
MicroRNA-132 (miR-132) has been demonstrated to affect multiple neuronal functions and its dysregulation is linked to several neurological disorders. We previously showed that acute Toxoplasma gondii infection induces miR-132 expression both in vitro and in vivo. To investigate the impact of chronic infection on miR-132, we infected mice with T. gondii PRU strain and performed assessment 5 months later in six brain regions (cortex, hypothalamus, striatum, cerebellum, olfactory bulb and hippocampus) by qPCR. We found that while acute infection of T. gondii increases the expression of miR-132, chronic infection has the opposite effect. The effect varied amongst different regions of the brain and presented in a sex-dependent manner, with females exhibiting more susceptibility than males. MiR-132 and brain-derived neurotrophic factor (BDNF, an inducer of miR-132) were not co-varies in the brain areas of infected mice. T. gondii DNA/RNA was found in all tested brain regions and a selective tropism towards the hippocampus, based on bradyzoite density, was observed in both males and females. However, the expressions of miR-132 or BDNF were poorly reflected by the density of T. gondii in brain areas. Our findings highlight the importance of investigating the miR-132-mediated neuronal function in mice infected with T. gondii.
Assuntos
Encéfalo/parasitologia , MicroRNAs/metabolismo , Toxoplasmose Animal/metabolismo , Toxoplasmose Cerebral/metabolismo , Animais , Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Regulação para Baixo , Feminino , Fibroblastos/parasitologia , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , Carga Parasitária , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Reversa , Fatores Sexuais , ToxoplasmaRESUMO
We developed a protocol to inactivate Toxoplasma gondii (T. gondii) tachyzoites employing 1 min of ultraviolet (UV) exposure. We show that this treatment completely inhibited parasite replication and cyst formation in vitro and in vivo but did not affect the induction of a robust IgG response in mice. We propose that our protocol can be used to study the contribution of the humoral immune response to rodent behavioral alterations following T. gondii infection.
Assuntos
Anticorpos Antiprotozoários/sangue , Imunoglobulina G/sangue , Toxoplasma/efeitos da radiação , Raios Ultravioleta , Animais , Anticorpos Antiprotozoários/biossíntese , Encéfalo/parasitologia , Membrana Celular/efeitos da radiação , Citocinas/genética , Citocinas/metabolismo , Imunoglobulina G/biossíntese , Cinética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA de Protozoário/análise , RNA Ribossômico 5S/análise , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Tempo , Toxoplasma/imunologia , Toxoplasma/fisiologiaRESUMO
Toxoplasma gondii is a protozoan parasite that infects a broad spectrum of hosts and can colonize many organs and cell types. The ability to reside within a wide range of different niches requires substantial adaptability to diverse microenvironments. Very little is known about how this parasite senses various milieus and adapts its metabolism to survive, replicate during the acute stage, and then differentiate to the chronic stage. T. gondii possesses a lysosome-like organelle known as the plant-like vacuolar compartment (PLVAC), which serves various functions, including digestion, ion storage and homeostasis, endocytosis, and autophagy. Lysosomes are critical for maintaining cellular health and function by degrading waste materials and recycling components. To supply the cell with the essential building blocks and energy sources required for the maintenance of its functions and structures, the digested solutes generated within the lysosome are transported into the cytosol by proteins embedded in the lysosomal membrane. Currently, a limited number of PLVAC transporters have been characterized, with TgCRT being the sole potential transporter of amino acids and small peptides identified thus far. To bridge this knowledge gap, we used lysosomal amino acid transporters from other organisms as queries to search the T. gondii proteome. This led to the identification of four potential amino acid transporters, which we have designated as TgAAT1-4. Assessing their expression and sub-cellular localization, we found that one of them, TgAAT1, localized to the PLVAC and is necessary for normal parasite extracellular survival and bradyzoite differentiation. Moreover, we present preliminary data showing the possible involvement of TgAAT1 in the PLVAC transport of arginine.IMPORTANCEToxoplasma gondii is a highly successful parasite infecting a broad range of warm-blooded organisms, including about one-third of all humans. Although Toxoplasma infections rarely result in symptomatic disease in individuals with a healthy immune system, the incredibly high number of persons infected, along with the risk of severe infection in immunocompromised patients and the potential link of chronic infection to mental disorders, makes this infection a significant public health concern. As a result, there is a pressing need for new treatment approaches that are both effective and well tolerated. The limitations in understanding how Toxoplasma gondii manages its metabolism to adapt to changing environments and triggers its transformation into bradyzoites have hindered the discovery of vulnerabilities in its metabolic pathways or nutrient acquisition mechanisms to identify new therapeutic targets. In this work, we have shown that the lysosome-like organelle plant-like vacuolar compartment (PLVAC), acting through the putative arginine transporter TgAAT1, plays a pivotal role in regulating the parasite's extracellular survival and differentiation into bradyzoites.
Assuntos
Parasitos , Toxoplasma , Animais , Humanos , Toxoplasma/metabolismo , Vacúolos/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Arginina/metabolismoRESUMO
The parasite Toxoplasma gondii persists in its hosts by converting from replicating tachyzoites to latent bradyzoites housed in tissue cysts. The molecular mechanisms that mediate T. gondii differentiation remain poorly understood. Through a mutagenesis screen, we identified translation initiation factor eIF1.2 as a critical factor for T. gondii differentiation. A F97L mutation in eIF1.2 or the genetic ablation of eIF1.2 (∆eif1.2) markedly impeded bradyzoite cyst formation in vitro and in vivo. We demonstrated, at single-molecule level, that the eIF1.2 F97L mutation impacts the scanning process of the ribosome preinitiation complex on a model mRNA. RNA sequencing and ribosome profiling experiments unveiled that ∆eif1.2 parasites are defective in upregulating bradyzoite induction factors BFD1 and BFD2 during stress-induced differentiation. Forced expression of BFD1 or BFD2 significantly restored differentiation in ∆eif1.2 parasites. Together, our findings suggest that eIF1.2 functions by regulating the translation of key differentiation factors necessary to establish chronic toxoplasmosis.
Assuntos
Toxoplasma , Toxoplasma/metabolismo , Toxoplasma/genética , Animais , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Toxoplasmose/parasitologia , Toxoplasmose/metabolismo , Camundongos , Mutação , Ribossomos/metabolismo , Biossíntese de Proteínas , Feminino , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Diferenciação Celular , HumanosRESUMO
Gene-environment interactions (GEIs) likely play significant roles in the pathogenesis of schizophrenia and underlie differences in pathological, behavioral, and clinical presentations of the disease. Findings from epidemiology and psychiatric genetics have assisted in the generation of animal models of GEI relevant to schizophrenia. These models may provide a foundation for elucidating the molecular, cellular, and circuitry mechanisms that mediate GEI in schizophrenia. Here we critically review current mouse models of GEI related to schizophrenia, describe directions for their improvement, and propose endophenotypes to provide a more tangible basis for molecular studies of pathways of GEI and facilitate the identification of novel therapeutic targets.
Assuntos
Modelos Animais de Doenças , Interação Gene-Ambiente , Camundongos , Esquizofrenia/genética , Animais , Encéfalo/fisiopatologia , Endofenótipos , Camundongos/genética , Esquizofrenia/etiologia , Esquizofrenia/fisiopatologiaAssuntos
Comportamento Animal , Chlorella/virologia , Cognição , Laringe/virologia , Memória , Mariposas/virologia , Phycodnaviridae , Animais , Feminino , Humanos , MasculinoRESUMO
Toxoplasma gondii is a protozoan parasite that infects a broad spectrum of hosts and can colonize many organs and cell types. The ability to reside within a wide range of different niches requires substantial adaptability to diverse microenvironments. Very little is known about how this parasite senses various milieus and adapts its metabolism to survive, replicate during the acute stage, and then differentiate to the chronic stage. Most eukaryotes, from yeast to mammals, rely on a nutrient sensing machinery involving the TORC complex as master regulator of cell growth and cell cycle progression. The lysosome functions as a signaling hub where TORC complex assembles and is activated by transceptors, which both sense and transport amino acids, including the arginine transceptor SLC38A9. While most of the TORC components are lost in T. gondii , indicating the evolution of a distinct nutrient sensing mechanism, the parasite's lysosomal plant-like vacuolar compartment (PLVAC) may still serve as a sensory platform for controlling parasite growth and differentiation. Using SLC38A9 to query the T. gondii proteome, we identified four putative amino acid transporters, termed TgAAT1-4, that structurally resemble the SLC38A9 arginine transceptor. Assessing their expression and sub-cellular localization, we found that one of them, TgAAT1, localized to the PLVAC and is necessary for normal parasite extracellular survival and bradyzoite differentiation. Moreover, we show that TgAAT1 is involved in the PLVAC efflux of arginine, an amino acid playing a key role in T. gondii differentiation, further supporting the hypothesis that TgAAT1 might play a role in nutrient sensing. IMPORTANCE: T. gondii is a highly successful parasite infecting a broad range of warm-blood organisms including about one third of all humans. Although Toxoplasma infections rarely result in symptomatic disease in individuals with a healthy immune system, the incredibly high number of persons infected along with the risk of severe infection in immunocompromised patients and the potential link of chronic infection to mental disorders make this infection a significant public health concern. As a result, there is a pressing need for new treatment approaches that are both effective and well-tolerated. The limitations in understanding how Toxoplasma gondii manages its metabolism to adapt to changing environments and triggers its transformation into bradyzoites have hindered the discovery of vulnerabilities in its metabolic pathways or nutrient acquisition mechanisms to identify new therapeutic targets. In this work, we have shown that the lysosome-like organelle PLVAC, acting through the putative arginine transporter TgAAT1, plays a pivotal role in regulating the parasite's extracellular survival and differentiation into bradyzoites.
RESUMO
The parasite Toxoplasma gondii persists in its hosts by converting from replicating tachyzoites to latent bradyzoites housed in tissue cysts. The molecular mechanisms that mediate T. gondii differentiation remain poorly understood. Through a mutagenesis screen, we identified translation initiation factor eIF1.2 as a critical factor for T. gondii differentiation. A F97L mutation in eIF1.2 or the genetic ablation of eIF1.2 (Δ eIF1.2 ) markedly impeded bradyzoite cyst formation in vitro and in vivo . We demonstrated, at single-molecule level, that the eIF1.2 F97L mutation impacts the scanning process of the ribosome preinitiation complex on a model mRNA. RNA sequencing and ribosome profiling experiments unveiled that Δ eIF1.2 parasites are defective in the upregulating bradyzoite induction factors BFD1 and BFD2 during stress-induced differentiation. Forced expression of BFD1 or BFD2 significantly restored differentiation in Δ eIF1.2 parasites. Together, our findings suggest that eIF1.2 functions by regulating the translation of key differentiation factors necessary to establish chronic toxoplasmosis.
RESUMO
Toxoplasma gondii is a protozoan parasite that persists in the central nervous system as intracellular chronic-stage bradyzoites that are encapsulated by a thick cyst wall. While the cyst wall separates bradyzoites from the host cytosol, it has been posited that small solutes can traverse the cyst wall to sustain bradyzoites. Recently, it was found that host cytosolic macromolecules can cross the parasitophorous vacuole and are ingested and digested by actively replicating acute-stage tachyzoites. However, the extent to which bradyzoites have an active ingestion pathway remained unknown. To interrogate this, we modified previously published protocols that look at tachyzoite acquisition and digestion of host proteins by measuring parasite accumulation of a host-expressed reporter protein after impairment of an endolysosomal protease (cathepsin protease L [CPL]). Using two cystogenic parasite strains (ME49 and Pru), we demonstrate that T. gondii bradyzoites can ingest host-derived cytosolic mCherry. Bradyzoites acquire host mCherry within 4 h of invasion and after cyst wall formation. This study provides direct evidence that host macromolecules can be internalized by T. gondii bradyzoites across the cyst wall in infected cells.IMPORTANCE Chronic infection of humans with Toxoplasma gondii is common, but little is known about how this intracellular parasite obtains the resources that it needs to persist indefinitely inside neurons and muscle cells. Here, we provide evidence that the chronic-stage form of T. gondii can internalize proteins from the cytosol of infected cells despite residing within an intracellular cyst that is surrounded by a cyst wall. We also show that accumulation of host-derived protein within the chronic-stage parasites is enhanced by disruption of a parasite protease, suggesting that such protein is normally degraded to generate peptides and amino acids. Taken together, our findings imply that chronic-stage T. gondii can ingest and digest host proteins, potentially to support its persistence.
Assuntos
Citosol/metabolismo , Interações Hospedeiro-Parasita , Proteínas Luminescentes/metabolismo , Toxoplasma/metabolismo , Animais , Células CHO , Cricetulus , Doxiciclina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos CBA , Proteína Vermelha FluorescenteRESUMO
Many of the world's warm-blooded species are chronically infected with Toxoplasma gondii tissue cysts, including an estimated one-third of the global human population. The cellular processes that permit long-term persistence within the cyst are largely unknown for T. gondii and related coccidian parasites that impact human and animal health. Herein, we show that genetic ablation of TgATG9 substantially reduces canonical autophagy and compromises bradyzoite viability. Transmission electron microscopy revealed numerous structural abnormalities occurring in ∆atg9 bradyzoites. Intriguingly, abnormal mitochondrial networks were observed in TgATG9-deficient bradyzoites, some of which contained numerous different cytoplasmic components and organelles. ∆atg9 bradyzoite fitness was drastically compromised in vitro and in mice, with very few brain cysts identified in mice 5 weeks post-infection. Taken together, our data suggests that TgATG9, and by extension autophagy, is critical for cellular homeostasis in bradyzoites and is necessary for long-term persistence within the cyst of this coccidian parasite.
Assuntos
Autofagia , Encéfalo/parasitologia , Proteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Toxoplasmose Cerebral/parasitologia , Animais , Encéfalo/patologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Parasita , Humanos , Estágios do Ciclo de Vida , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Camundongos Endogâmicos CBA , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas de Protozoários/genética , Proteínas de Protozoários/ultraestrutura , Fatores de Tempo , Toxoplasma/genética , Toxoplasma/patogenicidade , Toxoplasma/ultraestrutura , Toxoplasmose Cerebral/patologia , Vacúolos/genética , Vacúolos/metabolismo , Vacúolos/ultraestrutura , VirulênciaRESUMO
Toxoplasma gondii reportedly manipulates rodent behaviour to increase transmission to its definitive feline host. We compared the effects of mouse infection by two Type II strains of T. gondii, Prugniaud (PRU) and ME49, on attraction to cat odour, locomotor activity, anxiety, sensorimotor gating, and spatial working and recognition memory 2 months post-infection (mpi). Attraction to cat odour was reassessed 7 mpi. At 2 mpi, mice infected with either strain exhibited significantly more attraction to cat odour than uninfected animals did, but only PRU-infected mice exhibited this behaviour 7 mpi. PRU-infected mice had significantly greater body weights and hyperactivity, while ME49-infected mice exhibited impaired spatial working memory. No differences in parasite antibody titres were seen between PRU- and ME49-infected mice. The present data suggest the effect of T. gondii infection on mouse behaviour is parasite strain-dependent.
Assuntos
Comportamento Animal , Toxoplasma/classificação , Toxoplasmose Animal/psicologia , Animais , Gatos/urina , Feminino , Camundongos , Camundongos Endogâmicos BALB C , OdorantesRESUMO
The lysosome-like vacuolar compartment (VAC) is a major site of proteolysis in the intracellular parasite Toxoplasma gondii Previous studies have shown that genetic ablation of a VAC-residing cysteine protease, cathepsin protease L (CPL), resulted in the accumulation of undigested protein in the VAC and loss of parasite viability during the chronic stage of infection. However, since the maturation of another VAC localizing protease, cathepsin protease B (CPB), is dependent on CPL, it remained unknown whether these defects result directly from ablation of CPL or indirectly from a lack of CPB maturation. Likewise, although a previously described cathepsin D-like aspartyl protease 1 (ASP1) could also play a role in proteolysis, its definitive residence and function in the Toxoplasma endolysosomal system were not well defined. Here, we demonstrate that CPB is not necessary for protein turnover in the VAC and that CPB-deficient parasites have normal growth and viability in both the acute and chronic stages of infection. We also show that ASP1 depends on CPL for correct maturation, and it resides in the T. gondii VAC, where, similar to CPB, it plays a dispensable role in protein digestion. Taken together with previous work, our findings suggest that CPL is the dominant protease in a hierarchy of proteolytic enzymes within the VAC. This unusual lack of redundancy for CPL in T. gondii makes it a single exploitable target for disrupting chronic toxoplasmosis.IMPORTANCE Roughly one-third of the human population is chronically infected with the intracellular single-celled parasite Toxoplasma gondii, but little is known about how this organism persists inside people. Previous research suggested that a parasite proteolytic enzyme, termed cathepsin protease L, is important for Toxoplasma persistence; however, it remained possible that other associated proteolytic enzymes could also be involved in the long-term survival of the parasite during infection. Here, we show that two proteolytic enzymes associated with cathepsin protease L play dispensable roles and are dependent on cathepsin L to reach maturity, which differs from the corresponding enzymes in humans. These findings establish a divergent hierarchy of proteases and help focus attention principally on cathepsin protease L as a potential target for interrupting Toxoplasma chronic infection.
Assuntos
Ácido Aspártico Proteases/metabolismo , Catepsina B/metabolismo , Lisossomos/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Humanos , Estágios do Ciclo de Vida , Proteólise , Toxoplasma/crescimento & desenvolvimento , Vacúolos/metabolismoRESUMO
Toxoplasma gondii is a ubiquitous pathogen that can cause encephalitis, congenital defects, and ocular disease. T. gondii has also been implicated as a risk factor for mental illness in humans. The parasite persists in the brain as slow-growing bradyzoites contained within intracellular cysts. No treatments exist to eliminate this form of parasite. Although proteolytic degradation within the parasite lysosome-like vacuolar compartment (VAC) is critical for bradyzoite viability, whether other aspects of the VAC are important for parasite persistence remains unknown. An ortholog of Plasmodium falciparum chloroquine resistance transporter (CRT), TgCRT, has previously been identified in T. gondii To interrogate the function of TgCRT in chronic-stage bradyzoites and its role in persistence, we knocked out TgCRT in a cystogenic strain and assessed VAC size, VAC digestion of host-derived proteins and parasite autophagosomes, and the viability of in vitro and in vivo bradyzoites. We found that whereas parasites deficient in TgCRT exhibit normal digestion within the VAC, they display a markedly distended VAC and their viability is compromised both in vitro and in vivo Interestingly, impairing VAC proteolysis in TgCRT-deficient bradyzoites restored VAC size, consistent with a role for TgCRT as a transporter of products of digestion from the VAC. In conjunction with earlier studies, our current findings suggest a functional link between TgCRT and VAC proteolysis. This study provides further evidence of a crucial role for the VAC in bradyzoite persistence and a new potential VAC target to abate chronic Toxoplasma infection.IMPORTANCE Individuals chronically infected with the intracellular parasite Toxoplasma gondii are at risk of experiencing reactivated disease that can result in progressive loss of vision. No effective treatments exist for chronic toxoplasmosis due in part to a poor understanding of the biology underlying chronic infection and a lack of well-validated potential targets. We show here that a T. gondii transporter is functionally linked to protein digestion within the parasite lysosome-like organelle and that this transporter is necessary to sustain chronic infection in culture and in experimentally infected mice. Ablating the transporter results in severe bloating of the lysosome-like organelle. Together with earlier work, this study suggests the parasite's lysosome-like organelle is vital for parasite survival, thus rendering it a potential target for diminishing infection and reducing the risk of reactivated disease.