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1.
Life Sci ; 283: 119872, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34352261

RESUMO

The interaction of Toxoplasma gondii with the gastrointestinal tract of its host is highly regulated. Once ingested, the parasite crosses the epithelium without altering the permeability of the intestinal barrier. Nevertheless, many studies report alterations ranging from structural to functional damage in cells and tissues that make up the wall of the small and large intestine. Although the immune response to the parasite has been extensively studied, the role of serotonin (5-HT) in toxoplasmosis is poorly understood. Here we investigate the distribution of cells expressing 5-HT and its effects on cells and tissues of the jejunal wall of rats after 2, 3, or 7 days of T. gondii infection. KEY RESULTS: Our results show that transposition of the jejunal epithelium by T. gondii leads to ruptures in the basement membrane and activation of the immune system, as confirmed by the decrease in laminin immunostaining and the increase in the number of mast cells, respectively. CONCLUSIONS AND INFERENCES: We showed an increase in the number of enterochromaffin cells and mast cells expressing 5-HT in the jejunal wall. We also observed that the percentage of serotonergic mast cells increased in the total population. Thus, we can suggest that oral infection by T. gondii oocysts preferentially activates non-neuronal cells expressing 5-HT. Together, these results may explain both the changes in the extracellular matrix and the morphology of the enteric ganglia.


Assuntos
Células Enterocromafins , Jejuno , Oocistos/metabolismo , Serotonina/biossíntese , Toxoplasma/metabolismo , Toxoplasmose/metabolismo , Doença Aguda , Animais , Células Enterocromafins/metabolismo , Células Enterocromafins/parasitologia , Jejuno/metabolismo , Jejuno/parasitologia , Masculino , Ratos , Ratos Wistar
2.
Nat Commun ; 12(1): 4983, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404783

RESUMO

Parasites of the phylum Apicomplexa cause important diseases including malaria, cryptosporidiosis and toxoplasmosis. These intracellular pathogens inject the contents of an essential organelle, the rhoptry, into host cells to facilitate invasion and infection. However, the structure and mechanism of this eukaryotic secretion system remain elusive. Here, using cryo-electron tomography and subtomogram averaging, we report the conserved architecture of the rhoptry secretion system in the invasive stages of two evolutionarily distant apicomplexans, Cryptosporidium parvum and Toxoplasma gondii. In both species, we identify helical filaments, which appear to shape and compartmentalize the rhoptries, and an apical vesicle (AV), which facilitates docking of the rhoptry tip at the parasite's apical region with the help of an elaborate ultrastructure named the rhoptry secretory apparatus (RSA); the RSA anchors the AV at the parasite plasma membrane. Depletion of T. gondii Nd9, a protein required for rhoptry secretion, disrupts the RSA ultrastructure and AV-anchoring. Moreover, T. gondii contains a line of AV-like vesicles, which interact with a pair of microtubules and accumulate towards the AV, leading to a working model for AV-reloading and discharging of multiple rhoptries. Together, our analyses provide an ultrastructural framework to understand how these important parasites deliver effectors into host cells.


Assuntos
Organelas/metabolismo , Organelas/ultraestrutura , Parasitos/metabolismo , Parasitos/ultraestrutura , Proteínas de Protozoários/química , Animais , Evolução Biológica , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Criptosporidiose , Cryptosporidium , Cryptosporidium parvum/citologia , Cryptosporidium parvum/efeitos dos fármacos , Cryptosporidium parvum/metabolismo , Interações Hospedeiro-Parasita , Microtúbulos/ultraestrutura , Proteínas de Protozoários/metabolismo , Toxoplasma/citologia , Toxoplasma/efeitos dos fármacos , Toxoplasma/metabolismo , Toxoplasmose
3.
Front Cell Infect Microbiol ; 11: 728425, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34447699

RESUMO

The ability of an organism to sense and respond to environmental redox fluctuations relies on a signaling network that is incompletely understood in apicomplexan parasites such as Toxoplasma gondii. The impact of changes in redox upon the development of this intracellular parasite is not known. Here, we provide a revised collection of 58 genes containing domains related to canonical antioxidant function, with their encoded proteins widely dispersed throughout different cellular compartments. We demonstrate that addition of exogenous H2O2 to human fibroblasts infected with T. gondii triggers a Ca2+ flux in the cytosol of intracellular parasites that can induce egress. In line with existing models, egress triggered by exogenous H2O2 is reliant upon both Calcium-Dependent Protein Kinase 3 and diacylglycerol kinases. Finally, we show that the overexpression a glutaredoxin-roGFP2 redox sensor fusion protein in the parasitophorous vacuole severely impacts parasite replication. These data highlight the rich redox network that exists in T. gondii, evidencing a link between extracellular redox and intracellular Ca2+ signaling that can culminate in parasite egress. Our findings also indicate that the redox potential of the intracellular environment contributes to normal parasite growth. Combined, our findings highlight the important role of redox as an unexplored regulator of parasite biology.


Assuntos
Toxoplasma , Cálcio/metabolismo , Sinalização do Cálcio , Humanos , Peróxido de Hidrogênio , Oxirredução , Toxoplasma/metabolismo
4.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360709

RESUMO

Toxoplasma gondii (T. gondii) is an important human and veterinary pathogen causing life-threatening disease in immunocompromised patients. The UBL-UBA shuttle protein family are important components of the ubiquitin-proteasome system. Here, we identified a novel UBL-UBA shuttle protein DSK2b that is charactered by an N-terminal ubiquitin-like domain (UBL) and a C-terminal ubiquitin-associated domain (UBA). DSK2b was localized in the cytoplasm and nucleus. The deletion of dsk2b did not affect the degradation of ubiquitinated proteins, parasite growth in vitro or virulence in mice. The double-gene knockout of dsk2b and its paralogs dsk2a (ΔΔdsk2adsk2b) results in a significant accumulation of ubiquitinated proteins and the asynchronous division of T. gondii. The growth of ΔΔdsk2adsk2b was significantly inhibited in vitro, while virulence in mice was not attenuated. In addition, autophagy occurred in the ΔΔdsk2adsk2b, which was speculated to degrade the accumulated ubiquitinated proteins in the parasites. Overall, DSK2b is a novel UBL-UBA shuttle protein contributing to the degradation of ubiquitinated proteins and is important for the synchronous cell division of T. gondii.


Assuntos
Divisão Celular , Proteólise , Proteínas de Protozoários/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Toxoplasma/metabolismo , Ubiquitinação , Animais , Deleção de Genes , Humanos , Camundongos , Proteínas de Protozoários/genética , Receptores Citoplasmáticos e Nucleares/genética , Toxoplasma/genética , Toxoplasma/patogenicidade
5.
J Immunol ; 207(6): 1507-1512, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34400524

RESUMO

Resistance and tolerance are vital for survivability of the host-pathogen relationship. Virulence during Toxoplasma infection in mice is mediated by parasite kinase-dependent antagonism of IFN-γ-induced host resistance. Whether avirulence requires expression of parasite factors that induce host tolerance mechanisms or is a default status reflecting the absence of resistance-interfering factors is not known. In this study, we present evidence that avirulence in Toxoplasma requires parasite engagement of the scavenger receptor CD36. CD36 promotes macrophage tropism but is dispensable for the development of resistance mechanisms. Instead CD36 is critical for re-establishing tissue homeostasis and survival following the acute phase of infection. The CD36-binding capacity of T. gondii strains is negatively controlled by the virulence factor, ROP18. Thus, the absence of resistance-interfering virulence factors and the presence of tolerance-inducing avirulence factors are both required for long-term host-pathogen survival.


Assuntos
Antígenos CD36/deficiência , Antígenos CD36/metabolismo , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/parasitologia , Toxoplasma/metabolismo , Toxoplasma/patogenicidade , Toxoplasmose Animal/imunologia , Animais , Antígenos CD36/genética , Células CHO , Cricetulus , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Tolerância Imunológica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Protozoários/metabolismo , Células RAW 264.7 , Toxoplasmose Animal/metabolismo , Toxoplasmose Animal/parasitologia , Virulência/genética , Fatores de Virulência/metabolismo
6.
Nat Commun ; 12(1): 3788, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145271

RESUMO

Active host cell invasion by the obligate intracellular apicomplexan parasites relies on the formation of a moving junction, which connects parasite and host cell plasma membranes during entry. Invading Toxoplasma gondii tachyzoites secrete their rhoptry content and insert a complex of RON proteins on the cytoplasmic side of the host cell membrane providing an anchor to which the parasite tethers. Here we show that a rhoptry-resident kinase RON13 is a key virulence factor that plays a crucial role in host cell entry. Cryo-EM, kinase assays, phosphoproteomics and cellular analyses reveal that RON13 is a secretory pathway kinase of atypical structure that phosphorylates rhoptry proteins including the components of the RON complex. Ultimately, RON13 kinase activity controls host cell invasion by anchoring the moving junction at the parasite-host cell interface.


Assuntos
Membrana Celular/parasitologia , Proteínas de Protozoários/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Toxoplasma/metabolismo , Toxoplasmose/patologia , Transporte Biológico/fisiologia , Células Cultivadas , Interações Hospedeiro-Parasita , Humanos , Via Secretória/fisiologia , Fatores de Virulência
7.
Mol Biochem Parasitol ; 244: 111384, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34051228

RESUMO

A tetracycline-responsive transcription system (Tet-Off) adapted for use in Toxoplasma gondii (nicknamed TATi) is useful for molecular biological studies of this organism. Previous studies using TATi incorporated minimal promoters derived from the gene promoters for TgSAG1 or TgSAG4. The present study achieves improved activation and suppression of an integrated reporter gene in the absence and presence of anhydrotetracycline, respectively (p < 0.0001), by use of a newly derived minimal promoter based on the core promoter of TgGRA2. In comparison with the SAG1 minimal promoter, use of the GRA2 minimal promoter in stable transfectants has a 23-fold higher Signal to Noise Ratio for EYFP fluorescence in the absence or presence of anhydrotetracycline. We conclude that the performance of TATi for both activation and suppression of transcription can be markedly enhanced by incorporating a GRA2 minimal promoter.


Assuntos
Antígenos de Protozoários/genética , Proteínas de Bactérias/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Luminescentes/genética , Regiões Promotoras Genéticas , Proteínas de Protozoários/genética , Tetraciclinas/farmacologia , Toxoplasma/efeitos dos fármacos , Antígenos de Protozoários/metabolismo , Proteínas de Bactérias/metabolismo , Células Cultivadas , Clonagem Molecular/métodos , Fibroblastos/parasitologia , Expressão Gênica , Genes Reporter , Humanos , Proteínas Luminescentes/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismo , Transcrição Genética , Transfecção
8.
Nat Commun ; 12(1): 3065, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031406

RESUMO

In living cells, microtubules (MTs) play pleiotropic roles, which require very different mechanical properties. Unlike the dynamic MTs found in the cytoplasm of metazoan cells, the specialized cortical MTs from Toxoplasma gondii, a prevalent human pathogen, are extraordinarily stable and resistant to detergent and cold treatments. Using single-particle cryo-EM, we determine their ex vivo structure and identify three proteins (TrxL1, TrxL2 and SPM1) as bona fide microtubule inner proteins (MIPs). These three MIPs form a mesh on the luminal surface and simultaneously stabilize the tubulin lattice in both longitudinal and lateral directions. Consistent with previous observations, deletion of the identified MIPs compromises MT stability and integrity under challenges by chemical treatments. We also visualize a small molecule like density at the Taxol-binding site of ß-tubulin. Our results provide the structural basis to understand the stability of cortical MTs and suggest an evolutionarily conserved mechanism of MT stabilization from the inside.


Assuntos
Microscopia Crioeletrônica , Interações Hospedeiro-Parasita/fisiologia , Proteínas dos Microtúbulos/ultraestrutura , Microtúbulos/ultraestrutura , Toxoplasma/metabolismo , Sítios de Ligação , Sistemas CRISPR-Cas , Humanos , Proteínas dos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Paclitaxel/química , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
9.
Nat Commun ; 12(1): 2813, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001876

RESUMO

Apicomplexa are obligate intracellular parasites responsible for major human diseases. Their intracellular survival relies on intense lipid synthesis, which fuels membrane biogenesis. Parasite lipids are generated as an essential combination of fatty acids scavenged from the host and de novo synthesized within the parasite apicoplast. The molecular and metabolic mechanisms allowing regulation and channeling of these fatty acid fluxes for intracellular parasite survival are currently unknown. Here, we identify an essential phosphatidic acid phosphatase in Toxoplasma gondii, TgLIPIN, as the central metabolic nexus responsible for controlled lipid synthesis sustaining parasite development. Lipidomics reveal that TgLIPIN controls the synthesis of diacylglycerol and levels of phosphatidic acid that regulates the fine balance of lipids between storage and membrane biogenesis. Using fluxomic approaches, we uncover the first parasite host-scavenged lipidome and show that TgLIPIN prevents parasite death by 'lipotoxicity' through effective channeling of host-scavenged fatty acids to storage triacylglycerols and membrane phospholipids.


Assuntos
Membrana Celular/metabolismo , Lipidômica/métodos , Fosfatidato Fosfatase/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/parasitologia , Prepúcio do Pênis/citologia , Técnicas de Silenciamento de Genes , Homeostase/genética , Interações Hospedeiro-Parasita , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Fosfatidato Fosfatase/genética , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasma/ultraestrutura
10.
mSphere ; 6(2)2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33789945

RESUMO

The environmental stage of the apicomplexan Toxoplasma gondii oocyst is vital to its life cycle but largely understudied. Because oocysts are excreted only by infected felids, their availability for research is limited. We report the adaptation of an agarose-based method to immobilize minute amounts of oocysts to perform immunofluorescence assays. Agarose embedding allows high-resolution confocal microscopy imaging of antibodies binding to the oocyst surface as well as unprecedented imaging of intracellular sporocyst structures with Maclura pomifera agglutinin after on-slide permeabilization of the immobilized oocysts. To identify new possible molecules binding to the oocyst surface, we used this method to screen a library of C-type lectin receptor (CLR)-human IgG constant region fusion proteins from the group of related CLRs called the Dectin-1 cluster against oocysts. In addition to CLEC7A that was previously reported to decorate T. gondii oocysts, we present experimental evidence for specific binding of three additional CLRs to the surface of this stage. We discuss how these CLRs, known to be expressed on neutrophils, dendritic cells, or macrophages, could be involved in the early immune response by the host, such as oocyst antigen uptake in the intestine. In conclusion, we present a modified immunofluorescence assay technique that allows material-saving immunofluorescence microscopy with T. gondii oocysts in a higher resolution than previously published, which allowed us to describe three additional CLRs binding specifically to the oocyst surface.IMPORTANCE Knowledge of oocyst biology of Toxoplasma gondii is limited, not the least due to its limited availability. We describe a method that permits us to process minute amounts of oocysts for immunofluorescence microscopy without compromising their structural properties. This method allowed us to visualize internal structures of sporocysts by confocal microscopy in unprecedented quality. Moreover, the method can be used as a low- to medium-throughput method to screen for molecules interacting with oocysts, such as antibodies, or compounds causing structural damage to oocysts (i.e., disinfectants). Using this method, we screened a small library of C-type lectin receptors (CLRs) present on certain immune cells and found three CLRs able to decorate the oocyst wall of T. gondii and which were not known before to bind to oocysts. These tools will allow further study into oocyst wall composition and could also provoke experiments regarding immunological recognition of oocysts.


Assuntos
Lectinas Tipo C/metabolismo , Microscopia de Fluorescência/métodos , Oocistos/química , Oocistos/metabolismo , Toxoplasma/metabolismo , Oocistos/ultraestrutura
11.
Int J Mol Sci ; 22(5)2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33803262

RESUMO

In this study, we confirmed that the number of resident homeostatic microglia increases during chronic Toxoplasma gondii infection. Given that the progression of Alzheimer's disease (AD) worsens with the accumulation of amyloid ß (Aß) plaques, which are eliminated through microglial phagocytosis, we hypothesized that T. gondii-induced microglial proliferation would reduce AD progression. Therefore, we investigated the association between microglial proliferation and Aß plaque burden using brain tissues isolated from 5XFAD AD mice (AD group) and T. gondii-infected AD mice (AD + Toxo group). In the AD + Toxo group, amyloid plaque burden significantly decreased compared with the AD group; conversely, homeostatic microglial proliferation, and number of plaque-associated microglia significantly increased. As most plaque-associated microglia shifted to the disease-associated microglia (DAM) phenotype in both AD and AD + Toxo groups and underwent apoptosis after the lysosomal degradation of phagocytosed Aß plaques, this indicates that a sustained supply of homeostatic microglia is required for alleviating Aß plaque burden. Thus, chronic T. gondii infection can induce microglial proliferation in the brains of mice with progressed AD; a sustained supply of homeostatic microglia is a promising prospect for AD treatment.


Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Microglia , Toxoplasma/metabolismo , Toxoplasmose , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/parasitologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Microglia/parasitologia , Microglia/patologia , Toxoplasmose/genética , Toxoplasmose/metabolismo , Toxoplasmose/patologia
12.
PLoS Biol ; 19(3): e3001020, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33705377

RESUMO

Malaria is caused by unicellular Plasmodium parasites. Plasmodium relies on diverse microtubule cytoskeletal structures for its reproduction, multiplication, and dissemination. Due to the small size of this parasite, its cytoskeleton has been primarily observable by electron microscopy (EM). Here, we demonstrate that the nanoscale cytoskeleton organisation is within reach using ultrastructure expansion microscopy (U-ExM). In developing microgametocytes, U-ExM allows monitoring the dynamic assembly of axonemes and concomitant tubulin polyglutamylation in whole cells. In the invasive merozoite and ookinete forms, U-ExM unveils the diversity across Plasmodium stages and species of the subpellicular microtubule arrays that confer cell rigidity. In ookinetes, we additionally identify an apical tubulin ring (ATR) that colocalises with markers of the conoid in related apicomplexan parasites. This tubulin-containing structure was presumed to be lost in Plasmodium despite its crucial role in motility and invasion in other apicomplexans. Here, U-ExM reveals that a divergent and considerably reduced form of the conoid is actually conserved in Plasmodium species.


Assuntos
Citoesqueleto/ultraestrutura , Microtúbulos/ultraestrutura , Toxoplasma/ultraestrutura , Animais , Citoesqueleto/metabolismo , Malária/metabolismo , Malária/parasitologia , Microscopia Eletrônica/métodos , Microtúbulos/metabolismo , Parasitos , Plasmodium/metabolismo , Plasmodium/patogenicidade , Plasmodium/ultraestrutura , Toxoplasma/metabolismo , Toxoplasma/patogenicidade , Tubulina (Proteína)
13.
PLoS Biol ; 19(3): e3001081, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33705380

RESUMO

The apical complex is the instrument of invasion used by apicomplexan parasites, and the conoid is a conspicuous feature of this apparatus found throughout this phylum. The conoid, however, is believed to be heavily reduced or missing from Plasmodium species and other members of the class Aconoidasida. Relatively few conoid proteins have previously been identified, making it difficult to address how conserved this feature is throughout the phylum, and whether it is genuinely missing from some major groups. Moreover, parasites such as Plasmodium species cycle through 3 invasive forms, and there is the possibility of differential presence of the conoid between these stages. We have applied spatial proteomics and high-resolution microscopy to develop a more complete molecular inventory and understanding of the organisation of conoid-associated proteins in the model apicomplexan Toxoplasma gondii. These data revealed molecular conservation of all conoid substructures throughout Apicomplexa, including Plasmodium, and even in allied Myzozoa such as Chromera and dinoflagellates. We reporter-tagged and observed the expression and location of several conoid complex proteins in the malaria model P. berghei and revealed equivalent structures in all of its zoite forms, as well as evidence of molecular differentiation between blood-stage merozoites and the ookinetes and sporozoites of the mosquito vector. Collectively, we show that the conoid is a conserved apicomplexan element at the heart of the invasion mechanisms of these highly successful and often devastating parasites.


Assuntos
Apicomplexa/metabolismo , Plasmodium/metabolismo , Evolução Biológica , Citoesqueleto/metabolismo , Evolução Molecular , Malária/parasitologia , Mosquitos Vetores/metabolismo , Plasmodium/patogenicidade , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Toxoplasma/patogenicidade
14.
PLoS Pathog ; 17(3): e1009301, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33651838

RESUMO

The mitochondrial electron transport chain (mETC) and F1Fo-ATP synthase are of central importance for energy and metabolism in eukaryotic cells. The Apicomplexa, important pathogens of humans causing diseases such as toxoplasmosis and malaria, depend on their mETC in every known stage of their complicated life cycles. Here, using a complexome profiling proteomic approach, we have characterised the Toxoplasma mETC complexes and F1Fo-ATP synthase. We identified and assigned 60 proteins to complexes II, IV and F1Fo-ATP synthase of Toxoplasma, of which 16 have not been identified previously. Notably, our complexome profile elucidates the composition of the Toxoplasma complex III, the target of clinically used drugs such as atovaquone. We identified two new homologous subunits and two new parasite-specific subunits, one of which is broadly conserved in myzozoans. We demonstrate all four proteins are essential for complex III stability and parasite growth, and show their depletion leads to decreased mitochondrial potential, supporting their assignment as complex III subunits. Our study highlights the divergent subunit composition of the apicomplexan mETC and F1Fo-ATP synthase complexes and sets the stage for future structural and drug discovery studies.


Assuntos
Transporte de Elétrons/fisiologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Toxoplasma/metabolismo , Animais , Humanos , Parasitos/metabolismo , Proteômica/métodos , Proteínas de Protozoários/metabolismo , Toxoplasmose/metabolismo
15.
Parasitol Res ; 120(5): 1617-1626, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33655350

RESUMO

Lysine crotonylation (Kcr) is an evolutionally conserved post-translational modification (PTM) on histone proteins. However, information about Kcr and its involvement in the biology and metabolism of Toxoplasma gondii is limited. In the present study, a global Kcr proteome analysis using LC-MS/MS in combination with immune-affinity method was performed. A total of 12,152 Kcr sites distributed over 2719 crotonylated proteins were identified. Consistent with lysine acetylation and succinylation in Apicomplexa, Kcr was associated with various metabolic pathways, including carbon metabolism, pyrimidine metabolism, glycolysis, gluconeogenesis, and proteasome. Markedly, many stage-specific proteins, histones, and histone-modifying enzymes related to the stage transition were found to have Kcr sites, suggesting a potential involvement of Kcr in the parasite stage transformation. Most components of the apical secretory organelles were identified as crotonylated proteins which were associated with the attachment, invasion, and replication of T. gondii. These results expanded our understanding of Kcr proteome and proposed new hypotheses for further research of the Kcr roles in the pathobiology of T. gondii infection.


Assuntos
Histonas/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Acetilação , Cromatografia Líquida , Redes e Vias Metabólicas , Proteoma/metabolismo , Espectrometria de Massas em Tandem
16.
PLoS Pathog ; 17(2): e1009211, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33524071

RESUMO

The mitochondrion is critical for the survival of apicomplexan parasites. Several major anti-parasitic drugs, such as atovaquone and endochin-like quinolones, act through inhibition of the mitochondrial electron transport chain at the coenzyme Q:cytochrome c oxidoreductase complex (Complex III). Despite being an important drug target, the protein composition of Complex III of apicomplexan parasites has not been elucidated. Here, we undertake a mass spectrometry-based proteomic analysis of Complex III in the apicomplexan Toxoplasma gondii. Along with canonical subunits that are conserved across eukaryotic evolution, we identify several novel or highly divergent Complex III components that are conserved within the apicomplexan lineage. We demonstrate that one such subunit, which we term TgQCR11, is critical for parasite proliferation, mitochondrial oxygen consumption and Complex III activity, and establish that loss of this protein leads to defects in Complex III integrity. We conclude that the protein composition of Complex III in apicomplexans differs from that of the mammalian hosts that these parasites infect.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Toxoplasma/metabolismo , Animais , Western Blotting , Células Cultivadas , Complexo III da Cadeia de Transporte de Elétrons/química , Imunofluorescência , Humanos , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Smegmamorpha , Toxoplasma/genética
17.
FASEB J ; 35(2): e21352, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33543805

RESUMO

Toxoplasma gondii is an apicomplexan parasite, which has three unique secretory organelles: micronemes, rhoptries, and dense granules. Almost all the secreted proteins are transported through the endoplasmic reticulum (ER) and Golgi system to function in their respective destination by accurate targeting and packaging. Glutathione S-transferase (GST) is a supergene family enzyme that has multiple functions, which include regulation of cell proliferation and death signaling pathways, and participation in transportation and metabolism in mammal cells. However, the role of GST in Toxoplasma gondii has not been explained. In this study, we identified three GST proteins in T gondii, of which GST2 acts as a membrane protein that localizes to the Golgi-endosomal system and colocalizes with proteins involved in vesicle transport as well, including synaptobrevin, putative sortilin (VPS10), Rab5 and Rab6, which function as vesicle transport factors. Moreover, the loss of TgGST2 leads to Rab5 and Rab6 distribution of discrete puncta, and incorrect localization and decreased expression of several secretory proteins, and to significantly reduced invasion capacity and virulence to mice. Consistent with its relation to vesicle transport proteins, the distribution of TgGST2 relies on post-Golgi trafficking. Overall, our findings demonstrated that TgGST2 contributes to vesicle trafficking and plays a critical role in parasite lytic cycle.


Assuntos
Glutationa Transferase/metabolismo , Proteínas de Protozoários/metabolismo , Via Secretória , Toxoplasma/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Transporte Proteico , Proteínas R-SNARE/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo
18.
Cell Calcium ; 94: 102337, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33524795

RESUMO

Toxoplasma gondii is an obligate intracellular parasite and replicates inside a parasitophorous vacuole (PV) within the host cell. The membrane of the PV (PVM) contains pores that permits for equilibration of ions and small molecules between the host cytosol and the PV lumen. Ca2+ signaling is universal and both T. gondii and its mammalian host cell utilize Ca2+ signals to stimulate diverse cellular functions. Egress of T. gondii from host cells is an essential step for the infection cycle of T. gondii, and a cytosolic Ca2+ increase initiates a Ca2+ signaling cascade that culminates in the stimulation of motility and egress. In this work we demonstrate that intracellular T. gondii tachyzoites are able to take up Ca2+ from the host cytoplasm during host cell signaling events. Both intracellular and extracellular Ca2+ sources are important in reaching a threshold of parasite cytosolic Ca2+ needed for successful egress. Two peaks of Ca2+ were observed in egressing single parasites with the second peak resulting from Ca2+ entry. We patched infected host cells to allow the delivery of precise concentrations of Ca2+ for the stimulation of motility and egress. Using this approach of patching infected host cells, allowed us to determine that increasing the host cytosolic Ca2+ to a specific concentration can trigger egress, which is further accelerated by diminishing the concentration of potassium (K+).


Assuntos
Sinalização do Cálcio , Interações Hospedeiro-Patógeno , Potássio/metabolismo , Toxoplasma/metabolismo , Animais , Cálcio/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Células HeLa , Humanos , Espaço Intracelular/parasitologia , Modelos Biológicos , Parasitos/metabolismo
19.
mBio ; 12(1)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33500345

RESUMO

Toxoplasma gondii is an obligate intracellular parasite that persists in its vertebrate hosts in the form of dormant tissue cysts, which facilitate transmission through predation. The parasite must strike a balance that allows it to disseminate throughout its host without killing it, which requires the ability to properly counter host cell defenses. For example, oxidative stress encountered by Toxoplasma is suggested to impair parasite replication and dissemination. However, the strategies by which Toxoplasma mitigates oxidative stress are not yet clear. Among eukaryotes, environmental stresses induce the integrated stress response via phosphorylation of a translation initiation factor, eukaryotic initiation factor 2 (eIF2). Here, we show that the Toxoplasma eIF2 kinase TgIF2K-B is activated in response to oxidative stress and affords protection. Knockout of the TgIF2K-B gene, Δtgif2k-b, disrupted parasite responses to oxidative stresses and enhanced replication, diminishing the ability of the parasite to differentiate into tissue cysts. In addition, parasites lacking TgIF2K-B exhibited resistance to activated macrophages and showed greater virulence in an in vivo model of infection. Our results establish that TgIF2K-B is essential for Toxoplasma responses to oxidative stress, which are important for the parasite's ability to establish persistent infection in its host.IMPORTANCE Toxoplasma gondii is a single-celled parasite that infects nucleated cells of warm-blooded vertebrates, including one-third of the human population. The parasites are not cleared by the immune response and persist in the host by converting into a latent tissue cyst form. Development of tissue cysts can be triggered by cellular stresses, which activate a family of TgIF2 kinases to phosphorylate the eukaryotic translation initiation factor TgIF2α. Here, we establish that the TgIF2 kinase TgIF2K-B is activated by oxidative stress and is critical for maintaining oxidative balance in the parasite. Depletion of TgIF2K-B alters gene expression, leading to accelerated growth and a diminished ability to convert into tissue cysts. This study establishes that TgIF2K-B is essential for the parasite's oxidative stress response and its ability to persist in the host as a latent infection.


Assuntos
Interações Hospedeiro-Parasita , Estresse Oxidativo , Toxoplasma/metabolismo , Toxoplasma/patogenicidade , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , Fibroblastos/parasitologia , Prepúcio do Pênis/citologia , Técnicas de Inativação de Genes , Humanos , Masculino , Fosforilação , Estresse Fisiológico , Toxoplasma/enzimologia , Virulência
20.
mSphere ; 6(1)2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504659

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
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