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1.
Microbiol Res ; 230: 126352, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31634726

RESUMO

Rhoptry protein 18 (ROP18) is a major determinant of strain-specific virulence in Toxoplasma gondii. The kinase activity of ROP18 is required for acute virulence, while the aspartate in the catalytic loop of ROP18 is considered essential for phosphoryl transfer. We showed that a single amino acid mutation at the catalytic aspartate residue (D409A mutation) significantly altered ROP18 kinase activity in vitro, and abolished ROP18-mediated ATF6ß degradation. Furthermore, the investigated single amino acid mutation in ROP18 led to alternation of subcellular localization of ROP18 protein. Our findings demonstrate that a single amino acid mutation on the proton transport catalytic aspartic acid induced alternations associated with ROP18 protein.


Assuntos
Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Toxoplasma/enzimologia , Motivos de Aminoácidos , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Mutação de Sentido Incorreto , Transporte Proteico , Proteínas Serina-Treonina Quinases/química , Prótons , Toxoplasma/química , Toxoplasma/genética , Toxoplasma/metabolismo
2.
Eur J Med Chem ; 184: 111765, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31629163

RESUMO

Synthesis and investigation of anti-Toxoplasma gondii activity of novel thiazoles containing benzo [b]thiophene moiety are presented. Among the derivatives, compound 3k with adamantyl group shows exceptionally high potency against Me49 strain with IC50 (8.74 µM) value which is significantly lower than the activity of trimethoprim (IC50 39.23 µM). In addition, compounds 3a, 3b and 3k showed significant activity against RH strain (IC50 51.88-83.49 µM). The results of the cytotoxicity evaluation showed that Toxoplasma gondii growth was inhibited at non-cytotoxic concentrations for the mammalian L929 fibroblast (CC30 ∼ 880 µM). The most active compound 3k showed tyrosinase inhibition effect, with IC50 value of 328.5 µM. The binding energies calculated for compounds 3a-3e, 3k are strongly correlated with the experimentally determined values of tyrosinase inhibition activity. Moreover, the binding energies corresponding to the same ligands and calculated for both tyrosinase and tyrosine hydroxylase are also correlated with each other, suggesting that tyrosinase inhibitors may also have an inhibitory effect on tyrosine hydroxylase. Compounds 3j and 3k have also very strong antioxidant activity (IC50 15.9 and 15.5 µM), respectively, which is ten times higher than well-known antioxidant BHT.


Assuntos
Antioxidantes/farmacologia , Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Tiazóis/farmacologia , Tiofenos/farmacologia , Toxoplasma/efeitos dos fármacos , Animais , Antioxidantes/síntese química , Antioxidantes/química , Antiprotozoários/síntese química , Antiprotozoários/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/metabolismo , Relação Estrutura-Atividade , Tiazóis/química , Tiofenos/química , Toxoplasma/enzimologia , Toxoplasma/crescimento & desenvolvimento , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/metabolismo
3.
mSphere ; 4(4)2019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31366709

RESUMO

Toxoplasma gondii tachyzoites co-opt host cell functions through introduction of a large set of rhoptry- and dense granule-derived effector proteins. These effectors reach the host cytosol through different means: direct injection for rhoptry effectors and translocation across the parasitophorous vacuolar membrane (PVM) for dense granule (GRA) effectors. The machinery that translocates these GRA effectors has recently been partially elucidated, revealing three components, MYR1, MYR2, and MYR3. To determine whether other proteins might be involved, we returned to a library of mutants defective in GRA translocation and selected one with a partial defect, suggesting it might be in a gene encoding a new component of the machinery. Surprisingly, whole-genome sequencing revealed a missense mutation in a gene encoding a known rhoptry protein, a serine/threonine protein kinase known as ROP17. ROP17 resides on the host cytosol side of the PVM in infected cells and has previously been known for its activity in phosphorylating and thereby inactivating host immunity-related GTPases. Here, we show that null or catalytically dead mutants of ROP17 are defective in GRA translocation across the PVM but that translocation can be rescued "in trans" by ROP17 delivered by other tachyzoites infecting the same host cell. This strongly argues that ROP17's role in regulating GRA translocation is carried out on the host cytosolic side of the PVM, not within the parasites or lumen of the parasitophorous vacuole. This represents an entirely new way in which the different secretory compartments of Toxoplasma tachyzoites collaborate to modulate the host-parasite interaction.IMPORTANCE When Toxoplasma infects a cell, it establishes a protective parasitophorous vacuole surrounding it. While this vacuole provides protection, it also serves as a barrier to the export of parasite effector proteins that impact and take control of the host cell. Our discovery here that the parasite rhoptry protein ROP17 is necessary for export of these effector proteins provides a distinct, novel function for ROP17 apart from its known role in protecting the vacuole. This will enable future research into ways in which we can prevent the export of effector proteins, thereby preventing Toxoplasma from productively infecting its animal and human hosts.


Assuntos
Interações Hospedeiro-Parasita/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Vacúolos/parasitologia , Fatores de Virulência/metabolismo , Células Cultivadas , Humanos , Mutação de Sentido Incorreto , Proteínas de Protozoários/genética , Toxoplasma/genética , Translocação Genética , Fatores de Virulência/genética , Sequenciamento Completo do Genoma
4.
PLoS Biol ; 17(8): e3000364, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31430281

RESUMO

Many eukaryotic microbes have complex life cycles that include both sexual and asexual phases with strict species specificity. Whereas the asexual cycle of the protistan parasite Toxoplasma gondii can occur in any warm-blooded mammal, the sexual cycle is restricted to the feline intestine. The molecular determinants that identify cats as the definitive host for T. gondii are unknown. Here, we defined the mechanism of species specificity for T. gondii sexual development and break the species barrier to allow the sexual cycle to occur in mice. We determined that T. gondii sexual development occurs when cultured feline intestinal epithelial cells are supplemented with linoleic acid. Felines are the only mammals that lack delta-6-desaturase activity in their intestines, which is required for linoleic acid metabolism, resulting in systemic excess of linoleic acid. We found that inhibition of murine delta-6-desaturase and supplementation of their diet with linoleic acid allowed T. gondii sexual development in mice. This mechanism of species specificity is the first defined for a parasite sexual cycle. This work highlights how host diet and metabolism shape coevolution with microbes. The key to unlocking the species boundaries for other eukaryotic microbes may also rely on the lipid composition of their environments as we see increasing evidence for the importance of host lipid metabolism during parasitic lifecycles. Pregnant women are advised against handling cat litter, as maternal infection with T. gondii can be transmitted to the fetus with potentially lethal outcomes. Knowing the molecular components that create a conducive environment for T. gondii sexual reproduction will allow for development of therapeutics that prevent shedding of T. gondii parasites. Finally, given the current reliance on companion animals to study T. gondii sexual development, this work will allow the T. gondii field to use of alternative models in future studies.


Assuntos
Linoleoil-CoA Desaturase/metabolismo , Toxoplasma/enzimologia , Animais , Gatos , Especificidade de Hospedeiro , Interações Hospedeiro-Parasita , Intestinos/parasitologia , Estágios do Ciclo de Vida/fisiologia , Ácido Linoleico/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Parasitos/metabolismo , Desenvolvimento Sexual/fisiologia , Especificidade da Espécie , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/patogenicidade
5.
Exp Parasitol ; 203: 8-18, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31150653

RESUMO

Toxoplasma gondii is an important human and veterinary pathogen and the causative agent of toxoplasmosis, a potentially severe disease especially in immunocompromised or congenitally infected humans. Current therapeutic compounds are not well-tolerated, present increasing resistance, limited efficacy and require long periods of treatment. On this context, searching for new therapeutic targets is crucial to drug discovery. In this sense, recent works suggest that N-myristoyltransferase (NMT), the enzyme responsible for protein myristoylation that is essential in some parasites, could be the target of new anti-parasitic compounds. However, up to date there is no information on NMT and the extent of this modification in T. gondii. In this work, we decided to explore T. gondii genome in search of elements related with the N-myristoylation process. By a bioinformatics approach it was possible to identify a putative T. gondii NMT (TgNMT). This enzyme that is homologous to other parasitic NMTs, presents activity in vitro, is expressed in both intra- and extracellular parasites and interacts with predicted TgNMT substrates. Additionally, NMT activity seems to be important for the lytic cycle of Toxoplasma gondii. In parallel, an in silico myristoylome predicts 157 proteins to be affected by this modification. Myristoylated proteins would be affecting several metabolic functions with some of them being critical for the life cycle of this parasite. Together, these data indicate that TgNMT could be an interesting target of intervention for the treatment of toxoplasmosis.


Assuntos
Aciltransferases/metabolismo , Toxoplasma/metabolismo , Aciltransferases/antagonistas & inibidores , Aciltransferases/efeitos dos fármacos , Aciltransferases/genética , Sequência de Aminoácidos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Fibroblastos/parasitologia , Imunofluorescência , Prepúcio do Pênis/citologia , Prepúcio do Pênis/parasitologia , Humanos , Imunoprecipitação , Masculino , Filogenia , Alinhamento de Sequência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Toxoplasma/classificação , Toxoplasma/enzimologia , Toxoplasma/genética
6.
Life Sci Alliance ; 2(3)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31235476

RESUMO

cGMP signaling is one of the master regulators of diverse functions in eukaryotes; however, its architecture and functioning in protozoans remain poorly understood. Herein, we report an exclusive guanylate cyclase coupled with N-terminal P4-ATPase in a common parasitic protist, Toxoplasma gondii This bulky protein (477-kD), termed TgATPaseP-GC to fairly reflect its envisaged multifunctionality, localizes in the plasma membrane at the apical pole of the parasite, whereas the corresponding cGMP-dependent protein kinase (TgPKG) is distributed in the cytomembranes. TgATPaseP-GC is refractory to genetic deletion, and its CRISPR/Cas9-assisted disruption aborts the lytic cycle of T. gondii Besides, Cre/loxP-mediated knockdown of TgATPaseP-GC reduced the synthesis of cGMP and inhibited the parasite growth due to impairments in the motility-dependent egress and invasion events. Equally, repression of TgPKG by a similar strategy recapitulated phenotypes of the TgATPaseP-GC-depleted mutant. Notably, despite a temporally restricted function, TgATPaseP-GC is expressed constitutively throughout the lytic cycle, entailing a post-translational regulation of cGMP signaling. Not least, the occurrence of TgATPaseP-GC orthologs in several other alveolates implies a divergent functional repurposing of cGMP signaling in protozoans, and offers an excellent drug target against the parasitic protists.


Assuntos
Adenosina Trifosfatases/metabolismo , Guanilato Ciclase/química , Guanilato Ciclase/metabolismo , Domínios e Motivos de Interação entre Proteínas , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Ligação Proteica , Multimerização Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Toxoplasma/classificação , Toxoplasma/enzimologia , Toxoplasma/genética
7.
Parasit Vectors ; 12(1): 221, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068219

RESUMO

BACKGROUND: Protein arginine methylation is a prevalent post-translational modification. The protein arginine methyltransferase family (PRMT) is involved in many cellular processes in eukaryotes, including transcriptional regulation, epigenetic regulation, RNA metabolism, and DNA damage repair. Toxoplasma gondii, an opportunistic protozoan parasite, encodes five conserved PRMTs. PRMT5 is thought to be responsible for substantial PRMT activity in T. gondii; however, it has not yet been characterized. METHODS: We tagged the 3' end of the endogenous TgPRMT5 genomic locus with sequence encoding a 3X hemagglutinin (HA) epitope. IFA and WB were performed to check the expression and subcellular localization of TgPRMT5 in tachyzoites and bradyzoites. In vitro methylation assays were performed to determine whether endogenous TgPRMT5 has arginine methyltransferase activity. RESULTS: IFA and WB results showed that T. gondii PRMT5 (TgPRMT5) was localized in the cytoplasm in the tachyzoite stage; however, it shifts largely to the nuclear compartment in the bradyzoite stage. The in vitro methylation showed that TgPRMT5 has authentic type II PRMT activity and forms monomethylarginines and symmetric dimethylarginines. CONCLUSIONS: We determined the expression and cellular localization of TgPRMT5 in tachyzoites and bradyzoites and confirmed its type II PRMT activity. We demonstrated the major changes in expression and cellular localization of TgPRMT5 during the tachyzoite and bradyzoite stages in T. gondii. Our findings suggest that TgPRMT5 protein may be involved in tachyzoite-bradyzoite transformation.


Assuntos
Proteína-Arginina N-Metiltransferases/genética , Toxoplasma/enzimologia , Toxoplasma/genética , Citoplasma/química , Epigênese Genética , Estágios do Ciclo de Vida , Metilação , Proteína-Arginina N-Metiltransferases/metabolismo
8.
Acta Biochim Biophys Sin (Shanghai) ; 51(6): 615-626, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31139819

RESUMO

Few information of the function of stearoyl-coenzyme A (CoA) desaturase (SCD) in apicomplaxan parasite has been obtained. In this study, we retrieved a putative fatty acyl-CoA desaturase (TGGT1_238950) by a protein alignment with Plasmodium falciparum SCD in ToxoDB. A typical Δ9-desaturase domain was revealed in this protein. The putative desaturase was tagged with HA endogenously in Toxoplasma gondii, and the endoplasmic reticulum localization of the putative desaturase was revealed, which was consistent with the fatty acid desaturases in other organisms. Therefore, the TGGT1_238950 was designated T. gondii SCD. Based on CRISPR/Cas9 gene editing technology, SCD conditional knockout mutants in the T. gondii TATi strain were obtained. The growth in vitro and pathogenicity in mice of the mutants suggested that SCD might be dispensable for tachyzoite growth and proliferation. The SCD-overexpressing line was constructed to further explore SCD function. The portion of palmitoleic acid and oleic acid were increased in SCD-overexpressing parasites, compared with the RH parental strain, indicating that T. gondii indeed is competent for unsaturated fatty acid synthesis. The SCD-overexpressing tachyzoites propagated slower than the parental strain, with a decreased invasion capability and weaker pathogenicity in mice. The TgIF2α phosphorylation and the expression changes of several genes demonstrated that ER stress was triggered in the SCD-overexpressing parasites, which were more apt toward autophagy and apoptosis. The function of unsaturated fatty acid synthesis of TgSCD was consistent with our hypothesis. On the other hand, SCD might also be involved in tachyzoite autophagy and apoptosis.


Assuntos
Proteínas de Protozoários/imunologia , Estearoil-CoA Dessaturase/imunologia , Toxoplasma/enzimologia , Toxoplasmose/imunologia , Animais , Células Cultivadas , Clonagem Molecular , Humanos , Camundongos Endogâmicos BALB C , Mutação , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Análise de Sequência de DNA , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Análise de Sobrevida , Toxoplasma/genética , Toxoplasma/patogenicidade , Toxoplasmose/parasitologia
9.
Acta Trop ; 195: 35-43, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31004564

RESUMO

Toxoplasma gondii has evolved many successful strategies for immune evasion. However, the parasite-derived effectors involved in modulating NF-κB signalling pathway are largely unknown. T. gondii Cathepsin C1 (CPC1) is widely conserved among T. gondii strains and is important for T. gondii intracellular growth and proliferation. Our study showed that CPC1 protein could abrogate NF-κB activation after screening dense granule proteins. CPC1 suppressed NF-κB activation at or downstream of p65 and decreased the production of IL-1, IL-8, IL-6, IL-12, and TNF-α. Western blot analysis revealed that CPC1 inhibited phospho-p65 and CPC1 proteins primarily settled in cytoplasm. RNA sequencing analysis revealed that overexpression of CPC1 significantly upregulated erythropoietin (EPO), which can be induced by the hypoxia-inducible factor -1α (HIF-1α) during hypoxia. Furthermore, dual-luciferase reporter assays confirmed that CPC1 upregulated HIF-1α. Finally, both the knockdown of EPO and restriction of HIF-1α partially eliminated the suppression impact of CPC1 on the NF-κB signalling pathway. Our study identified a previously unrecognized role of CPC1 in the negative regulation of NF-κB activation through positive regulation of the HIF-1α/EPO axis. For the first time, CPC1 was shown to play an important role in immune evasion during T. gondii infection.


Assuntos
Catepsina C/fisiologia , Eritropoetina/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , NF-kappa B/fisiologia , Toxoplasma/imunologia , Células HEK293 , Humanos , Evasão da Resposta Imune , Transdução de Sinais/fisiologia , Toxoplasma/enzimologia
10.
mBio ; 10(2)2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30914506

RESUMO

As the protozoan parasite Toxoplasma gondii disseminates through its host, it responds to environmental changes by altering its gene expression, metabolism, and other processes. Oxygen is one variable environmental factor, and properly adapting to changes in oxygen levels is critical to prevent the accumulation of reactive oxygen species and other cytotoxic factors. Thus, oxygen-sensing proteins are important, and among these, 2-oxoglutarate-dependent prolyl hydroxylases are highly conserved throughout evolution. Toxoplasma expresses two such enzymes, TgPHYa, which regulates the SCF-ubiquitin ligase complex, and TgPHYb. To characterize TgPHYb, we created a Toxoplasma strain that conditionally expresses TgPHYb and report that TgPHYb is required for optimal parasite growth under normal growth conditions. However, exposing TgPHYb-depleted parasites to extracellular stress leads to severe decreases in parasite invasion, which is likely due to decreased abundance of parasite adhesins. Adhesin protein abundance is reduced in TgPHYb-depleted parasites as a result of inactivation of the protein synthesis elongation factor eEF2 that is accompanied by decreased rates of translational elongation. In contrast to most other oxygen-sensing proteins that mediate cellular responses to low O2, TgPHYb is specifically required for parasite growth and protein synthesis at high, but not low, O2 tensions as well as resistance to reactive oxygen species. In vivo, reduced TgPHYb expression leads to lower parasite burdens in oxygen-rich tissues. Taken together, these data identify TgPHYb as a sensor of high O2 levels, in contrast to TgPHYa, which supports the parasite at low O2 IMPORTANCE Because oxygen plays a key role in the growth of many organisms, cells must know how much oxygen is available. O2-sensing proteins are therefore critical cellular factors, and prolyl hydroxylases are the best-studied type of O2-sensing proteins. In general, prolyl hydroxylases trigger cellular responses to decreased oxygen availability. But, how does a cell react to high levels of oxygen? Using the protozoan parasite Toxoplasma gondii, we discovered a prolyl hydroxylase that allows the parasite to grow at elevated oxygen levels and does so by regulating protein synthesis. Loss of this enzyme also reduces parasite burden in oxygen-rich tissues, indicating that sensing both high and low levels of oxygen impacts the growth and physiology of Toxoplasma.


Assuntos
Regulação da Expressão Gênica , Estresse Oxidativo , Elongação Traducional da Cadeia Peptídica , Prolil Hidroxilases/metabolismo , Estresse Fisiológico , Toxoplasma/enzimologia , Toxoplasma/fisiologia , Moléculas de Adesão Celular/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/metabolismo
11.
Parasit Vectors ; 12(1): 98, 2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867024

RESUMO

BACKGROUND: Rhoptry protein 18 (ROP18) is a key virulence factor of Toxoplasma gondii. The host's immune responses mediated by immune-related GTPases (IRGs) could be blocked by ROP18's kinase activity. ROP18 also interacts with various substrates, such as activating transcription factor 6 beta (ATF6ß) and affects multiple physiological functions within host cells, thereby inducing intense virulence. In this study, competitive inhibitors targeted to ROP18 were subjected to virtual screening based on the principle of structure-based drug design (SBDD). METHODS: The preparation of the ROP18 structure was conducted using the "Structure Prepare" function of Molecular Operating Environment (MOE) software. The ATP-binding pocket was selected as the starting point for virtual screening. Construction of the pharmacophore model used Extended Hückel Theory (EHT) half-quantitative measurement and construction, as well as the characteristics of Type I kinase inhibitors. The pharmacophore model of ROP18 was imported into the Specs database for small molecule similarity screening using EHT pharmacophore measurement. Hit compounds were selected using the functions of London dG and generalized-born volume integral/weighted surface area (GBVI/WSA) scoring. The top 100 hits were analyzed by molecular docking and structure activity relationships (SAR) analysis. RESULTS: The final pharmacophore comprised three typical characteristics: three hydrogen bond acceptors/donors, two ring aromatic features occupying the hydrophobic core, and one cation group feature targeted to the terminus of ATP. A total of 1314 hit compounds analogous to ROP18 pharmacophore were passed through the Specs. After two rounds of docking, 25 out of 100 hits were identified as belonging to two main scaffold types: phthalimide ring structure, thiazole ring and styrene structure. Additionally, the screen also identified 13 inhibitors with distinct scaffold types. The docking models and SAR analysis demonstrated that these hits could engage in multiple hydrogen bonds, salt bridges halogen bonds, and hydrophobic interactions with ROP18, and para-position halo substituents on the benzene ring may enhance their affinity scoring. CONCLUSIONS: A pharmacophore against the ROP18 ATP-binding pocket was successfully constructed, and 25 representative inhibitors from 15 scaffold clusters were screened using the Specs database. Our results provide useful scaffold types for the chemical inhibition of ROP18 or alternative conformations to develop new anti-toxoplasmosis drug leads.


Assuntos
Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Toxoplasma/enzimologia , Desenho de Fármacos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Software , Relação Estrutura-Atividade , Toxoplasma/genética , Toxoplasma/patogenicidade , Virulência , Fatores de Virulência
12.
J Biol Chem ; 294(14): 5720-5734, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30723156

RESUMO

The Plasmodium falciparum ATPase PfATP4 is the target of a diverse range of antimalarial compounds, including the clinical drug candidate cipargamin. PfATP4 was originally annotated as a Ca2+ transporter, but recent evidence suggests that it is a Na+ efflux pump, extruding Na+ in exchange for H+ Here we demonstrate that ATP4 proteins belong to a clade of P-type ATPases that are restricted to apicomplexans and their closest relatives. We employed a variety of genetic and physiological approaches to investigate the ATP4 protein of the apicomplexan Toxoplasma gondii, TgATP4. We show that TgATP4 is a plasma membrane protein. Knockdown of TgATP4 had no effect on resting pH or Ca2+ but rendered parasites unable to regulate their cytosolic Na+ concentration ([Na+]cyt). PfATP4 inhibitors caused an increase in [Na+]cyt and a cytosolic alkalinization in WT but not TgATP4 knockdown parasites. Parasites in which TgATP4 was knocked down or disrupted exhibited a growth defect, attributable to reduced viability of extracellular parasites. Parasites in which TgATP4 had been disrupted showed reduced virulence in mice. These results provide evidence for ATP4 proteins playing a key conserved role in Na+ regulation in apicomplexan parasites.


Assuntos
Membrana Celular/enzimologia , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Animais , Membrana Celular/genética , Citoplasma/genética , Citoplasma/metabolismo , Feminino , Técnicas de Silenciamento de Genes , ATPase Trocadora de Hidrogênio-Potássio/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genética , Sódio/metabolismo , Toxoplasma/genética , Toxoplasma/patogenicidade
13.
Parasitol Res ; 118(1): 289-306, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30506516

RESUMO

Toxoplasma gondii can infect all nucleated cells from warm-blooded organisms. After infection, Toxoplasma spreads throughout the body and migrates across biological barriers, such as the intestinal and blood-brain barriers, as well as the placenta in pregnant women. The mechanisms for parasite dissemination are still unknown; however, proteases could play a role as a virulence factor. The aim of this study was to detect and to characterize proteases in whole-cell extracts and in excretion/secretion products from tachyzoites of the RH strain isolated from infected mice. Both fractions were analyzed by gelatin and casein zymography and by azocasein degradation. The biochemical characterization of proteases included standardization of optimal conditions for their activation, such as pH, the presence of cofactors, and a reducing agent. In both fractions, we detected at least nine gelatin-degrading metalloproteases in the range of 50 to 290 kDa. The proteases present in the excretion/secretion products were found as soluble proteins and not associated with exosome-like vesicles or other secretory vesicles. Moreover, by using casein zymography, it was possible to detect three serine proteases. Exposure of MDCK cells to excretion/secretion products modified the organization of the cell monolayer, and this effect was reverted after washing thoroughly with PBS and inhibition by metalloprotease and serine protease inhibitors. Proteomic analysis of excretion/secretion products identified 19 proteases. These findings suggest that tachyzoites of a highly virulent strain of Toxoplasma use a battery of proteases to modify the epithelium, probably as a strategy to facilitate their tissue dissemination.


Assuntos
Células Epiteliais/parasitologia , Metaloproteases/metabolismo , Proteínas de Protozoários/metabolismo , Serina Proteases/metabolismo , Toxoplasma/enzimologia , Toxoplasmose/parasitologia , Animais , Feminino , Humanos , Metaloproteases/genética , Camundongos , Gravidez , Proteômica , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasma/crescimento & desenvolvimento , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
14.
Int J Biol Macromol ; 122: 636-643, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30391427

RESUMO

The exposed hydrophobic patches of protein are widely detected through the binding by the fluorescent probes such as 1-anilino-8-naphthalene sulfonate (ANS), Nile Red (NR) and 1-(N-phenylamino) naphthalene, N-(1-Naphthyl) aniline (1NPN). Interestingly, at pH4, where the Toxoplasma gondii Ferredoxin-NADP(+) reductase (TgFNR) is stable, an exclusive binding and fluorescence emission was observed for ANS. To understand the underlying difference in the binding of ANS, NR and 1NPN; their effect on the protein structure was studied in detail. ANS was found to interact with TgFNR via electrostatic as well as hydrophobic interactions at pH4. NR and 1NPN did not show any such binding to TgFNR in the similar conditions, however showed strong hydrophobic interaction in the presence of NaCl or DSS (2, 2-dimethyl-2-silapentane-5-sulfonate). The subsequent structural studies suggest that ANS, NaCl and DSS induced partial unfolding of TgFNR by modulating ionic interactions of the enzyme, leading to the exposure of buried hydrophobic patches amicable for the binding by NR and 1NPN. The induced unfolding of TgFNR by ANS is unique and thus cautions to use the fluorescent dye as simple indicator to probe the exposed hydrophobic patches of the protein or its folding intermediates.


Assuntos
Naftalenossulfonato de Anilina/metabolismo , Naftalenossulfonato de Anilina/farmacologia , Ferredoxinas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , NADP/metabolismo , Oxirredutases/metabolismo , Toxoplasma/enzimologia , Oxirredutases/química , Ligação Proteica , Desdobramento de Proteína/efeitos dos fármacos
15.
Cell Host Microbe ; 24(6): 804-816.e6, 2018 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-30449726

RESUMO

Apicomplexan parasites rely on cyclic nucleotide-dependent kinases for host cell infection, yet the mechanisms that control their activation remain unknown. Here we show that an apically localized guanylate cyclase (GC) controls microneme secretion and lytic growth in the model apicomplexan Toxoplasma gondii. Cell-permeable cGMP reversed the block in microneme secretion seen in a knockdown of TgGC, linking its function to production of cGMP. TgGC possesses an N-terminal P-type ATPase domain fused to a C-terminal heterodimeric guanylate cyclase domain, an architecture found only in Apicomplexa and related protists. Complementation with a panel of mutants revealed a critical requirement for the P-type ATPase domain for maximum GC function. We further demonstrate that knockdown of TgGC in vivo protects mice from lethal infection by blocking parasite expansion and dissemination. Collectively, this work demonstrates that cGMP-mediated signaling in Toxoplasma relies on a multi-domain architecture, which may serve a conserved role in related parasites.


Assuntos
GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Toxoplasmose/parasitologia , Animais , Feminino , Fibroblastos , Técnicas de Silenciamento de Genes , Guanilato Ciclase/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , ATPases do Tipo-P/metabolismo , Proteínas de Protozoários/genética , Toxoplasma/genética , Virulência/genética
16.
mBio ; 9(5)2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30377279

RESUMO

Toxoplasma gondii infects approximately 30% of the world's population, causing disease primarily during pregnancy and in individuals with weakened immune systems. Toxoplasma secretes and exports effector proteins that modulate the host during infection, and several of these proteins are processed by the Golgi-associated aspartyl protease 5 (ASP5). Here, we identify ASP5 substrates by selectively enriching N-terminally derived peptides from wild-type and Δasp5 parasites. We reveal more than 2,000 unique Toxoplasma N-terminal peptides, mapping to both natural N termini and protease cleavage sites. Several of these peptides mapped directly downstream of the characterized ASP5 cleavage site, arginine-arginine-leucine (RRL). We validate candidates as true ASP5 substrates, revealing they are not processed in parasites lacking ASP5 or in wild-type parasites following mutation of the motif from RRL to ARL. All identified ASP5 substrates are dense granule proteins, and interestingly, none appear to be exported, thus differing from the analogous system in related Plasmodium spp. Instead we show that the majority of substrates reside within the parasitophorous vacuole (PV), and its membrane (the PVM), including two kinases and one phosphatase. We show that genetic deletion of WNG2 leads to attenuation in a mouse model, suggesting that this putative kinase is a new virulence factor in Toxoplasma Collectively, these data constitute the first in-depth analyses of ASP5 substrates and shed new light on the role of ASP5 as a maturase of dense granule proteins during the Toxoplasma lytic cycle.IMPORTANCE Toxoplasma gondii is one of the most successful human parasites. Central to its success is the arsenal of virulence proteins introduced into the infected host cell. Several of these virulence proteins require direct maturation by the aspartyl protease ASP5, and all require ASP5 for translocation into the host cell, yet the true number of ASP5 substrates and complete repertoire of effectors is currently unknown. Here we selectively enrich N-terminally derived peptides using Terminal Amine Isotopic Labeling of Substrates (TAILS) and use quantitative proteomics to reveal novel ASP5 substrates. We identify, using two different enrichment techniques, new ASP5 substrates and their specific cleavage sites. ASP5 substrates include two kinases and one phosphatase that reside at the host-parasite interface, which are important for infection.


Assuntos
Ácido Aspártico Proteases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Toxoplasma/metabolismo , Ácido Aspártico Proteases/genética , Células Cultivadas , Fibroblastos/parasitologia , Deleção de Genes , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Protozoários/genética , Toxoplasma/genética , Vacúolos/metabolismo , Vacúolos/parasitologia
17.
Artigo em Inglês | MEDLINE | ID: mdl-30345257

RESUMO

Toxoplasma gondii, an Apicomplexan parasite, causes significant morbidity and mortality, including severe disease in immunocompromised hosts and devastating congenital disease, with no effective treatment for the bradyzoite stage. To address this, we used the Tropical Disease Research database, crystallography, molecular modeling, and antisense to identify and characterize a range of potential therapeutic targets for toxoplasmosis. Phosphoglycerate mutase II (PGMII), nucleoside diphosphate kinase (NDK), ribulose phosphate 3-epimerase (RPE), ribose-5-phosphate isomerase (RPI), and ornithine aminotransferase (OAT) were structurally characterized. Crystallography revealed insights into the overall structure, protein oligomeric states and molecular details of active sites important for ligand recognition. Literature and molecular modeling suggested potential inhibitors and druggability. The targets were further studied with vivoPMO to interrupt enzyme synthesis, identifying the targets as potentially important to parasitic replication and, therefore, of therapeutic interest. Targeted vivoPMO resulted in statistically significant perturbation of parasite replication without concomitant host cell toxicity, consistent with a previous CRISPR/Cas9 screen showing PGM, RPE, and RPI contribute to parasite fitness. PGM, RPE, and RPI have the greatest promise for affecting replication in tachyzoites. These targets are shared between other medically important parasites and may have wider therapeutic potential.


Assuntos
Enzimas/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Toxoplasma/enzimologia , Toxoplasma/fisiologia , Cristalografia por Raios X , Enzimas/química , Enzimas/genética , Técnicas de Silenciamento de Genes , Modelos Moleculares , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Toxoplasma/crescimento & desenvolvimento
18.
mBio ; 9(5)2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30279285

RESUMO

Invasion of host cells by apicomplexan parasites such as Toxoplasma gondii is critical for their infectivity and pathogenesis. In Toxoplasma, secretion of essential egress, motility, and invasion-related proteins from microneme organelles is regulated by oscillations of intracellular Ca2+ Later stages of invasion are considered Ca2+ independent, including the secretion of proteins required for host cell entry and remodeling from the parasite's rhoptries. We identified a family of three Toxoplasma proteins with homology to the ferlin family of double C2 domain-containing Ca2+ sensors. In humans and model organisms, such Ca2+ sensors orchestrate Ca2+-dependent exocytic membrane fusion with the plasma membrane. Here we focus on one ferlin that is conserved across the Apicomplexa, T. gondii FER2 (TgFER2). Unexpectedly, conditionally TgFER2-depleted parasites secreted their micronemes normally and were completely motile. However, these parasites were unable to invade host cells and were therefore not viable. Knockdown of TgFER2 prevented rhoptry secretion, and these parasites failed to form the moving junction at the parasite-host interface necessary for host cell invasion. Collectively, these data demonstrate the requirement of TgFER2 for rhoptry secretion in Toxoplasma tachyzoites and suggest a possible Ca2+ dependence of rhoptry secretion. These findings provide the first mechanistic insights into this critical yet poorly understood aspect of apicomplexan host cell invasion.IMPORTANCE Apicomplexan protozoan parasites, such as those causing malaria and toxoplasmosis, must invade the cells of their hosts in order to establish a pathogenic infection. Timely release of proteins from a series of apical organelles is required for invasion. Neither the vesicular fusion events that underlie secretion nor the observed reliance of the various processes on changes in intracellular calcium concentrations is completely understood. We identified a group of three proteins with strong homology to the calcium-sensing ferlin family, which are known to be involved in protein secretion in other organisms. Surprisingly, decreasing the amounts of one of these proteins (TgFER2) did not have any effect on the typically calcium-dependent steps in invasion. Instead, TgFER2 was essential for the release of proteins from organelles called rhoptries. These data provide a tantalizing first look at the mechanisms controlling the very poorly understood process of rhoptry secretion, which is essential for the parasite's infection cycle.


Assuntos
Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Linhagem Celular , Técnicas de Silenciamento de Genes , Genoma de Protozoário , Interações Hospedeiro-Parasita , Humanos , Proteínas de Protozoários/genética , Toxoplasma/genética
19.
PLoS Biol ; 16(9): e2005642, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30208022

RESUMO

The phylum Apicomplexa comprises a group of obligate intracellular parasites that alternate between intracellular replicating stages and actively motile extracellular forms that move through tissue. Parasite cytosolic Ca2+ signalling activates motility, but how this is switched off after invasion is complete to allow for replication to begin is not understood. Here, we show that the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A catalytic subunit 1 (PKAc1) of Toxoplasma is responsible for suppression of Ca2+ signalling upon host cell invasion. We demonstrate that PKAc1 is sequestered to the parasite periphery by dual acylation of PKA regulatory subunit 1 (PKAr1). Upon genetic depletion of PKAc1 we show that newly invaded parasites exit host cells shortly thereafter, in a perforin-like protein 1 (PLP-1)-dependent fashion. Furthermore, we demonstrate that loss of PKAc1 prevents rapid down-regulation of cytosolic [Ca2+] levels shortly after invasion. We also provide evidence that loss of PKAc1 sensitises parasites to cyclic GMP (cGMP)-induced Ca2+ signalling, thus demonstrating a functional link between cAMP and these other signalling modalities. Together, this work provides a new paradigm in understanding how Toxoplasma and related apicomplexan parasites regulate infectivity.


Assuntos
Sinalização do Cálcio , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Toxoplasma/enzimologia , Acilação , Animais , Cálcio/metabolismo , AMP Cíclico/metabolismo , Citosol/metabolismo , Fibroblastos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Estágios do Ciclo de Vida , Camundongos , Parasitos/enzimologia , Parasitos/crescimento & desenvolvimento , Subunidades Proteicas/metabolismo , Proteínas de Protozoários , Transdução de Sinais , Toxoplasma/crescimento & desenvolvimento
20.
Elife ; 72018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30204085

RESUMO

The mitochondrial ATP synthase is a macromolecular motor that uses the proton gradient to generate ATP. Proper ATP synthase function requires a stator linking the catalytic and rotary portions of the complex. However, sequence-based searches fail to identify genes encoding stator subunits in apicomplexan parasites like Toxoplasma gondii or the related organisms that cause malaria. Here, we identify 11 previously unknown subunits from the Toxoplasma ATP synthase, which lack homologs outside the phylum. Modeling suggests that two of them, ICAP2 and ICAP18, are distantly related to mammalian stator subunits. Our analysis shows that both proteins form part of the ATP synthase complex. Depletion of ICAP2 leads to aberrant mitochondrial morphology, decreased oxygen consumption, and disassembly of the complex, consistent with its role as an essential component of the Toxoplasma ATP synthase. Our findings highlight divergent features of the central metabolic machinery in apicomplexans, which may reveal new therapeutic opportunities.


Assuntos
ATPases Mitocondriais Próton-Translocadoras/metabolismo , Subunidades Proteicas/metabolismo , Toxoplasma/enzimologia , Sequência de Aminoácidos , Animais , Bovinos , Sequência Conservada , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/química , Conformação Proteica , Subunidades Proteicas/química , Proteínas de Protozoários/metabolismo , Homologia Estrutural de Proteína , Toxoplasma/crescimento & desenvolvimento
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