RESUMO
Toxoplasma gondii is a parasite that can invade any cell in the human body. Here, we implemented and described an ex vivo model with human peripheral blood mononuclear cells (PBMCs) without using culture supplements/antibiotics and without cryopreserved cells (EXMOWS) to study the interactions between T. gondii and human cells. To establish the EXMOWS, three independent tests were carried out. Firstly, blood samples from 5 individuals were included to assess the viability and adherence of PBMCs in plate culture. In a second trial, blood samples from three seropositive and two seronegative individuals for T. gondii were used to evaluate human PBMCs cells: parasites, multiplicity of infection (MOI) 1:1, 1:3 and 1:5 at different times post infection (1 h, 6 h and 24 h). The possible immunomodulatory effect of the infection for this EXMOWS were evaluated in a third trial where HFF cells were infected with T. gondii and co-cultured with PBMCs obtained from anti-Toxoplasma IgG positive and IgG negative individuals. One hour was enough time for T. gondii infection of human PBMCs and 2 h was the minimum incubation time to guarantee adherence before carrying out any infection assay. A minimum of 1:3 MOI was necessary to guarantee efficient infection in human PBMCs with T. gondii RH-GFP. All protocols, including PBMCs isolation and stimulation, should be conducted the same day. This EXMOWS can be adapted to study the early stages of interaction with other microorganisms of human interest, without need of using cryopreservation and supplements/antibiotics.
Assuntos
Interações Hospedeiro-Parasita/fisiologia , Leucócitos Mononucleares/parasitologia , Toxoplasma/fisiologia , Adulto , Análise de Variância , Sobrevivência Celular , Células Cultivadas , Fibroblastos , Prepúcio do Pênis/citologia , Humanos , Imunoglobulina G/sangue , Masculino , RNA de Protozoário/química , RNA de Protozoário/isolamento & purificação , Adulto JovemRESUMO
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/genéticaRESUMO
Toxoplasma gondii infects a wide range of hosts worldwide, including humans and domesticated animals causing toxoplasmosis disease. Recently, exosomes, small extracellular vesicles (EV) that contain nucleic acids, proteins, and lipids derived from their original cells were linked with disease protection. The effect of EVs derived from T. gondii on the immune response and its relevance in a physiological context is unknown. Here we disclose the first proteomic profiling of T. gondii EVs compared to EVs isolated from a human foreskin fibroblast infected cell line cultured in a vesicle-free medium. Our results reveal a broad range of canonical exosomes proteins. Data are available via ProteomeXchange with the identifier PXD004895.
Assuntos
Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Proteômica/métodos , Toxoplasma/metabolismo , Toxoplasmose/parasitologia , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/parasitologia , Prepúcio do Pênis/citologia , Prepúcio do Pênis/metabolismo , Prepúcio do Pênis/parasitologia , Humanos , Masculino , Toxoplasmose/metabolismoRESUMO
Pathological scar tissues and normal skin tissues were differentiated by screening for differentially expressed genes in pathologic scar tissues via gene expression microarray. The differentially expressed gene data was analyzed by gene ontology and pathway analyses. There were 5001 up- or down-regulated genes in 2-fold differentially expressed genes, 956 up- or down-regulated genes in 5-fold differentially expressed genes, and 114 up- or down-regulated genes in 20-fold differentially expressed genes. Therefore, significant differences were observed in the gene expression in pathological scar tissues and normal foreskin tissues. The development of pathological scar tissues has been correlated to changes in multiple genes and pathways, which are believed to form a dynamic network connection.
Assuntos
Cicatriz/genética , Foliculite/genética , Furunculose/genética , Proteínas/genética , Adulto , Cicatriz/etiologia , Cicatriz/metabolismo , Cicatriz/patologia , Foliculite/complicações , Foliculite/metabolismo , Foliculite/patologia , Prepúcio do Pênis/citologia , Prepúcio do Pênis/metabolismo , Furunculose/complicações , Furunculose/metabolismo , Furunculose/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Humanos , Masculino , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas/metabolismoRESUMO
The efficacy of three amino-terpenyl naphthoquinones and the alkaloid liriodenine were examined against tachyzoites and tissues cysts of the RH and EGS strains, respectively. Monolayers of 2C4 fibroblasts infected with tachyzoites of the RH strain were incubated with different concentrations of the compounds for 48 h. Specifically, 7-(4-methyl-3-pentenyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-5), 6-(4-methyl-3-pentenyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-6), 6-(4-methylpentyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-11), and 8 h-benzo[g]-1,3-benzodioxolo[6,5,4-de]quinolin-8-one,9Cl-1,2-methylene dioxiaporfina (liriodenine) inhibited intracellular replication of T. gondii. The IC(50) values obtained for compounds QUI-5 and QUI-6 were 69.35 and 172.81 µM (i.e., 21.4 and 53.4 µg/mL), respectively. The naphthoquinone QUI-11 and liriodenine significantly inhibited intracellular replication of T. gondii. The IC(50) values obtained with these experiments were 0.32 and 0.07 µM (i.e., 0.1 and 0.02 µg/mL), respectively. Compounds QUI-5, QUI-6, QUI-11 and liriodenine demonstrated lower toxicity for 2C4 fibroblasts compared to atovaquone. In addition, cysts isolated from the brains of mice chronically infected with the EGS strain were exposed to the compounds. Infectivity of the cysts after incubation with the compounds was assessed by infection of mice. The data obtained showed that in vitro incubation with QUI-6, QUI-11 and liriodenine inhibited the infectivity of the bradyzoites. This activity was time- and concentration-dependent.
Assuntos
Aporfinas/farmacologia , Coccidiostáticos/farmacologia , Fibroblastos/parasitologia , Naftoquinonas/farmacologia , Toxoplasma/efeitos dos fármacos , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Aporfinas/química , Atovaquona/química , Atovaquona/farmacologia , Células Cultivadas , Coccidiostáticos/química , Feminino , Fibroblastos/efeitos dos fármacos , Prepúcio do Pênis/citologia , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Naftoquinonas/química , Relação Estrutura-Atividade , Sulfadiazina/química , Sulfadiazina/farmacologia , Toxoplasma/patogenicidade , Toxoplasmose Animal/tratamento farmacológico , Toxoplasmose Animal/parasitologiaRESUMO
Considering that the treatment for toxoplasmosis is based on drugs that show limited efficacy due to their substantial side effects, the purpose of the present study was to evaluate the effects of Artemisia annua on in vitro and in vivo Toxoplasma gondii infection. A. annua infusion was prepared from dried herb and tested in human foreskin fibroblasts (HFF) or mice that were infected with the parasite and compared with sulfadiazine treatment. For in vitro experiments, treatment was done on parasite before HFF infection or on cells previously infected with T. gondii and the inhibitory concentration (IC(50)) values for each treatment condition were determined. Viability of HFF cells in the presence of different concentrations of A. annua infusion and sulfadiazine was above 72%, even when the highest concentrations from both treatments were tested. Also, the treatment of T. gondii tachyzoites with A. annua infusion before infection in HFF cells showed a dose-response inhibitory curve that reached up to 75% of inhibition, similarly to the results observed when parasites were treated with sulfadiazine. In vivo experiments with a cystogenic T. gondii strain demonstrated an effective control of infection using A. annua infusion. In conclusion, our results indicate that A. annua infusion is useful to control T. gondii infection, due to its low toxicity and its inhibitory action directly against the parasite, resulting in a well tolerated therapeutic tool.
Assuntos
Artemisia annua/química , Fitoterapia , Extratos Vegetais/farmacologia , Toxoplasma/efeitos dos fármacos , Toxoplasmose Animal/tratamento farmacológico , Animais , Bioensaio , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Coccidiostáticos/farmacologia , Coccidiostáticos/uso terapêutico , Citocinas/análise , Feminino , Fibroblastos , Prepúcio do Pênis/citologia , Humanos , Imuno-Histoquímica , Macrófagos Peritoneais/química , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nitritos/análise , Extratos Vegetais/uso terapêutico , Extratos Vegetais/toxicidade , Reação em Cadeia da Polimerase , Sigmodontinae , Sulfadiazina/farmacologia , Sulfadiazina/uso terapêutico , Toxoplasma/isolamento & purificaçãoRESUMO
Fludarabine (FLU), an analogue of adenosine, interferes with DNA synthesis and inhibits the chain elongation leading to replication arrest and DNA double strand break (DSB) formation. Mammalian cells use two main pathways of DSB repair to maintain genomic stability: homologous recombination (HR) and non-homologous end joining (NHEJ). The aim of the present work was to evaluate the repair pathways employed in the restoration of DSB formed following replication arrest induced by FLU in mammalian cells. Replication inhibition was induced in human lymphocytes and fibroblasts by FLU. DSB occurred in a dose-dependent manner on early/middle S-phase cells, as detected by gammaH2AX foci formation. To test whether conservative HR participates in FLU-induced DSB repair, we measured the kinetics of Rad51 nuclear foci formation in human fibroblasts. There was no significant induction of Rad51 foci after FLU treatment. To further confirm these results, we analyzed the frequency of sister chromatid exchanges (SCE) in both human cells. We did not find increased frequencies of SCE after FLU treatment. To assess the participation of NHEJ pathway in the repair of FLU-induced damage, we used two chemical inhibitors of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), vanillin and wortmannin. Human fibroblasts pretreated with DNA-PKcs inhibitors showed increased levels of chromosome breakages and became more sensitive to cell death. An active role of NHEJ pathway was also suggested from the analysis of Chinese hamster cell lines. XR-C1 (DNA-PKcs-deficient) and XR-V15B (Ku80-deficient) cells showed hypersensitivity to FLU as evidenced by the increased frequency of chromosome aberrations, decreased mitotic index and impaired survival rates. In contrast, CL-V4B (Rad51C-deficient) and V-C8 (Brca2-deficient) cell lines displayed a FLU-resistant phenotype. Together, our results suggest a major role for NHEJ repair in the preservation of genome integrity against FLU-induced DSB in mammalian cells.