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1.
Exp Parasitol ; 224: 108100, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33744229

RESUMO

Chagas disease and leishmaniasis are neglected diseases caused by parasites of the Trypanosomatidae family and together they affect millions of people in the five continents. The treatment of Chagas disease is based on benznidazole, whereas for leishmaniasis few drugs are available, such as amphotericin B and miltefosine. In both cases, the current treatment is not entirely efficient due to toxicity or side effects. Encouraged by the need to discover valid targets and new treatment options, we evaluated 8 furan compounds against Trypanosoma cruzi and Leishmania amazonensis, considering their effects against proliferation, infection, and ultrastructure. Many of them were able to impair T. cruzi and L. amazonensis proliferation, as well as cause ultrastructural alterations, such as Golgi apparatus disorganization, autophagosome formation, and mitochondrial swelling. Taken together, the results obtained so far make these compounds eligible for further steps of chemotherapy study.


Assuntos
Furanos/farmacologia , Leishmania mexicana/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Linhagem Celular , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Cromatografia em Camada Delgada , Doenças Endêmicas , Furanos/química , Humanos , Concentração Inibidora 50 , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/ultraestrutura , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/parasitologia , Macrófagos , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Varredura , Simulação de Acoplamento Molecular , Doenças Negligenciadas/tratamento farmacológico , Doenças Negligenciadas/parasitologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/ultraestrutura
2.
Exp Parasitol ; 221: 108061, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33383023

RESUMO

Chagas disease (CD) caused by Trypanosoma cruzi remains a serious public health problem in Latin America. The available treatment is limited to two old drugs, benznidazole (Bz) and nifurtimox, which exhibit limited efficacy and trigger side effects, justifying the search for new therapies. Also, more accurate and sensitive experimental protocols for drug discovery programs are necessary to shrink the translational gaps found among pre-clinical and clinical trials. Presently, cardiac spheroids were used to evaluate host cell cytotoxicity and anti-T.cruzi activity of benznidazole, exploring its effect on the release of inflammatory mediators. Bz presented low toxic profile on 3D matrices (LC50 > 200 µM) and high potency in vitro (EC50 = 0.99 µM) evidenced by qPCR analysis of T.cruzi-infected cardiac spheroids. Flow cytometry appraisal of inflammatory mediators released at the cellular supernatant showed increases in IL - 6 and TNF contents (≈190 and ≈ 25-fold) in parasitized spheroids as compared to uninfected cultures. Bz at 10 µM suppressed parasite load (92%) concomitantly decreasing in IL-6 (36%) and TNF (68%). Our findings corroborate the successful use of 3D cardiac matrices for in vitro identification of novel anti-parasitic agents and potential impact in host cell physiology.


Assuntos
Nitroimidazóis/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Imageamento Tridimensional , Camundongos , Microscopia de Fluorescência , Conformação Molecular , Esferoides Celulares , Trypanosoma cruzi/crescimento & desenvolvimento
3.
Exp Parasitol ; 217: 107962, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32763249

RESUMO

Trypanosoma cruzi is a parasitic protozoan that infects various species of domestic and wild animals, triatomine bugs and humans. It is the etiological agent of American trypanosomiasis, also known as Chagas Disease, which affects about 17 million people in Latin America and is emerging elsewhere in the world. Iron (Fe) is a crucial micronutrient for almost all cells, acting as a cofactor for several metabolic enzymes. T. cruzi has a high requirement for Fe, using heminic and non-heminic Fe for growth and differentiation. Fe occurs in the oxidized (Fe3+) form in aerobic environments and needs to be reduced to Fe2+ before it enters cells. Fe-reductase, located in the plasma membranes of some organisms, catalyzes the Fe3+⇒ Fe2+ conversion. In the present study we found an amino acid sequence in silico that allowed us to identify a novel 35 kDa protein in T. cruzi with two transmembrane domains in the C-terminal region containing His residues that are conserved in the Ferric Reductase Domain Superfamily and are required for catalyzing Fe3+ reduction. Accordingly, we named this protein TcFR. Intact epimastigotes from the T. cruzi DM28c strain reduced the artificial Fe3+-containing substrate potassium ferricyanide in a cell density-dependent manner, following Michaelis-Menten kinetics. The TcFR activity was more than eightfold higher in a plasma membrane-enriched fraction than in whole homogenates, and this increase was consistent with the intensity of the 35 kDa band on Western blotting images obtained using anti-NOX5 raised against the human antigen. Immunofluorescence experiments demonstrated TcFR on the parasite surface. That TcFR is part of a catalytic complex allowing T. cruzi to take up Fe from the medium was confirmed by experiments in which DM28c was assayed after culturing in Fe-depleted medium: (i) proliferation during the stationary growth phase was five times slower; (ii) the relative expression of TcFR (qPCR) was 50% greater; (iii) intact cells had 120% higher Fe-reductase activity. This ensemble of results indicates that TcFR is a conserved enzyme in T. cruzi, and its catalytic properties are modulated in order to respond to external Fe fluctuations.


Assuntos
FMN Redutase/metabolismo , Ferro/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Membrana Celular/enzimologia , Doença de Chagas/parasitologia , Colorimetria , FMN Redutase/análise , FMN Redutase/química , Imunofluorescência , Humanos , Filogenia , Distribuição de Poisson , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Trypanosoma cruzi/classificação , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismo , Regulação para Cima
4.
Chem Biol Interact ; 330: 109165, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32771326

RESUMO

The effect of N-geranyl-ethane-1,2-diamine dihydochloride (GIB24), a synthetic diamine, was assayed against different developmental forms of the parasitic protozoan Trypanosoma cruzi (strain Dm28c). The compound was effective against culture epimastigote forms (IC50/24h = 5.64 µM; SI = 16.4) and intracellular amastigotes (IC50/24h = 12.89 µM; SI = 7.18), as detected by the MTT methodology and by cell counting, respectively. Incubation of epimastigotes for 6h with 6 µM GIB24 (IC50/24h value) resulted in significant dissipation of the mitochondrial membrane potential, prior to permeabilization of the plasma membrane. Rounded epimastigotes with cell size reduction were observed by scanning electron microscopy. These morpho-physiological changes induced by GIB24 suggest an incidental death process. Treatment of infected Vero cells did not prevent the intracellular amastigotes from completing the intracellular cycle. However, there was a decrease in the number of released parasites, increasing the ratio amastigotes/trypomastigotes. Proteomic analysis of 15 µM GIB24 resistant epimastigotes indicated that the compound acts mainly on mitochondrial components involved in the Krebs cycle and in maintaining the oxidative homeostasis of the parasites. Our data suggest that GIB24 is active against the main morphological forms of T. cruzi.


Assuntos
Diaminas/farmacologia , Resistência a Medicamentos , Espaço Intracelular/efeitos dos fármacos , Proteômica , Terpenos/química , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Chlorocebus aethiops , Diaminas/química , Espaço Intracelular/parasitologia , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/metabolismo , Células Vero
5.
Parasitol Res ; 119(9): 2775-2781, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32737590

RESUMO

Triatoma platensis is occasionally found coexisting with Triatoma infestans in chicken coops in Argentina. Some authors have reported the presence of hybrid specimens of both species in chicken coops and other peridomestic habitats. Given the coexistence of T. infestans with T. platensis and the possibility of generating fertile hybrids, it is important to evaluate the vectorial competence of these hybrids. The objective of this study was to record the dynamics of feeding-defecation behavior in fifth-stage nymphs and adults of hybrids between both species and to compare it with T. platensis and T. infestans. Three experimental groups were formed separated by stage and sex: Hybrid group, T. infestans group, and T. platensis group. During feeding, the following variables were recorded for each group: (i) blood meal size, (ii) feeding time, (iii) number of defecations during feeding, and (iv) number of defecations at 10 and 30 min after feeding. The results indicate that adults and fifth-instar nymphs of hybrids have a feeding and defecation behavior similar to T. infestans: they achieve feeding in a short time and first defecation occurs during or just after feeding. Nevertheless, hybrid's ingestion of blood occurs at higher velocity and they require higher blood intake to provoke early defecations. Considering the blood ingestion velocity, the amount of blood ingested, and the short time required for the production of the first defecation, the results of this study suggest that hybrid can be a competent Trypanosoma cruzi vector.


Assuntos
Defecação/fisiologia , Comportamento Alimentar/fisiologia , Insetos Vetores/fisiologia , Triatoma/fisiologia , Animais , Argentina , Doença de Chagas/transmissão , Galinhas/parasitologia , Ecossistema , Fezes/parasitologia , Feminino , Masculino , Ninfa/crescimento & desenvolvimento , Trypanosoma cruzi/crescimento & desenvolvimento
6.
Exp Parasitol ; 215: 107930, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32464221

RESUMO

Trypanosoma cruzi, the etiological agent of Chagas disease, is responsible for the infection of millions of people worldwide and it is a public health problem, without an effective cure. Four fragments with antimicrobial potential from the hemocyanin of Penaeus monodon shrimp were identified using a computer software AMPA. The present study aimed to evaluate the antichagasic effect of these four peptides (Hmc364-382, Hmc666-678, Hmc185-197 and Hmc476-498). The peptides were tested against the epimastigote, trypomastigote and amastigote forms of Trypanosoma cruzi Y strain (benznidazole-resistant strain) and cytotoxicity in mammalian cells was evaluated against LLC-MK2 lineage cells. Two fragments (Hmc364-382, Hmc666-678) showed activity against the epimastigote and trypomastigote forms and their selectivity index (SI) was calculated. The Hmc364-382 peptide was considered the most promising (SI > 50) one and it was used for further studies, using flow cytometry analyses with specific molecular probes and scanning electron microscopy (SEM). Hmc364-382 was able to induce cell death in T. cruzi through necrosis, observed by loss of membrane integrity in flow cytometry analyses and pore formation in SEM. Overall, Hmc364-382 open perspectives to the development of new antichagasic agents.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Hemocianinas/farmacologia , Penaeidae/química , Trypanosoma cruzi/efeitos dos fármacos , Animais , Peptídeos Catiônicos Antimicrobianos/toxicidade , Linhagem Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doença de Chagas/tratamento farmacológico , Citometria de Fluxo , Hemocianinas/toxicidade , Concentração Inibidora 50 , Macaca mulatta , Microscopia Eletrônica de Varredura , Fatores de Tempo , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/ultraestrutura
7.
PLoS Negl Trop Dis ; 14(5): e0008262, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32469928

RESUMO

Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion. We have recently described a significant increase in the tyrosine nitration levels of histones H2A and H4 when trypomastigotes are incubated with components of the ECM. In this work, we used chromatin immunoprecipitation (ChIP) with an anti-nitrotyrosine antibody followed by mass spectrometry to identify nitrated DNA binding proteins in T. cruzi and to detect alterations in nitration levels induced upon parasite incubation with the ECM. Histone H1, H2B, H2A and H3 were detected among the 9 most abundant nitrated DNA binding proteins using this proteomic approach. One nitrated tyrosine residue (Y29) was identified in Histone H2B in the MS/MS spectrum. In addition, we observed a significant increase in the nitration levels of histones H1, H2B, H2A and H4 upon parasite incubation with ECM. Finally, we used ChIP-Seq to map global changes in the DNA binding profile of nitrated proteins. We observed a significant change in the binding pattern of nitrated proteins to DNA after parasite incubation with ECM. This work provides the first global profile of nitrated DNA binding proteins in T. cruzi and additional evidence for modification in the nitration profile of histones upon parasite incubation with ECM. Our data also indicate that the parasite interaction with the ECM induces alterations in chromatin structure, possibly affecting nuclear functions.


Assuntos
Matriz Extracelular/parasitologia , Histonas/análise , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/análise , Trypanosoma cruzi/química , Trypanosoma cruzi/crescimento & desenvolvimento , Imunoprecipitação da Cromatina , Matriz Extracelular/metabolismo , Histonas/metabolismo , Espectrometria de Massas , Nitrosação , Proteômica , Proteínas de Protozoários/metabolismo , Tirosina/análogos & derivados , Tirosina/imunologia
8.
PLoS Negl Trop Dis ; 14(3): e0008068, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32163414

RESUMO

Trypanosoma cruzi parasites utilise de novo pyrimidine biosynthesis to produce DNA and survive within mammalian host cells. This pathway can be hijacked to assess the replication of intracellular parasites with the exogenous addition of a DNA specific probe. To identify suitable probe compounds for this application, a collection of pyrimidine nucleoside analogues was assessed for incorporation into T. cruzi intracellular amastigote DNA using image-based technology and script-based analysis. Associated mammalian cell toxicity of these compounds was also determined against both the parasite host cells (3T3 cells) and HEK293 cells. Incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into parasite DNA was the most effective of the probes tested, with minimal growth inhibition observed following either two or four hours EdU exposure. EdU was subsequently utilised as a DNA probe, followed by visualisation with click chemistry to a fluorescent azide, to assess the impact of drugs and compounds with previously demonstrated activity against T. cruzi parasites, on parasite replication. The inhibitory profiles of these molecules highlight the benefit of this approach for identifying surviving parasites post-treatment in vitro and classifying compounds as either fast or slow-acting. F-ara-EdU resulted in <50% activity observed against T. cruzi amastigotes following 48 hours incubation, at 73 µM. Collectively, this supports the further development of pyrimidine nucleosides as chemical probes to investigate replication of the parasite T. cruzi.


Assuntos
Antiprotozoários/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Testes de Sensibilidade Parasitária/métodos , Nucleosídeos de Pirimidina/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimento , Células 3T3 , Animais , Antiprotozoários/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Nucleosídeos de Pirimidina/toxicidade
9.
PLoS Negl Trop Dis ; 14(3): e0008007, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32196491

RESUMO

Investigations into intracellular replication and differentiation of Trypanosoma cruzi within the mammalian host have been restricted by limitations in our ability to detect parasitized cells throughout the course of infection. We have overcome this problem by generating genetically modified parasites that express a bioluminescent/fluorescent fusion protein. By combining in vivo imaging and confocal microscopy, this has enabled us to routinely visualise murine infections at the level of individual host cells. These studies reveal that intracellular parasite replication is an asynchronous process, irrespective of tissue location or disease stage. Furthermore, using TUNEL assays and EdU labelling, we demonstrate that within individual infected cells, replication of both mitochondrial (kDNA) and nuclear genomes is not co-ordinated within the parasite population, and that replicating amastigotes and non-replicating trypomastigotes can co-exist in the same cell. Finally, we report the presence of distinct non-canonical morphological forms of T. cruzi in the mammalian host. These appear to represent transitional forms in the amastigote to trypomastigote differentiation process. Therefore, the intracellular life-cycle of T. cruzi in vivo is more complex than previously realised, with potential implications for our understanding of disease pathogenesis, immune evasion and drug development. Dissecting the mechanisms involved will be an important experimental challenge.


Assuntos
Doença de Chagas/parasitologia , Replicação do DNA , Estágios do Ciclo de Vida , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Modelos Animais de Doenças , Feminino , Genes Reporter , Microscopia Intravital/métodos , Camundongos SCID , Microscopia Confocal/métodos , Coloração e Rotulagem/métodos , Trypanosoma cruzi/genética
10.
PLoS Negl Trop Dis ; 14(1): e0007481, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961864

RESUMO

BACKGROUND: Crystal violet (CV) was used for several years in blood banks to eliminate the parasite Trypanosoma cruzi in endemic areas in order to prevent transfusion-transmitted Chagas disease. One mechanism of action described for CV involves inhibition of proline uptake. In T. cruzi, proline is essential for host cell infection and intracellular differentiation among other processes, and can be obtained through the proline permease TcAAAP069. METHODOLOGY/PRINCIPAL FINDINGS: CV inhibited proline transporter TcAAAP069 and parasites overexpressing this permease were 47-fold more sensitive to this compound than control parasites. Using CV as reference molecule, loratadine, cyproheptadine, olanzapine and clofazimine were identified as structurally related compounds to CV (structural analogues) by in silico drug repurposing through a similarity-based virtual screening protocol. All these already-approved drugs for clinical use inhibited TcAAAP069 activity with different efficacies and also presented trypanocidal action in epimastigotes, trypomastigotes and amastigotes of the Y, CL Brener and Dm28c T. cruzi strains. Finally, a synergistic effect between benznidazole and the CV chemical analogues was evidenced by combination and dose-reduction indexes values in epimastigotes and trypomastigotes of the Y strain. CONCLUSIONS/SIGNIFICANCE: Loratadine, cyproheptadine and clofazimine inhibit TcAAAP069 proline transporter and also present trypanocidal effect against all T. cruzi life stages in strains from three different DTUs. These CV structural analogues could be a starting point to design therapeutic alternatives to treat Chagas disease by finding new indications for old drugs. This approach, called drug repurposing is a recommended strategy by the World Health Organization to treat neglected diseases, like Chagas disease, and combination therapy may improve the possibility of success of repositioned drugs.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/antagonistas & inibidores , Violeta Genciana/química , Violeta Genciana/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Doença de Chagas/parasitologia , Clofazimina/farmacologia , Simulação por Computador , Reposicionamento de Medicamentos , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Loratadina/farmacologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Tripanossomicidas/química , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismo
11.
PLoS Negl Trop Dis ; 14(1): e0007945, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31895927

RESUMO

Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite, Trypanosoma cruzi, and is transmitted by triatomine insects during its blood meal. Proliferative epimastigotes forms thrive inside the insects in the presence of heme (iron protoporphyrin IX), an abundant product of blood digestion, however little is known about the metabolic outcome of this signaling molecule in the parasite. Trypanosomatids exhibit unusual gene transcription employing a polycistronic transcription mechanism through trans-splicing that regulates its life cycle. Using the Deep Seq transcriptome sequencing we characterized the heme induced transcriptome of epimastigotes and determined that most of the upregulated genes were related to glucose metabolism inside the glycosomes. These results were supported by the upregulation of glycosomal isoforms of PEPCK and fumarate reductase of heme-treated parasites, implying that the fermentation process was favored. Moreover, the downregulation of mitochondrial gene enzymes in the presence of heme also supported the hypothesis that heme shifts the parasite glycosomal glucose metabolism towards aerobic fermentation. These results are examples of the environmental metabolic plasticity inside the vector supporting ATP production, promoting epimastigotes proliferation and survival.


Assuntos
Perfilação da Expressão Gênica , Heme/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismo , Animais , Doença de Chagas/metabolismo , Genes Mitocondriais , Glucose/metabolismo , Insetos Vetores/parasitologia , Microcorpos/metabolismo , Transdução de Sinais , Transcrição Genética , Triatominae/parasitologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimento
12.
Folia Microbiol (Praha) ; 65(2): 323-328, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31250361

RESUMO

Endophytic fungi live inside vegetal tissues without causing damage to the host plant and may provide lead compounds for drug discovery. The co-culture of two or more endophytic fungi can trigger silent gene clusters, which could lead to the isolation of bioactive compounds. In this study, two endophytic strains isolated from Handroanthus impetiginosus leaves, identified as Talaromyces purpurogenus H4 and Phanerochaete sp. H2, were grown in mixed and axenic cultures. The meroterpenoid austin was detected only in the extracts from the mixed culture. Once isolated, austin displayed very interesting trypanocidal activity, with an IC50 value of 36.6 ± 1.2 µg/mL against Trypanosoma cruzi in the epimastigote form. The results obtained highlight the importance of the co-culturing of endophytic fungi to obtain natural bioactive products. The findings also enhance our understanding of the ecological relationships between endophytic fungi.


Assuntos
Endófitos/crescimento & desenvolvimento , Tabebuia/microbiologia , Talaromyces/crescimento & desenvolvimento , Talaromyces/metabolismo , Tripanossomicidas/metabolismo , Técnicas de Cocultura , Endófitos/química , Endófitos/genética , Phanerochaete/química , Phanerochaete/genética , Phanerochaete/crescimento & desenvolvimento , Phanerochaete/metabolismo , Folhas de Planta/microbiologia , Talaromyces/química , Talaromyces/genética , Terpenos/análise , Terpenos/metabolismo , Terpenos/farmacologia , Tripanossomicidas/análise , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimento
13.
Genomics ; 112(1): 990-997, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31229555

RESUMO

Trypanosoma cruzi is the etiologic agent of Chagas disease, a life-threatening disease that affects different tissues. Within its mammalian host, T. cruzi develops molecular strategies for successful invasion of different cell types and adaptation to the intracellular environment. Conversely, the host cell responds to the infection by activating intracellular pathways to control parasite replication. Here, we reviewed genome-wide expression studies based on microarray and RNA-seq data from both parasite and host genes generated from animal models of infection as well as from Chagas disease patients. As expected, analyses of T. cruzi genes highlighted changes related to parasite energy metabolism and cell surface molecules, whereas host cell transcriptome emphasized the role of immune response genes. Besides allowing a better understanding of mechanisms behind the pathogenesis of Chagas disease, these studies provide essential information for the development of new therapies as well as biomarkers for diagnosis and assessment of disease progression.


Assuntos
Doença de Chagas/genética , Transcriptoma , Trypanosoma cruzi/genética , Animais , Cardiomiopatia Chagásica/genética , Doença de Chagas/parasitologia , Fibroblastos/metabolismo , Fibroblastos/parasitologia , Humanos , Estágios do Ciclo de Vida , Camundongos , RNA não Traduzido/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismo
14.
BMC Pharmacol Toxicol ; 20(Suppl 1): 76, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31852548

RESUMO

BACKGROUND: Chagas disease (CD) is a tropical parasitic disease. Although the number of people infected is very high, the only drugs available to treat CD, nifurtimox (Nfx) and benznidazole, are highly toxic, particularly in the chronic stage of the disease. Coumarins are a large class of compounds that display a wide range of interesting biological properties, such as antiparasitic. Hence, the aim of this work is to find a good antitrypanosomal drug with less toxicity. The use of simple organism models has become increasingly attractive for planning and simplifying efficient drug discovery. Within these models, Caenorhabditis elegans has emerged as a convenient and versatile tool with significant advantages for the toxicological potential identification for new compounds. METHODS: Trypanocidal activity: Forty-two 4-methylamino-coumarins were assayed against the epimastigote form of Trypanosoma cruzi (Tulahuen 2 strain) by inhibitory concentration 50% (IC50). Toxicity assays: Lethal dose 50% (LD50) and Body Area were determined by Caenorhabditis elegans N2 strain (wild type) after acute exposure. Structure-activity relationship: A classificatory model was built using 3D descriptors. RESULTS: Two of these coumarins demonstrated near equipotency to Nifurtimox (IC50 = 5.0 ± 1 µM), with values of: 11 h (LaSOM 266), (IC50 = 6.4 ± 1 µM) and 11 g (LaSOM 231), (IC50 = 8.2 ± 2.3 µM). In C. elegans it was possible to observe that Nfx showed greater toxicity in both the LD50 assay and the evaluation of the development of worms. It is possible to observe that the efficacy between Nfx and the synthesized compounds (11 h and 11 g) are similar. On the other hand, the toxicity of Nfx is approximately three times higher than that of the compounds. Results from the QSAR-3D study indicate that the volume and hydrophobicity of the substituents have a significant impact on the trypanocidal activities for derivatives that cause more than 50% of inhibition. These results show that the C. elegans model is efficient for screening potentially toxic compounds. CONCLUSION: Two coumarins (11 h and 11 g) showed activity against T. cruzi epimastigote similar to Nifurtimox, however with lower toxicity in both LD50 and development of C. elegans assays. These two compounds may be a feasible starting point for the development of new trypanocidal drugs.


Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Cumarínicos/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Cumarínicos/síntese química , Cumarínicos/química , Cumarínicos/toxicidade , Concentração Inibidora 50 , Dose Letal Mediana , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Tripanossomicidas/síntese química , Tripanossomicidas/química , Tripanossomicidas/toxicidade , Trypanosoma cruzi/crescimento & desenvolvimento
15.
Exp Parasitol ; 206: 107730, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31494215

RESUMO

Phospholipids are the main component of membranes and are responsible for cell integrity. Alkylphospholipid analogues (APs) were first designed as antitumoral agents and were later tested against different cell types. Trypanosoma cruzi, the Chagas disease etiological agent, is sensitive to APs (edelfosine, miltefosine and ilmofosine) in vitro. We investigated the effect of synthetic ring substituted AP against epimastigotes, amastigotes and trypomastigotes. TCAN26, could inhibit the in vitro growth of epimastigotes and amastigotes with the 50% inhibitory concentrations (IC50) in the nanomolar range. Trypomastigotes lysis was also induced with 24-h treatment and a LC50 of 2.3 µM. Ultrastructural analysis by electron microscopy demonstrated that TCAN26 mainly affected the parasite's membranes leading to mitochondrial and Golgi cisternae swelling, membrane blebs, and autophagic figures in the different parasite developmental stages. While the Golgi of the parasites was significantly affected, the Golgi complex of the host cells remained normal suggesting a specific mechanism of action. In summary, our results suggest that TCAN 26 is a potent and selective inhibitor of T. cruzi growth probably due to disturbances of phospholipid biosynthesis.


Assuntos
Adamantano/farmacologia , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Adamantano/química , Animais , Antiprotozoários/química , Antiprotozoários/farmacologia , Autofagia/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Complexo de Golgi/efeitos dos fármacos , Concentração Inibidora 50 , Dose Letal Mediana , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Mitocôndrias/efeitos dos fármacos , Fosforilcolina/química , Tripanossomicidas/química , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/ultraestrutura
16.
Nat Rev Microbiol ; 17(10): 607-620, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31444481

RESUMO

Antimicrobial treatment failure threatens our ability to control infections. In addition to antimicrobial resistance, treatment failures are increasingly understood to derive from cells that survive drug treatment without selection of genetically heritable mutations. Parasitic protozoa, such as Plasmodium species that cause malaria, Toxoplasma gondii and kinetoplastid protozoa, including Trypanosoma cruzi and Leishmania spp., cause millions of deaths globally. These organisms can evolve drug resistance and they also exhibit phenotypic diversity, including the formation of quiescent or dormant forms that contribute to the establishment of long-term infections that are refractory to drug treatment, which we refer to as 'persister-like cells'. In this Review, we discuss protozoan persister-like cells that have been linked to persistent infections and discuss their impact on therapeutic outcomes following drug treatment.


Assuntos
Antiprotozoários/farmacologia , Tolerância a Medicamentos , Leishmania/efeitos dos fármacos , Plasmodium/efeitos dos fármacos , Toxoplasma/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Variação Biológica da População , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Humanos , Leishmania/crescimento & desenvolvimento , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Malária/tratamento farmacológico , Malária/parasitologia , Plasmodium/crescimento & desenvolvimento , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose/tratamento farmacológico , Toxoplasmose/parasitologia , Falha de Tratamento , Trypanosoma cruzi/crescimento & desenvolvimento
17.
Exp Cell Res ; 383(2): 111560, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31437457

RESUMO

In higher eukaryotic cells, pertubations in ER environment, called ER stress, usually activate unfolded protein response (UPR) pathway in an attempt to re-stablish the ER homeostasis and prevent cell death. Because trypanosomatids appear to lack the classical UPR, it is not clear how these parasites respond to ER stress. Thus, the aim of this work was to evaluate the effects of ER stressors tunicamycin (TM) or dithiothreitol (DTT) on Trypanosoma cruzi. The TM treatment showed strong trypanostatic effect. At 2.5 µg/mL of TM, the mRNA levels of both binding protein (BiP) and calreticulin (CRT) increased significantly, whereas the protein levels of BiP remained stable. TM treatment induced ultrastructural changes compatible with an autophagic process. The DTT treatment inhibited the cell growth, induced drastic morphological changes, mitochondrial membrane depolarization and increased ROS production. The expression of BiP apparently was not affected by DTT, whereas the mRNA levels of BiP and CRT were significantly reduced. Our results suggest that TM induces autophagy/ER-phagy without causing substantial injury to the parasite. Conversely, the DTT treatment seems to rupture the mitochondrion homeostasis leading to parasite death. The comprehension of the mechanisms behind the susceptibility of T. cruzi to ER stress open perspectives for the development of chemotherapeutic agents addressed to these pathways.


Assuntos
Ditiotreitol/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Tunicamicina/farmacologia , Calreticulina/genética , Calreticulina/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Testes de Sensibilidade Parasitária , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/ultraestrutura , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética
18.
J Vis Exp ; (149)2019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31424432

RESUMO

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas' disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.


Assuntos
Sistemas CRISPR-Cas , Doença de Chagas/parasitologia , Técnicas de Inativação de Genes/métodos , Estágios do Ciclo de Vida/fisiologia , Proteínas de Protozoários/antagonistas & inibidores , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/genética , Animais , Doença de Chagas/genética , Fibroblastos/metabolismo , Fibroblastos/parasitologia , Edição de Genes , Humanos , Proteínas de Protozoários/genética , Trypanosoma cruzi/metabolismo
19.
Parasitol Res ; 118(9): 2609-2619, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31267245

RESUMO

Trypanosoma cruzi is the etiological agent of Chagas disease. These parasites undergo dramatic morphological and physiological changes during their life cycle. The human-infective metacyclic trypomastigotes differentiate from epimastigotes inside the midgut of the Triatominae insect vector. Our group has shown that the saliva and feces of Rhodnius prolixus contains a lysophospholipid, lysophosphatidylcholine (LPC), which modulates several aspects of T. cruzi infection in macrophages. LPC hydrolysis by a specific lysophospholipase D, autotaxin (ATX), generates lysophosphatidic acid (LPA). These bioactive lysophospholipids are multisignaling molecules and are found in human plasma ingested by the insect during blood feeding. Here, we show the role of LPC and LPA in T. cruzi proliferation and differentiation. Both lysophospholipids are able to induce parasite proliferation. We observed an increase in parasite growth with different fatty acyl chains, such as C18:0, C16:0, or C18:1 LPC. The dynamics of LPC and LPA effect on parasite proliferation was evaluated in vivo through a time- and space-dependent strategy in the vector gut. LPC but not LPA was also able to affect parasite metacyclogenesis. Finally, we determined LPA and LPC distribution in the parasite itself. Such bioactive lipids are associated with reservosomes of T. cruzi. To the best of our knowledge, this is the first study to suggest the role of surrounding bioactive lipids ingested during blood feeding in the control of parasite transmission.


Assuntos
Doença de Chagas/parasitologia , Metabolismo dos Lipídeos , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismo , Animais , Doença de Chagas/transmissão , Humanos , Insetos Vetores/parasitologia , Estágios do Ciclo de Vida , Lipídeos/química , Rhodnius/parasitologia
20.
Exp Parasitol ; 204: 107724, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31279930

RESUMO

Only two drugs are currently available for the treatment of Chagas disease and their effectiveness are unsatisfactory. Photorhabdus luminescens and Xenorhabdus nematophila, two enteric bacteria highly pathogenic to a broad range of insects, have been studied as potential source for bioactive metabolites against protozoa causing neglected tropical diseases. Therefore, we tested the in vitro anti-Trypanosoma cruzi activity of secreted metabolites from these bacteria. The conditioned medium of X. nematophila and P. luminescens showed significant parasiticidal activity in a concentration-dependent manner (IC50XN = 0.34 mg/mL, IC50PL = 1.0 mg/mL). The parasiticidal compound was identified as a small molecule stable to heating and pH changes ranging from 2 to 12. Moreover, anti-Trypanosoma molecules secreted by both bacteria stimulate the trypanocidal activity of macrophages by a mechanism independent of nitric oxide. Summarizing, our studies reveal that P. luminescens and X. nematophila are potential sources of putative novel drugs against Chagas disease.


Assuntos
Proteínas de Bactérias/farmacologia , Photorhabdus/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Xenorhabdus/química , Análise de Variância , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/uso terapêutico , Bioensaio , Doença de Chagas/tratamento farmacológico , Meios de Cultivo Condicionados , Endopeptidase K/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Temperatura , Tripanossomicidas/efeitos adversos , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/crescimento & desenvolvimento
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