RESUMEN

Biofilm formation is a major health concern and studies have been pursued to find compounds able to prevent biofilm establishment and remove pre-existing biofilms. While biosurfactants (BS) have been well-known for possessing antibiofilm activities, bioemulsifiers (BE) are still scarcely explored for this purpose. The present study aimed to evaluate the bioemulsifying properties of cell-free supernatants produced by Bacillaceae and Vibrio strains isolated from marine sponges and investigate their antiadhesive and antibiofilm activities against different pathogenic Gram-positive and Gram-negative bacteria. The BE production by the marine strains was confirmed by the emulsion test, drop-collapsing, oil-displacement, cell hydrophobicity and hemolysis assays. Notably, Bacillus cereus 64BHI1101 displayed remarkable emulsifying activity and the ultrastructure analysis of its BE extract (BE64-1) revealed the presence of structures typically observed in macromolecules composed of polysaccharides and proteins. BE64-1 showed notable antiadhesive and antibiofilm activities against Staphylococcus aureus, with a reduction of adherence of up to 100 % and a dispersion of biofilm of 80 %, without affecting its growth. BE64-1 also showed inhibition of Staphylococcus epidermidis and Escherichia coli biofilm formation and adhesion. Thus, this study provides a starting point for exploring the antiadhesive and antibiofilm activities of BE from sponge-associated bacteria, which could serve as a valuable tool for future research to combat S. aureus biofilms.

Asunto(s)

Adhesión Bacteriana , Biopelículas , Emulsionantes , Poríferos , Staphylococcus aureus , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Poríferos/microbiología , Animales , Adhesión Bacteriana/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/fisiología , Emulsionantes/farmacología , Emulsionantes/química , Staphylococcus epidermidis/efectos de los fármacos , Staphylococcus epidermidis/fisiología , Escherichia coli/efectos de los fármacos , Escherichia coli/fisiología , Interacciones Hidrofóbicas e Hidrofílicas , Antibacterianos/farmacología , Bacillus cereus/efectos de los fármacos , Bacillus cereus/fisiología , Hemólisis , Tensoactivos/farmacología , Tensoactivos/metabolismo , Vibrio/efectos de los fármacos , Vibrio/fisiología , Vibrio/metabolismo , Pruebas de Sensibilidad Microbiana , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/fisiología

RESUMEN

The main goal of this work was to evaluate the therapeutic potential of green superparamagnetic iron oxide nanoparticles (SPIONs) produced with coconut water for treating cutaneous leishmaniasis caused by Leishmania amazonensis. Optical and electron microscopy techniques were used to evaluate the effects on cell proliferation, infectivity percentage, and ultrastructure. SPIONs were internalized by both parasite stages, randomly distributed in the cytosol and located mainly in membrane-bound compartments. The selectivity index for intracellular amastigotes was more than 240 times higher compared to current drugs used to treat the disease. The synthesized SPIONs showed promising activity against Leishmania and can be considered a strong candidate for a new therapeutic approach for treating leishmaniases.

RESUMEN

Searching for new alternatives for treating leishmaniasis, we present the synthesis, characterization, and biological evaluation against Leishmania amazonensis of the new ZnCl2(H3)2 complex. H3 is 22-hydrazone-imidazoline-2-yl-chol-5-ene-3ß-ol, a well-known bioactive molecule functioning as a sterol Δ24-sterol methyl transferase (24-SMT) inhibitor. The ZnCl2(H3)2 complex was characterized by infrared, UV-vis, molar conductance measurements, elemental analysis, mass spectrometry, and NMR experiments. The biological results showed that the free ligand H3 and ZnCl2(H3)2 significantly inhibited the growth of promastigotes and intracellular amastigotes. The IC50 values found for H3 and ZnCl2(H3)2 were 5.2 µM and 2.5 µM for promastigotes, and 543 nM and 32 nM for intracellular amastigotes, respectively. Thus, the ZnCl2(H3)2 complex proved to be seventeen times more potent than the free ligand H3 against the intracellular amastigote, the clinically relevant stage. Furthermore, cytotoxicity assays and determination of selectivity index (SI) revealed that ZnCl2(H3)2 (CC50 = 5 µΜ, SI = 156) is more selective than H3 (CC50 = 10 µΜ, SI = 20). Furthermore, as H3 is a specific inhibitor of the 24-SMT, free sterol analysis was performed. The results showed that H3 was not only able to induce depletion of endogenous parasite sterols (episterol and 5-dehydroepisterol) and their replacement by 24-desalkyl sterols (cholesta-5,7,24-trien-3ß-ol and cholesta-7,24-dien-3ß-ol) but also its zinc derivative resulting in a loss of cell viability. Using electron microscopy, studies on the fine ultrastructure of the parasites showed significant differences between the control cells and parasites treated with H3 and ZnCl2(H3)2. The inhibitors induced membrane wrinkle, mitochondrial injury, and abnormal chromatin condensation changes that are more intense in the cells treated with ZnCl2(H3)2.

RESUMEN

Snake venoms are complex cocktails of non-toxic and toxic molecules that work synergistically for the envenoming outcome. Alongside the immediate consequences, chronic manifestations and long-term sequelae can occur. Recently, extracellular vesicles (EVs) were found in snake venom. EVs mediate cellular communication through long distances, delivering proteins and nucleic acids that modulate the recipient cell's function. However, the biological roles of snake venom EVs, including possible cross-organism communication, are still unknown. This knowledge may expand the understanding of envenoming mechanisms. In the present study, we isolated and characterized the EVs from Bothrops jararaca venom (Bj-EVs), giving insights into their biological roles. Fresh venom was submitted to differential centrifugation, resulting in two EV populations with typical morphology and size range. Several conserved EV markers and a subset of venom related EV markers, represented mainly by processing enzymes, were identified by proteomic analysis. The most abundant protein family observed in Bj-EVs was 5'-nucleotidase, known to be immunosuppressive and a low abundant and ubiquitous toxin in snake venoms. Additionally, we demonstrated that mammalian cells efficiently internalize Bj-EVs. The commercial antibothropic antivenom partially recognizes Bj-EVs and inhibits cellular EV uptake. Based on the proteomic results and the in vitro interaction assays using macrophages and muscle cells, we propose that Bj-EVs may be involved not only in venom production and processing but also in host immune modulation and long-term effects of envenoming.

Asunto(s)

Bothrops , Venenos de Crotálidos , Vesículas Extracelulares , Animales , Venenos de Crotálidos/química , Proteómica , Proteínas , Venenos de Serpiente , Mamíferos

RESUMEN

Leishmaniasis is a neglected disease caused by protozoan parasites of the Leishmania genus. Benzylamines are a class of compounds selectively designed to inhibit the squalene synthase (SQS) that catalyzes the first committed reaction on the sterol biosynthesis pathway. Herein, we studied seven new benzylamines (SBC 37-43) against Leishmania amazonensis. After the first screening of cell viability, two inhibitors (SBC 39 and SBC 40) were selected. Against intracellular amastigotes, SBC 39 and SBC 40 presented selectivity indexes of 117.7 and 180, respectively, indicating high selectivity. Analysis of the sterol composition revealed a depletion of endogenous 24-alkylated sterols such as episterol and 5-dehydroepisterol, with a concomitant accumulation of fecosterol, implying a disturbance in cellular lipid content. This result suggests a blockade of de novo sterol synthesis at the level of SQS and C-5 desaturase. Furthermore, physiological analysis and electron microscopy revealed three main alterations: (1) in the mitochondrion; (2) the presence of lipid bodies and autophagosomes; and (3) the appearance of projections in the plasma membrane. In conclusion, our results support the notion that benzylamines have a potent effect against Leishmania amazonensis and should be an exciting novel pharmaceutical lead for developing new chemotherapeutic alternatives to treat leishmaniasis.

Asunto(s)

Leishmania mexicana , Leishmania , Bencilaminas/farmacología , Farnesil Difosfato Farnesil Transferasa/metabolismo , Estrés Oxidativo , Esteroles/metabolismo

RESUMEN

The new complexes Zn(ITZ)2Cl2 (1) and Zn(ITZ)2(OH)2 (2) were synthetized by a reaction of itraconazole with their respective zinc salts under reflux. These Zn-ITZ complexes were characterized by elemental analyses, molar conductivity, mass spectrometry, 1H and 13C{1H} nuclear magnetic resonance, and UV-vis and infrared spectroscopies. The antiparasitic and antifungal activity of Zn-ITZ complexes was evaluated against three protozoans of medical importance, namely, Leishmania amazonensis, Trypanosoma cruzi, and Toxoplasma gondii, and two fungi, namely, Sporothrix brasiliensis and Sporothrix schenckii The Zn-ITZ complexes exhibited a broad spectrum of action, with antiparasitic and antifungal activity in low concentrations. The strategy of combining zinc with ITZ was efficient to enhance ITZ activity since Zn-ITZ-complexes were more active than the azole alone. This study opens perspectives for future applications of these Zn-ITZ complexes in the treatment of parasitic diseases and sporotrichosis.

Asunto(s)

Antifúngicos/farmacología , Antiparasitarios/farmacología , Itraconazol/farmacología , Zinc/farmacología , Leishmania/efectos de los fármacos , Leishmania mexicana/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Pruebas de Sensibilidad Parasitaria , Sporothrix/efectos de los fármacos , Toxoplasma/efectos de los fármacos , Trypanosoma cruzi/efectos de los fármacos

RESUMEN

Kinetoplast DNA (kDNA) bearing unusual mitochondrion of trypanosomatid parasites offers a new paradigm in chemotherapy modality. Topoisomerase II of Leishmania donovani (LdTopII), a key enzyme associated with kDNA replication, is emerging as a potential drug target. However, mode of action of LdTopII targeted compounds in the parasites at sub-cellular level remains largely unknown. Previously, we reported that an isobenzofuranone derivative, namely 3,5-bis(4-chlorophenyl)-7-hydroxyisobenzofuran-1(3H)-one (JVPH3), targets LdTopII and induces apoptosis-like cell death in L. donovani. Here, we elucidate the phenotypic changes and the events occurring at sub-cellular level caused by JVPH3 in L. donovani. In addition, we have evaluated the cytotoxicity and ultrastructural alterations caused by JVPH3 in two brazilian trypanosomatid pathogens viz. L. amazonensis and Trypanosoma cruzi. Despite killing these parasites, JVPH3 caused significantly different phenotypes in L. donovani and L. amazonensis. More than 90% population of parasites showed altered morphology. Mitochondrion was a major target organelle subsequently causing kinetoplast network disorganization in Leishmania. Altered mitochondrial architecture was evident in 75-80% Leishmania population being investigated. Quantification of mitochondrial function using JC-1 fluorophore to measure a possible mitochondrial membrane depolarization further confirmed the mitochondrion as an essential target of the JVPH3 corroborating with the phenotype observed by electron microscopy. However, the impact of JVPH3 was lesser on T. cruzi than Leishmania. The molecule caused mitochondrial alteration in 40% population of the epimastigotes being investigated. To our knowledge, this is the first report to evaluate the proliferation pattern and ultrastructural alterations caused in Brazilian kinetoplastid pathogens by a synthetic LdTopII inhibitor previously established to have promising in vivo activity against Indian strain of L. donovani.

Asunto(s)

ADN-Topoisomerasas de Tipo II/metabolismo , Leishmania donovani/enzimología , Leishmania/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Inhibidores de Topoisomerasa II/farmacología , Trypanosoma cruzi/efectos de los fármacos , Apoptosis/efectos de los fármacos , Biocatálisis/efectos de los fármacos , ADN de Cinetoplasto/metabolismo , Leishmania/metabolismo , Leishmania/ultraestructura , Leishmania donovani/ultraestructura , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/ultraestructura

RESUMEN

INTRODUCTION: Cancer-associated thrombosis is one of the major causes of worse prognosis among tumor-bearing patients. Extracellular vesicles derived from cancer cells, which can be divided mainly into microvesicles and exosomes, can participate in several tumor progression phenomena. Tumor-derived microvesicles positive for tissue factor (TF) have been associated with thrombotic risk in certain cancer types. Cancer cell-derived exosomes, however, have not. In this study we evaluated the capacity of extracellular vesicles (EVs, containing both microvesicles and exosomes) derived from breast-cancer cell lines in promoting platelet activation, aggregation and plasma coagulation, in experiments that access both TF-dependent and -independent activities. MATERIALS AND METHODS: EVs were isolated from the conditioned media of two human mammary carcinoma cell lines: MDA-MB-231 (highly invasive) and MCF-7 (less invasive). TF-independent EV/platelet interaction, platelet P-selectin exposure and aggregation were evaluated. Western blotting, plasma clotting and platelet aggregation in the presence of plasma were performed for the measurement of TF-dependent activity in EVs. RESULTS: Interaction between MDA-MB-231 EVs and washed platelets led to increased platelet P-selectin exposure and platelet aggregation compared to MCF-7 EVs. MDA-MB-231 EVs had higher TF protein levels and TF-dependent procoagulant activity than MCF-7 EVs. Consequently, TF-dependent platelet aggregation was also induced by MDA-MB-231 EVs, but not by MCF-7 EVs. CONCLUSION: Our results suggest that MDA-MB-231 EVs induce TF-independent platelet activation and aggregation, as well as TF-dependent plasma clotting and platelet aggregation by means of thrombin generation. In this context, aggressive breast cancer-derived EVs may contribute to cancer-associated thrombosis.

Asunto(s)

Neoplasias de la Mama/genética , Vesículas Extracelulares/metabolismo , Tromboplastina/metabolismo , Trombosis/etiología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Vesículas Extracelulares/patología , Femenino , Humanos , Activación Plaquetaria , Agregación Plaquetaria , Trombosis/patología

RESUMEN

Treatment of leishmaniasis involves the use of antimonials, miltefosine, amphotericin B or pentamidine. However, the side effects of these drugs and the reports of drug-resistant parasites demonstrate the need for new treatments that are safer and more efficacious. Histone deacetylase inhibitors are a new class of compounds with potential to treat leishmaniasis. Herein, we evaluated the effects of KH-TFMDI, a novel histone deacetylase inhibitor, on Leishmania amazonensis promastigotes and intracellular amastigotes. The IC50 values of this compound for promastigotes and intracellular amastigotes were 1.976 and 1.148 µM, respectively, after 72 h of treatment. Microscopic analyses revealed that promastigotes became elongated and thinner in response to KH-TFMDI, indicating changes in cytoskeleton organization. Immunofluorescence microscopy, western blotting and flow cytometry using an anti-acetylated tubulin antibody revealed an increase in the expression of acetylated tubulin. Furthermore, transmission electron microscopy revealed several ultrastructural changes, such as (a) mitochondrial swelling, followed by the formation of many vesicles inside the matrix; (b) presence of lipid bodies randomly distributed through the cytoplasm; (c) abnormal chromatin condensation; and (d) formation of blebs on the plasma membrane. Physiological studies for mitochondrial function, flow cytometry with propidium iodide and TUNEL assay confirmed the alterations in the mitochondrial metabolism, cell cycle, and DNA fragmentation, respectively, which could result to cell death by mechanisms related to apoptosis-like. All these together indicate that histone deacetylases are promising targets for the development of new drugs to treat Leishmania, and KH-TFMDI is a promising drug candidate that should be tested in vivo.

Asunto(s)

Compuestos de Bencilideno/farmacología , Muerte Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Indoles/farmacología , Leishmania/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Sirtuinas/antagonistas & inhibidores , Animales , Antiparasitarios/farmacología , Antiparasitarios/toxicidad , Apoptosis/efectos de los fármacos , Compuestos de Bencilideno/toxicidad , Puntos de Control del Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citoesqueleto/metabolismo , Inhibidores de Histona Desacetilasas/toxicidad , Indoles/toxicidad , Concentración 50 Inhibidora , Leishmania/citología , Leishmania/crecimiento & desarrollo , Leishmania/ultraestructura , Leishmaniasis/tratamiento farmacológico , Leishmaniasis/parasitología , Gotas Lipídicas/efectos de los fármacos , Gotas Lipídicas/metabolismo , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Estrés Oxidativo/efectos de los fármacos

RESUMEN

Indole alkaloids possess a large spectrum of biological activities including anti-protozoal action. Here we report for the first time that voacamine, isolated from the plant Tabernaemontana coronaria, is an antiprotozoal agent effective against a large array of trypanosomatid parasites including Indian strain of Leishmania donovani and Brazilian strains of Leishmania amazonensis and Trypanosoma cruzi. It inhibits the relaxation activity of topoisomerase IB of L. donovani (LdTop1B) and stabilizes the cleavable complex. Voacamine is probably the first LdTop1B-specific poison to act uncompetitively. It has no impact on human topoisomerase I and II up to 200µM concentrations. The study also provides a thorough insight into ultrastructural alterations induced in three kinetoplastid parasites by a specific inhibitor of LdTop1B. Voacamine is also effective against intracellular amastigotes of different drug unresponsive field isolates of Leishmania donovani obtained from endemic zones of India severely affected with visceral leishmaniasis. Most importantly, this is the first report demonstrating the efficacy of a compound to reduce the burden of drug resistant parasites, unresponsive to SAG, amphotericin B and miltefosine, in experimental BALB/c mice model of visceral leishmaniasis. The findings cumulatively provide a strong evidence that voacamine can be a promising drug candidate against trypanosomatid infections.

Asunto(s)

Antiprotozoarios/farmacología , ADN-Topoisomerasas de Tipo I/metabolismo , Ibogaína/análogos & derivados , Leishmania donovani/efectos de los fármacos , Leishmania mexicana/efectos de los fármacos , Inhibidores de Topoisomerasa I/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Antiprotozoarios/administración & dosificación , Antiprotozoarios/aislamiento & purificación , Antiprotozoarios/uso terapéutico , Forma de la Célula/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/genética , Relación Dosis-Respuesta a Droga , Resistencia a Múltiples Medicamentos , Estabilidad de Enzimas/efectos de los fármacos , Femenino , Ibogaína/administración & dosificación , Ibogaína/aislamiento & purificación , Ibogaína/farmacología , Ibogaína/uso terapéutico , Leishmania donovani/enzimología , Leishmania donovani/crecimiento & desarrollo , Leishmania donovani/ultraestructura , Leishmania mexicana/enzimología , Leishmania mexicana/crecimiento & desarrollo , Leishmania mexicana/ultraestructura , Leishmaniasis Visceral/tratamiento farmacológico , Leishmaniasis Visceral/parasitología , Dosificación Letal Mediana , Ratones Endogámicos BALB C , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Corteza de la Planta/química , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tabernaemontana/química , Inhibidores de Topoisomerasa I/administración & dosificación , Inhibidores de Topoisomerasa I/aislamiento & purificación , Inhibidores de Topoisomerasa I/uso terapéutico , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/ultraestructura

RESUMEN

BACKGROUND: Leishmaniasis is a neglected vector-borne tropical disease caused by Leishmania protozoa that are transmitted to mammalian hosts by infected sand flies. Infection is associated with distinct clinical manifestations that include cutaneous, mucocutaneous and visceral lesions. Visceral leishmaniasis (VL) is the most severe form of the disease and is considered second in terms of mortality and fourth in terms of morbidity among tropical diseases. IFN-γ-producing T cells are involved in protection against the disease. METHODS: CD43⁺/⁺ and CD43⁻/⁻ mice on a C57BL/6 background were intravenously injected with 5 × 10 7 amastigotes of Leishmania (L.) infantum chagasi, and 30 days after infection the clinical signs of disease were examined; the splenocytes were isolated and assayed for cytokine production; and the livers were removed for phenotypic analysis of T cell subsets by flow cytometry. RESULTS: We report that mice lacking CD43 display increased susceptibility to infection by Leishmania (L.) infantum chagasi, with higher parasite burdens than wild-type mice. The increased susceptibility of CD43⁻/⁻ mice were associated with a weakened delayed hypersensitivity response and reduced levels of IgG2a antibodies to leishmania antigens. We further showed that expression of CD43 defines a major intrahepatic CD4⁺ and CD8⁺ T cell subsets with pro-inflammatory phenotypes and leads to increased levels of IFN-γ secretion by activated splenocytes. CONCLUSIONS: Our findings point to a role of CD43 in the development of host resistance to visceral leishmaniasis.

Asunto(s)

Leishmania infantum/inmunología , Leishmaniasis Visceral/inmunología , Leucosialina/inmunología , Psychodidae/parasitología , Subgrupos de Linfocitos T/inmunología , Animales , Citocinas/genética , Susceptibilidad a Enfermedades , Femenino , Leishmaniasis Visceral/parasitología , Ratones , Ratones Endogámicos C57BL

RESUMEN

Phytomonas serpens are flagellates in the family Trypanosomatidae that parasitise the tomato plant (Solanum lycopersicum L.), which results in fruits with low commercial value. The tomato glycoalkaloid tomatine and its aglycone tomatidine inhibit the growth of P. serpens in axenic cultures. Tomatine, like many other saponins, induces permeabilisation of the cell membrane and a loss of cell content, including the cytosolic enzyme pyruvate kinase. In contrast, tomatidine does not cause permeabilisation of membranes, but instead provokes morphological changes, including vacuolisation. Phytomonas treated with tomatidine show an increased accumulation of labelled neutral lipids (BODYPY-palmitic), a notable decrease in the amount of C24-alkylated sterols and an increase in zymosterol content. These results are consistent with the inhibition of 24-sterol methyltransferase (SMT), which is an important enzyme that is responsible for the methylation of sterols at the 24 position. We propose that the main target of tomatidine is the sterols biosynthetic pathway, specifically, inhibition of the 24-SMT. Altogether, the results obtained in the present paper suggest a more general effect of alkaloids in trypanosomatids, which opens potential therapeutic possibilities for the treatment of the diseases caused by these pathogens.

Asunto(s)

Proliferación Celular/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Tomatina/análogos & derivados , Tomatina/farmacología , Trypanosomatina/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Colesterol/análisis , Solanum lycopersicum/parasitología , Metiltransferasas/efectos de los fármacos , Microscopía Electrónica de Transmisión , Enfermedades de las Plantas/parasitología , Esteroles/análisis , Esteroles/biosíntesis , Trypanosomatina/metabolismo , Trypanosomatina/ultraestructura

RESUMEN

Phytomonas serpens are flagellates in the family Trypanosomatidae that parasitise the tomato plant (Solanum lycopersicum L.), which results in fruits with low commercial value. The tomato glycoalkaloid tomatine and its aglycone tomatidine inhibit the growth of P. serpens in axenic cultures. Tomatine, like many other saponins, induces permeabilisation of the cell membrane and a loss of cell content, including the cytosolic enzyme pyruvate kinase. In contrast, tomatidine does not cause permeabilisation of membranes, but instead provokes morphological changes, including vacuolisation. Phytomonas treated with tomatidine show an increased accumulation of labelled neutral lipids (BODYPY-palmitic), a notable decrease in the amount of C24-alkylated sterols and an increase in zymosterol content. These results are consistent with the inhibition of 24-sterol methyltransferase (SMT), which is an important enzyme that is responsible for the methylation of sterols at the 24 position. We propose that the main target of tomatidine is the sterols biosynthetic pathway, specifically, inhibition of the 24-SMT. Altogether, the results obtained in the present paper suggest a more general effect of alkaloids in trypanosomatids, which opens potential therapeutic possibilities for the treatment of the diseases caused by these pathogens.

Asunto(s)

Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Cateterismo/métodos , Obstrucción Intestinal/patología , Obstrucción Intestinal/terapia , Intestino Delgado/patología , Biopsia , Constricción Patológica , Endoscopía del Sistema Digestivo , Resultado del Tratamiento

RESUMEN

Autophagy is a central process behind the cellular remodeling that occurs during differentiation of Leishmania, yet the cargo of the protozoan parasite's autophagosome is unknown. We have identified glycosomes, peroxisome-like organelles that uniquely compartmentalize glycolytic and other metabolic enzymes in Leishmania and other kinetoplastid parasitic protozoa, as autophagosome cargo. It has been proposed that the number of glycosomes and their content change during the Leishmania life cycle as a key adaptation to the different environments encountered. Quantification of RFP-SQL-labeled glycosomes showed that promastigotes of L. major possess ~20 glycosomes per cell, whereas amastigotes contain ~10. Glycosome numbers were significantly greater in promastigotes and amastigotes of autophagy-defective L. major Δatg5 mutants, implicating autophagy in glycosome homeostasis and providing a partial explanation for the previously observed growth and virulence defects of these mutants. Use of GFP-ATG8 to label autophagosomes showed glycosomes to be cargo in ~15% of them; glycosome-containing autophagosomes were trafficked to the lysosome for degradation. The number of autophagosomes increased 10-fold during differentiation, yet the percentage of glycosome-containing autophagosomes remained constant. This indicates that increased turnover of glycosomes was due to an overall increase in autophagy, rather than an upregulation of autophagosomes containing this cargo. Mitophagy of the single mitochondrion was not observed in L. major during normal growth or differentiation; however, mitochondrial remnants resulting from stress-induced fragmentation colocalized with autophagosomes and lysosomes, indicating that autophagy is used to recycle these damaged organelles. These data show that autophagy in Leishmania has a central role not only in maintaining cellular homeostasis and recycling damaged organelles but crucially in the adaptation to environmental change through the turnover of glycosomes.

Asunto(s)

Autofagia/genética , Leishmania major , Microcuerpos/metabolismo , Fagosomas/metabolismo , Animales , Técnicas de Cultivo de Célula/métodos , Estadios del Ciclo de Vida/genética , Lisosomas/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Peroxisomas/metabolismo

RESUMEN

Leishmania and Trypanosoma belong to the Trypanosomatidae family and cause important human infections such as leishmaniasis, Chagas disease, and sleeping sickness. Leishmaniasis, caused by protozoa belonging to Leishmania, affects about 12 million people worldwide and can present different clinical manifestations, i.e., visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), and post-kala-azar dermal leishmaniasis (PKDL). Chagas disease, also known as American trypanosomiasis, is caused by Trypanosoma cruzi and is mainly prevalent in Latin America but is increasingly occurring in the United States, Canada, and Europe. Sleeping sickness or human African trypanosomiasis (HAT), caused by two sub-species of Trypanosoma brucei (i.e., T. b. rhodesiense and T. b. gambiense), occurs only in sub-Saharan Africa countries. These pathogenic trypanosomatids alternate between invertebrate and vertebrate hosts throughout their lifecycles, and different developmental stages can live inside the host cells and circulate in the bloodstream or in the insect gut. Trypanosomatids have a classical eukaryotic ultrastructural organization with some of the same main organelles found in mammalian host cells, while also containing special structures and organelles that are absent in other eukaryotic organisms. For example, the mitochondrion is ramified and contains a region known as the kinetoplast, which houses the mitochondrial DNA. Also, the glycosomes are specialized peroxisomes containing glycolytic pathway enzymes. Moreover, a layer of subpellicular microtubules confers mechanic rigidity to the cell. Some of these structures have been investigated to determine their function and identify potential enzymes and metabolic pathways that may constitute targets for new chemotherapeutic drugs.

Asunto(s)

Trypanosoma/fisiología , Tripanosomiasis/epidemiología , Humanos , Estadios del Ciclo de Vida , Trypanosoma/ultraestructura , Tripanosomiasis/fisiopatología

RESUMEN

Leishmaniasis, caused by protozoan parasites of the Leishmania genus, is one of the most prevalent neglected tropical diseases. It is endemic in 98 countries, causing considerable morbidity and mortality. Pentavalent antimonials are the first line of treatment for leishmaniasis except in India. In resistant cases, miltefosine, amphotericin B and pentamidine are used. These treatments are unsatisfactory due to toxicity, limited efficacy, high cost and difficult administration. Thus, there is an urgent need to develop drugs that are efficacious, safe, and more accessible to patients. Trypanosomatids, including Leishmania spp. and Trypanosoma cruzi, have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. Inhibition of ergosterol biosynthesis is increasingly recognized as a promising target for the development of new chemotherapeutic agents. The aim of this work was to investigate the antiproliferative, physiological and ultrastructural effects against Leishmania amazonensis of itraconazole (ITZ) and posaconazole (POSA), two azole antifungal agents that inhibit sterol C14α-demethylase (CYP51). Antiproliferative studies demonstrated potent activity of POSA and ITZ: for promastigotes, the IC50 values were 2.74 µM and 0.44 µM for POSA and ITZ, respectively, and for intracellular amastigotes, the corresponding values were 1.63 µM and 0.08 µM, for both stages after 72 h of treatment. Physiological studies revealed that both inhibitors induced a collapse of the mitochondrial membrane potential (ΔΨm), which was consistent with ultrastructural alterations in the mitochondrion. Intense mitochondrial swelling, disorganization and rupture of mitochondrial membranes were observed by transmission electron microscopy. In addition, accumulation of lipid bodies, appearance of autophagosome-like structures and alterations in the kinetoplast were also observed. In conclusion, our results indicate that ITZ and POSA are potent inhibitors of L. amazonensis and suggest that these drugs could represent novel therapies for the treatment of leishmaniasis, either alone or in combination with other agents.

Asunto(s)

Antiprotozoarios/farmacología , Inhibidores Enzimáticos/farmacología , Itraconazol/farmacología , Leishmania mexicana/efectos de los fármacos , Estadios del Ciclo de Vida/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Triazoles/farmacología , Antifúngicos/farmacología , Reposicionamiento de Medicamentos , Concentración 50 Inhibidora , Leishmania mexicana/enzimología , Leishmania mexicana/ultraestructura , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Microscopía Electrónica de Transmisión , Mitocondrias/enzimología , Mitocondrias/ultraestructura , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Esterol 14-Desmetilasa/metabolismo

RESUMEN

Parasitic protozoa of the Leishmania genus cause leishmaniasis, an important complex of tropical diseases that affect about 12 million people around the world. The drugs used to treat leishmaniasis are pentavalent antimonials, miltefosine, amphotericin B and pentamidine. In the present study, we evaluated the effect of a novel alkyl phosphocholine-dinitroaniline hybrid molecule, TC95, against Leishmania amazonensis promastigotes and intracellular amastigotes. Antiproliferative assays indicated that TC95 is a potent inhibitor of promastigotes and intracellular amastigotes with IC50 values of 2.6 and 1.2 µM, respectively. Fluorescence microscopy with anti-α-tubulin antibody revealed changes in the cytoskeleton, whilst scanning electron microscopy showed alterations in the shape, plasma membrane, length of the flagellum, and cell cycle. Flow cytometry confirmed the cell cycle arrest mainly in G1 phase, however a significant population appeared in sub G0/G1 and super-G2. The alterations in the plasma membrane integrity were confirmed by fluorometric analysis using Sytox Blue. Transmission electron microscopy also revealed an accumulation of lipid bodies, confirmed by fluorescence microscopy and fluorometric analysis using Nile Red. Important lesions were also observed in organelles such as mitochondrion, endoplasmic reticulum and Golgi complex. In summary, our study suggests that TC95, an alkyl phosphocholine-trifluralin hybrid molecule, is a promising novel compound against L. amazonensis.

Asunto(s)

Compuestos de Anilina/farmacología , Antiprotozoarios/farmacología , Leishmania mexicana/efectos de los fármacos , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacología , Trifluralina/análogos & derivados , Trifluralina/farmacología , Compuestos de Anilina/química , Compuestos de Anilina/toxicidad , Animales , Antiprotozoarios/química , Antiprotozoarios/toxicidad , Puntos de Control del Ciclo Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citometría de Flujo , Histocitoquímica , Humanos , Cuerpos de Inclusión/efectos de los fármacos , Concentración 50 Inhibidora , Leishmania mexicana/citología , Leishmania mexicana/ultraestructura , Leishmaniasis Cutánea Difusa/parasitología , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Microscopía de Interferencia , Fosforilcolina/química , Fosforilcolina/toxicidad , Trifluralina/química , Trifluralina/toxicidad

RESUMEN

Cancer cells display high proliferation rates and survival provided by high glycolysis, chemoresistance and radioresistance, metabolic features that appear to be activated with malignancy, and seemed to have arisen as early in evolution as in unicellular/prokaryotic organisms. Based on these assumptions, we hypothesize that aggressive phenotypes found in malignant cells may be related to acquired unicellular behavior, launched within a tumor when viral and prokaryotic homologs are overexpressed performing likely robust functions. The ensemble of these expressed viral and prokaryotic close homologs in the proteome of a tumor tissue gives them advantage over normal cells. To assess the hypothesis validity, sequences of human proteins involved in apoptosis, energetic metabolism, cell mobility and adhesion, chemo- and radio-resistance were aligned to homologs present in other life forms, excluding all eukaryotes, using PSI-BLAST, with further corroboration from data available in the literature. The analysis revealed that selected sequences of proteins involved in apoptosis and tumor suppression (as p53 and pRB) scored non-significant (E-value>0.001) with prokaryotic homologs; on the other hand, human proteins involved in cellular chemo- and radio-resistance scored highly significant with prokaryotic and viral homologs (as catalase, E-value=zero). We inferred that such upregulated and/or functionally activated proteins in aggressive malignant cells represent a toolbox of modern human homologs evolved from a similar key set that have granted survival of ancient prokaryotes against extremely harsh environments. According to what has been discussed along this analysis, high mutation rates usually hit hotspots in important conserved protein domains, allowing uncontrolled expansion of more resistant, death-evading malignant clones. That is the case of point mutations in key viral proteins affording viruses escape to chemotherapy, and human homologs of such retroviral proteins (as Ras, Akt and EGFR) can elicit the same phenotype. Furthermore, a corollary to this hypothesis presumes that target-directed anti-cancer therapy should target human protein domains of low similarity to prokaryotic homologs for a well-succeeded anti-cancer therapy.

Asunto(s)

Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Genes Bacterianos/genética , Genes Relacionados con las Neoplasias/genética , Fenotipo , Células Procariotas/química , Apoptosis/genética , Adhesión Celular/genética , Movimiento Celular/genética , Biología Computacional , Metabolismo Energético/genética , Humanos , Modelos Biológicos , Células Procariotas/metabolismo , Alineación de Secuencia , Homología de Secuencia

RESUMEN

Leishmaniasis is one of the most serious worldwide diseases caused by protozoan parasites of the Leishmania genus, affecting millions of people around the world. All currently available treatments present severe toxic side effects, require long-term compliance, cause serious side effects and are uncomfortable for patients. Leishmania amazonensis, a species endemic to Brazil, causes severe localised or diffuse skin lesions in humans. Owing to the unsatisfactory nature of the currently available chemotherapies, new approaches have been assessed for improved therapeutic intervention strategies against leishmaniasis. Miltefosine is an alkylphospholipid analogue that exhibits potent activity against the different clinical manifestations of leishmaniasis. Thus, the aim of this study was to investigate the long-term efficacy of miltefosine in BALB/c mice infected with L. amazonensis owing to the lack of a profound study demonstrating its dose-dependent and long-term effects. It was observed that animals treated with 20-50 mg/kg/day of miltefosine exhibited a significant dose-dependent reduction in lesion size; furthermore, in mice receiving higher doses, lesions disappeared after the end of treatment. To confirm a possible parasitological cure, mice up to 250 days after the end of treatment were analysed. No lesions or presence of parasite DNA were found in mice treated with 30, 40 and 50 mg/kg/day of miltefosine. In summary, these results show that miltefosine may be used to treat cutaneous leishmaniasis caused by L. amazonensis, alone or as combination therapy.

Asunto(s)

Antiprotozoarios/farmacología , Leishmania/patogenicidad , Leishmaniasis Cutánea/tratamiento farmacológico , Fosforilcolina/análogos & derivados , Animales , Antiprotozoarios/administración & dosificación , Colorantes Azulados/química , ADN Protozoario/química , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Leishmania/química , Leishmania/genética , Leishmaniasis Cutánea/parasitología , Meglumina/farmacología , Antimoniato de Meglumina , Ratones , Ratones Endogámicos BALB C , Compuestos Organometálicos/farmacología , Carga de Parásitos , Fosforilcolina/administración & dosificación , Fosforilcolina/farmacología , Factores de Tiempo , Úlcera/tratamiento farmacológico , Úlcera/parasitología

RESUMEN

Amiodarone (AMIO), the most frequently antiarrhythmic drug used for the symptomatic treatment of chronic Chagas' disease patients with cardiac compromise, has recently been shown to have also specific activity against fungi, Trypanosoma cruzi and Leishmania. In this work, we characterized the effects of AMIO on proliferation, mitochondrial physiology, and ultrastructure of Leishmania amazonensis promastigotes and intracellular amastigotes. The IC(50) values were 4.21 and 0.46 µM against promastigotes and intracellular amastigotes, respectively, indicating high selectivity for the clinically relevant stage. We also found that treatment with AMIO leads to a collapse of the mitochondrial membrane potential (ΔΨm) and to an increase in the production of reactive oxygen species, in a dose-dependent manner. Fluorescence microscopy of cells labeled with JC-1, a marker for mitochondrial energization, and transmission electron microscopy confirmed severe alterations of the mitochondrion, including intense swelling and modification of its membranes. Other ultrastructural alterations included (1) presence of numerous lipid-storage bodies, (2) presence of large autophagosomes containing part of the cytoplasm and membrane profiles, sometimes in close association with the mitochondrion and endoplasmic reticulum, and (3) alterations in the chromatin condensation and plasma membrane integrity. Taken together, our results indicate that AMIO is a potent inhibitor of L. amazonensis growth, acting through irreversible alterations in the mitochondrial structure and function, which lead to cell death by necrosis, apoptosis and/or autophagy.