HSP70 of Leishmania amazonensis alters resistance to different stresses and mitochondrial bioenergetics

The 70 kDa heat shock protein (HSP70) is a molecular chaperone that assists the parasite Leishmania in returning to homeostasis after being subjected to different types of stress during its life cycle. In the present study, we evaluated the effects of HSP70 transfection of L. amazonensis promastigotes (pTEX-HSP70) in terms of morphology, resistance, infectivity and mitochondrial bioenergetics. The pTEX-HSP70 promastigotes showed no ultrastructural morphological changes compared to control parasites. Interestingly, the pTEX-HSP70 promastigotes are resistant to heat shock, H2O2-induced oxidative stress and hyperbaric environments. Regarding the bioenergetics parameters, the pTEX-HSP70 parasites had higher respiratory rates and released less H2O2 than the control parasites. Nevertheless, the infectivity capacity of the parasites did not change, as verified by the infection of murine peritoneal macrophages and human macrophages, as well as the infection of BALB/c mice. Together, these results indicate that the overexpression of HSP70 protects L. amazonensis from stress, but does not interfere with its infective capacity.

Antileishmanial activity of diterpene acids in copaiba oil

Leishmaniasis is a neglected tropical disease. According to the World Health Organization, there are approximately 1.5-two million new cases of cutaneous leishmaniasis each year worldwide. Chemotherapy against leishmaniasis is based on pentavalent antimonials, which were developed more than a century ago. The goals of this study were to investigate the antileishmanial activity of diterpene acids in copaiba oil, as well as some possible targets of their action against Leishmania amazonensis. Methyl copalate and agathic, hydroxycopalic, kaurenoic, pinifolic and polyaltic acids isolated from Copaifera officinales oleoresins were utilised. Ultrastructural changes and the specific organelle targets of diterpenes were investigated with electron microscopy and flow cytometry, respectively. All compounds had some level of activity against L. amazonensis. Hydroxycopalic acid and methyl copalate demonstrated the most activity against promastigotes and had 50% inhibitory concentration (IC50) values of 2.5 and 6.0 µg/mL, respectively. However, pinifolic and kaurenoic acid demonstrated the most activity against axenic amastigote and had IC50 values of 3.5 and 4.0 µg/mL, respectively. Agathic, kaurenoic and pinifolic acid caused significant increases in plasma membrane permeability and mitochondrial membrane depolarisation of the protozoan. In conclusion, copaiba oil and its diterpene acids should be explored for the development of new antileishmanial drugs.

Estudo da atividade biológica de produtos naturais de macroalgas do litoral nordestino sobre Leishmania amazonensis / Study of the biological activity of natural products from macroalgae of the northeastern coast against Leishmania amazonensis

O tratamento utilizado para a leishmaniose apresenta efeitos colaterais graves, exige acompanhamento médico e prolongado tempo de terapia. Assim, a prospecção de novos compostos contra esta doença ainda se faz necessária. As macroalgas possuem grande variedade de moléculas bioativas. No presente trabalho foi avaliada a atividade leishmanicida, a citotoxicidade, a produção de óxido nítrico (NO) e os possíveis alvos dos extratos de macroalgas. Foi avaliado o efeito de 10 extratos sobre o crescimento de formas promastigotas de L. amazonensis. A citotoxicidade em células de mamíferos foi avaliada através do método do MTT e o índice de seletividade foi determinado. A produção de NO por macrófagos foi avaliada pelo reagente de Griess. A análise ultraestrutural foi realizada por microscopia eletrônica. Para análise dos efeitos dos extratos sobre a membrana e o potencial de membrana mitocondrial células tratadas e controles foram submetidas à marcação com iodeto de propídio (IP) e rodamina 123. Os dados mostraram que todos os extratos inibiram o crescimento de formas promastigotas e apresentaram baixa toxidade para células de mamífero. Canistrocarpus cervicornis (CC), Dictyota mertensii (DM) e Laurencia dendroidea (LD) foram as mais efetivas e mais seletivas contra formas promastigotas entre todas as algas testadas. Estes extratos também inibiram a infecção e índice de sobrevivência de formas amastigotas no interior de macrófagos. Esses extratos aumentaram a produção de NO em relação às células controles. Alterações compatíveis com a perda de viabilidade e morte celular foram observadas por microscopia eletrônica. Células tratadas apresentaram discreto aumento no número de células IP+. Os extratos induziram alterações significativas no potencial de membrana mitocondrial. A baixa toxicidade às células de mamíferos e a atividade leishmanicida apresentadas apontam para a utilização dos extratos de CC, DM e LD como agentes promissores para o tratamento da leishmaniose cutânea.

Estudio ultraestructural de la fagocitosis de promastigotes y amastigotes de Leishmania mexicana por la línea de células dendríticas FSDC / Phagocytosis of promastigotes and amastigotes of Leishmania mexicana by the FSDC

Introducción. Las células dendríticas están presentes en la mayoría de los tejidos, ellas capturan y presentan antígenos para activar a los linfocitos T. Objetivo. Se describe ultraestructuralmente la fagocitosis de Leishmania mexicana por la línea de células dendríticas FSDC, una línea de células de Langerhans obtenida de la epidermis fetal de ratón, e inmortalizada por la transducción retroviral del oncogen v-myc. Materiales y métodos. Se obtuvieron amastigotes de la lesión de ratones Balb/c y promastigotes a partir del cultivo (24°C) de la lesión. Las FSDC se cultivaron con los parásitos en una proporción de 5 parásitos por célula, en medio IMDM, durante 24 horas. Los cultivos infectados y los controles se procesaron para microscopía electrónica de transmisión. Se hicieron cortes semifinos contrastados con azul de toluidina para evaluar porcentaje de fagocitosis y finos, contrastados con acetato de uranilo y citrato de plomo. Resultados. El 13,42 por ciento de las FSDC fagocitaron promastigotes; de ellas el 8 por ciento contenían un parásito y el restante 5,2 por ciento fagocitó dos o más. El 20 por ciento de las FSDC fagocitaron amastigotes; 10 por ciento contenían un parásito y 10 por ciento dos o más. Ultraestructuralmente se observaron promastigotes en contacto con las células por el flagelo o por el polo posterior. Los fagosomas que contenían promastigotes eran organelos estrechos con uno ó dos parásitos. Los que contenían amastigotes eran de gran tamaño (8 µm) con uno o varios parásitos, libres o adosados a la membrana del fagosoma por su polo posterior. Conclusión. La infección de las FSDC se caracterizó por una baja tasa de células infectadas al ser expuestas a promastigotes o amastigotes. La vacuola parasitofora presentó características similares a las de los macrófagos. En su mayoría las FSDC presentaban 1 a 3 parásitos por célula. Las observaciones plantean la necesidad de estudiar la relación entre capacidad de fagocitosis y función...

Introduction. Dendritic cells, which capture and present antigen to activate unprimed T cell, are found in most tissues. Objective. This work describes the ultrastructure of Leishmania mexicana phagocytosis by the fetal skin dendritic cell (FSDC) line, a Langerhans cell line isolated from mouse fetal epidermis immortalized by retroviral transduction of the v-myc oncogene. Materials and methods. Leishmania amastigotes were obtained from mouse (BALB/c) lesion and promastigotes from culture ( 24°C) of the lesion. FSDC cells were cultured with parasites (5 parasites per cell) using IMDM medium, during 24 hours. Control and infected cultures were processed for transmission electron microscopy. Semi-thin sections counterstained with toluidine blue to evaluate phagocytosis and thin sections counterstained with uranyl acetate and lead citrate were made. Results. 13.42% of the FSDC phagocytosed promastigotes; 8% contained a single parasite and 5.2% phagocytosed 2 or more. 20% of the FSDC phagocytosed amastigotes; 10% contained a single parasite and 10% phagocytosed 2 or more. Ultrastructurally, promastigotes in contact with FSDC by the flagellum or the posterior pole were observed. The parasitophorous vacuoles harbouring promastigotes were small organelles containing one or two parasites each. Parasitophorous vacuoles containing amastigotes were larger (8µm diameter) with one or several parasites free or attached to the vacuole at the posterior pole. Conclusion. The low rate of infected FSDC cells was characteristic and the parasitophorous vacuole showed similar characteristics to those observed in macrophages. The parasite density in the infected cells was 1 to 3 parasites per cell. These observations highlight the need to study the relationship between phagocytic capacity and function.

Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes

Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania) amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME) as substrate, phenylmethylsulphone fluoride (PMSF) and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK) as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate), but also in a crude plasma membrane fraction (2.0-fold). Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP), with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

Efecto in vitro de una sustancia alcaloidea aislada de Amphimedon viridis (Porifera) sobre promastigotes de Leishmania mexicana / In vitro effect of an isolated alkaloid substance from Amphimedon viridis (Porifea) on promastigots of Leishmania mexicana

The dose dependent antiproliferative effect of an alkaloidal substance extracted from the sponge Amphimedon viridis was tested on Leishmania mexicana promastigotes. Sponges were collected in Isla Larga, Venezuela (10 degrees 20' 20" - 10 degrees 24" N, 64 degrees 19' - 64 degrees 22' W), cut and dipped in methanol for vacum filtering extraction every 24 hr. The aqueous extract was separated by chromatography over silica gel. The parasites were from the Venezuelan NR strain. Their growth rate was reduced by 50% with a dose of 10 microg/ml in 48 hr, whilst concentrations of 30 and 40 microg/ml induce leishmanicidal action after 110 and 20 min, respectively. Lysis is preceded by an immediate increase in cellular volume associated with progressive damage of cellular content and the destruction of organelles. These findings suggest that one important factor associated with the antiproliferative effect of this alkaloidal substance on L. mexicana promastigotes is the loss of the plasma membrane selective permeability.

Ultraestructura del núcleo mitótico en Leishmania mexicana ssp.: reconstrucción tridimensional del huso mitótico y placas densas / Ultrastructure of the mitotic nuclei in Leishmania mexicana ssp.: tridimensional reconstruction of the mitotic spindle and dense plaques

Se estudió mediante cortes ultrafinos seriados, la ultraestructura del núcleo mitótico en una especie del complejo Leishmania mexicana. Al inicio de la división nuclear, un grupo de seis placas densa se localiza en la región ecuatorial del núcleo y un huso microtubular se forma entre dos polos opuestos. El huso mitótico es completamente intranuclear, con la membrana nuclear presente en todo el proceso de la división. Los huso polares están formados por aproximadamente (zona de superposición) por aproximadamente 100 microtúbulos. No se observó centros organizadores de microtúbulos en relación con el huso. Las placas y hemiplacas apareciaron en asociación con grupos de microtúbulos, que finalizan en ellas o pasan tangencialmente. Esto sugiere que el huso tiene un especial significado en la ffisiologia del desplazamiento de las hemiplacas durante la separación de los genomios. Al inicio del estado de elongación, las placas se dividen en mitades y cada grupo emigra a un polo opuesto. Se concluye que las placas juegan un papel similar a de los cinetocoros y así Leishmania mexicana tendría seis unidades cromosomales. Los eventos mitóticos en esta especie son esencialmente similares a los observados en Trypanosoma cruzi

Ultrastructure of the mitotic nucleus in Leishmania mexicana sp. tridimensional reconstruction of the mitotic sindle and dense plaques

Se ha estudiado mediante cortes ultrafinos seriados la ultraestructura del núcleo mitótico en una especie del complejo Leishmania mexicana. Cambios en el núcleo interfásico y en los cuatro estados de la división son descritos. Al inicio de la división nuclear, un grupo de seis placas densas se localizan en la región ecuatorial del núcleo y un huso microtubular se forma entre dos polos opuestos. El huso mitótico es completamente intranuclear, con las membrana nuclear presente en todo el proceso de la división. Los husos polares están formados por aproximadamente 50 microtúbulos, y el ecuatoial (zona de superposición) por aproximadamente 100 mucrotúbulos. No se observaron centros organizadores de microtúbulos en relación con el huso. Las placas y hemiplacas fueron obaservadas en asociación con grupos de microtúbulos, que finalizan en ellas o pasan tangencialmente. Esto sugiere que el Huso tiene un especial significado en la fisiología del desplazamiento de las hemiplacas durante la separación de los genomios. Al inicio del estado elongacional, las placas se dividen en mitades y cada grupo emigra a un polo opuesto. Se concluye que las placas juegan un papel similar al de los cinetocoros y así Leishmania mexicana tendría seis unidades cormosomales. Los eventos mitóticos en esta especie son esencialmente similares a los observados en Trypanosoma cruzi