Is the mitochondrion a promising drug target in trypanosomatids?

The trypanosomatids Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are etiological agents of important neglected tropical diseases, affecting millions of people worldwide, and the drugs available for these diseases present several limitations. Novel efficient and nontoxic drugs are necessary as an alternative to the current chemotherapy. The unique mitochondrion of trypanosomatids and its peculiar features turn this organelle a potential drug target. Several phenotypic studies describe the damage in the parasite mitochondrial ultrastructure, but the molecular target is unknown. Few reports demonstrated the electron transport system (ETS) as a target due to the high similarities to mammalian orthologues, hence ETS is not a good candidate for drug intervention. On the other hand, antioxidant enzymes, such as trypanothione reductase, and an alternative oxidase (AOX) seem to be interesting targets; however no high active inhibitors were developed up to now. Finally, due to the remarkable differences to mammalian machinery, together with the high biological importance for the parasite survival, the mitochondrial import system stands out as a very promising target in trypanosomatids. Archaic translocase of the outer membrane (ATOM) and translocase of the inner membrane (TIM) complexes, which mediate both protein and tRNA import, composed by specific subunits of these parasites, could be excellent candidates, deserving studies focused on the development of specific drugs.

Trypanosoma janseni n. sp. (Trypanosomatida: Trypanosomatidae) isolated from Didelphis aurita (Mammalia: Didelphidae) in the Atlantic Rainforest of Rio de Janeiro, Brazil: integrative taxonomy and phylogeography within the Trypanosoma cruzi clade

BACKGROUND Didelphis spp. are a South American marsupial species that are among the most ancient hosts for the Trypanosoma spp. OBJECTIVES We characterise a new species (Trypanosoma janseni n. sp.) isolated from the spleen and liver tissues of Didelphis aurita in the Atlantic Rainforest of Rio de Janeiro, Brazil. METHODS The parasites were isolated and a growth curve was performed in NNN and Schneider's media containing 10% foetal bovine serum. Parasite morphology was evaluated via light microscopy on Giemsa-stained culture smears, as well as scanning and transmission electron microscopy. Molecular taxonomy was based on a partial region (737-bp) of the small subunit (18S) ribosomal RNA gene and 708 bp of the nuclear marker, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. Maximum likelihood and Bayesian inference methods were used to perform a species coalescent analysis and to generate individual and concatenated gene trees. Divergence times among species that belong to the T. cruzi clade were also inferred. FINDINGS In vitro growth curves demonstrated a very short log phase, achieving a maximum growth rate at day 3 followed by a sharp decline. Only epimastigote forms were observed under light and scanning microscopy. Transmission electron microscopy analysis showed structures typical to Trypanosoma spp., except one structure that presented as single-membraned, usually grouped in stacks of three or four. Phylogeography analyses confirmed the distinct species status of T. janseni n. sp. within the T. cruzi clade. Trypanosoma janseni n. sp. clusters with T. wauwau in a well-supported clade, which is exclusive and monophyletic. The separation of the South American T. wauwau + T. janseni coincides with the separation of the Southern Super Continent. CONCLUSIONS This clade is a sister group of the trypanosomes found in Australian marsupials and its discovery sheds light on the initial diversification process based on what we currently know about the T. cruzi clade.

Susceptibility of Phytomonas serpens to calpain inhibitors in vitro: interference on the proliferation, ultrastructure, cysteine peptidase expression and interaction with the invertebrate host

A pleiotropic response to the calpain inhibitor MDL28170 was detected in the tomato parasite Phytomonas serpens. Ultrastructural studies revealed that MDL28170 caused mitochondrial swelling, shortening of flagellum and disruption of trans Golgi network. This effect was correlated to the inhibition in processing of cruzipain-like molecules, which presented an increase in expression paralleled by decreased proteolytic activity. Concomitantly, a calcium-dependent cysteine peptidase was detected in the parasite extract, the activity of which was repressed by pre-incubation of parasites with MDL28170. Flow cytometry and Western blotting analyses revealed the differential expression of calpain-like proteins (CALPs) in response to the pre-incubation of parasites with the MDL28170, and confocal fluorescence microscopy confirmed their surface location. The interaction of promastigotes with explanted salivary glands of the insect Oncopeltus fasciatus was reduced when parasites were pre-treated with MDL28170, which was correlated to reduced levels of surface cruzipain-like and gp63-like molecules. Treatment of parasites with anti-Drosophila melanogaster (Dm) calpain antibody also decreased the adhesion process. Additionally, parasites recovered from the interaction process presented higher levels of surface cruzipain-like and gp63-like molecules, with similar levels of CALPs cross-reactive to anti-Dm-calpain antibody. The results confirm the importance of exploring the use of calpain inhibitors in studying parasites’ physiology.

Naftoquinonas e derivados: estudo da atividade, mecanismo de ação e indução de morte celular em Trypanosoma cruzi / Naphthoquinones and derivatives: study of the activity, mechanism of action and induction of cellular death in Trypanosoma cruzi

Através de hibridização molecular e explorando a eletrofilicidade de carbonilas 1,2-quinoidal, naftoquinonas foram acopladas a [1,2,3]-triazóis ou a arilaminas, originando uma série de derivados que foram ensaiados sobre tripomastigotas sangüíneas de Trypanosoma cruzi. Quatro naftofuranoquinonas foram mais ativas que benznidazol, a droga referência. Três outras naftofuranoquinonas, previamente selecionadas com base na atividade sobre tripomastigotas, inibiram a proliferação de amastigotas intracelulares e apresentaram baixa toxicidade para a célula hospedeira. Análises ultra-estrutural, bioquímica e por citometria de fluxo de parasitos tratados apontaram para a mitocôndria como o alvo inicial, com inibição de consumo de oxigênio mitocondrial, e, em epimastigotas também um significativo aumento na geração de H2O2. As moléculas híbridas de quinonas com grupo triazol ou arilamino apresentam propriedades redox, consistindo assim um interessante ponto de partida para estudos de química medicinal direcionados a quimioterapia da doença de chagas. Três naftoimidazóis derivados de beta-lapachona apresentando grupamentos aromáticos ligados ao anel imidazólico (N1, N2 e N3) foram anteriormente selecionados com base na atividade sobre tripomastigotas. Neste contexto foram investigados os mecanismos de morte envolvidos no efeito tripanocoda destes naftoimidazóis. Em tripomastigotas tratados ocorreram alterações no cinetoplasto, blebing da membrana e fragmentação de DNA. Em tripomastigotas, N2 induziu exposição de fosfatidilserina - característica de apoptose em mamíferos, enquanto necrose foi detectada em parasitos tratados com N1 ou N2. Estes dois fenótipos de morte não foram observados em epimastigotas tratados. N3 levou em epimastigotas a liberação do citocromo c no citosol, que não foi bloqueado por pré-incubação com o inibidor de pancaspases zVAD.fmk, sugerindo que metacaspases não participam deste processo. Análise ultra-estrutural revelou em epimastigotas...