Desenvolvimento de um teste colorimétrico para triagem da atividade leishmanicida de compostos utilizando Leishmania amazonensis expressando a enzima beta-galactosidase

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Programa de Pós-graduação em Biotecnologia e Biociências, Florianópolis, 2013No presente trabalho transfectamos uma cepa de Leishmania amazonensis com um plasmídeo contendo o gene da ?-galactosidase, visando estabelecer um ensaio colorimétrico para triagem de compostos ativos contra amastigotas intracelulares de leishmania. A transfecção não alterou o crescimento de promastigotas em cultura, a infectividade de células THP-1 e de camundongos Balb/c. A atividade da enzima foi diretamente proporcional ao número de parasitos. A inibição do crescimento dos parasitos intracelulares pelo fármaco de referência Anfotericina B, determinada tanto pelo método colorimétrico quanto por contagem microscópica dos parasitos, produziu resultados semelhantes, validando o ensaio. O teste colorimétrico também foi aplicado com sucesso para avaliar a atividade de uma classe de moléculas análogas e derivadas do ácido gálico. O estudo da correlação estrutura atividade mostrou que estas moléculas possuem uma atividade leishmanicida pouco seletiva. Algumas melhorias como a transfecção integrativa e estável, bem como padronização deste método com outras espécies de Leishmania ainda são desejáveis. Contudo, o ensaio com L. amazonensis expressando ?-galactosidase foi robusto, sensível, reprodutível e rápido na avaliação da atividade leishmanicida de compostos <br

Branched late-steps of the cytosolic iron-sulphur cluster assembly machinery of Trypanosoma brucei

Support from the Czech Grant Agency (16-18699S to JL) and partial funding by CAPES/Science without Borders (BEX1333/13-5 to MLT) is kindly acknowledged. We are grateful to the University of St. Andrews mass spectrometry facility for collecting and processing MS data and to other members of the TKS and SM groups for their assistance with this project. Work of RL and AJP was supported by the Deutsche Forschungsgemeinschaft (Koselleck grant (to RL) and SPP 1927) and a Coordenação de aperfeiçoamento de pessoal de nivel superior (CAPES - 1333/2013-05) for the financial support to this project. We acknowledge networking support from the COST Action FeSBioNet (Contract CA15133). ERD Funds, The Czech Ministry of Education, project OPVVV 16_019/0000759 to JL.Fe-S clusters are ubiquitous cofactors of proteins involved in a variety of essential cellular processes. The biogenesis of Fe-S clusters in the cytosol and their insertion into proteins is accomplished through the cytosolic iron-sulphur protein assembly (CIA) machinery. The early- and middle-acting modules of the CIA pathway concerned with the assembly and trafficking of Fe-S clusters have been previously characterised in the parasitic protist Trypanosoma brucei. In this study, we applied proteomic and genetic approaches to gain insights into the network of protein-protein interactions of the late-acting CIA targeting complex in T. brucei. All components of the canonical CIA machinery are present in T. brucei including, as in humans, two distinct CIA2 homologues TbCIA2A and TbCIA2B. These two proteins are found interacting with TbCIA1, yet the interaction is mutually exclusive, as determined by mass spectrometry. Ablation of most of the components of the CIA targeting complex by RNAi led to impaired cell growth in vitro, with the exception of TbCIA2A in procyclic form (PCF) trypanosomes. Depletion of the CIA-targeting complex was accompanied by reduced levels of protein-bound cytosolic iron and decreased activity of an Fe-S dependent enzyme in PCF trypanosomes. We demonstrate that the C-terminal domain of TbMMS19 acts as a docking site for TbCIA2B and TbCIA1, forming a trimeric complex that also interacts with target Fe-S apo-proteins and the middle-acting CIA component TbNAR1.Publisher PDFPeer reviewe

Anti-Infective Potential of Marine Invertebrates and Seaweeds from the Brazilian Coast

This manuscript describes the evaluation of anti-infective potential in vitro of organic extracts from nine sponges, one ascidian, two octocorals, one bryozoan, and 27 seaweed species collected along the Brazilian coast. Antimicrobial activity was tested against Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922) and Candida albicans (ATCC 10231) by the disk diffusion method. Antiprotozoal activity was evaluated against Leishmania braziliensis (MHOM/BR/96/LSC96-H3) promastigotes and Trypanosoma cruzi (MHOM/BR/00/Y) epimastigotes by MTT assay. Activity against intracellular amastigotes of T. cruzi and L. brasiliensis in murine macrophages was also evaluated. Antiviral activity was tested against Herpes Simplex Virus type 1 (HSV-1, KOS strain) by the plaque number reduction assay (IC50). Cytotoxicity on VERO cells was evaluated by the MTT assay (CC50). The results were expressed as SI = CC50/IC50. The most promising antimicrobial results were obtained against S. aureus and C. albicans with Dragmacidon reticulatum. Among the seaweeds, only Osmundaria obtusiloba showed moderate activity against P. aeruginosa. Concerning antiprotozoal activity, Bugula neritina, Carijoa riseii, Dragmaxia anomala and Haliclona (Halichoclona) sp. showed the most interesting results, mainly against extracellular promastigote forms of L. braziliensis (66, 35.9, 97.2, and 43.6% inhibition, respectively). Moreover, six species of seaweeds Anadyomene saldanhae, Caulerpa cupressoides, Canistrocarpus cervicornis, Dictyota sp., Ochtodes secundiramea, and Padina sp. showed promising results against L. braziliensis (87.9, 51.7, 85.9, 93.3, 99.7, and 80.9% inhibition, respectively), and only Dictyota sp. was effective against T. cruzi (60.4% inhibition). Finally, the antiherpes activity was also evaluated, with Haliclona (Halichoclona) sp. and Petromica citrina showing the best results (SI = 11.9 and SI > 5, respectively). All the active extracts deserve special attention in further studies to chemically characterize the bioactive compounds, and to perform more refined biological assays