Calpains of Leishmania braziliensis: genome analysis, differential expression, and functional analysis

BACKGROUND Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome. OBJECTIVES Here, we characterised calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. METHODS/FINDINGS In total, 34 predicted calpain-like genes were identified. After domain structure evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in the procyclic stage, (ii) one augmented gene in the metacyclic stage, and (iii) one procyclic-exclusive transcript. Western blot analysis revealed that an antibody against a consensus-conserved peptide reacted with multiple calpain-like proteins, which is consistent with the multi-gene family characteristic. Flow cytometry and immunocytochemistry analyses revealed the presence of calpain-like molecules mainly in the cytoplasm, to a lesser extent in the plasma membrane, and negligible levels in the nucleus, which are all consistent with calpain localisation. Eventually, the calpain inhibitor MDL28170 was used for functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultra-structural alterations conceivable with autophagy, and (iv) an enhanced expression of the virulence factor GP63. CONCLUSION This report adds novel insights into the domain structure, expression, and localisation of L. braziliensis calpain-like molecules.

Prospecção de novas drogas contra a leishmaniose cutânea: avaliação da atividade biológica da lectina de Oreochromis niloticus / Prospecting for new drugs against cutaneousleishmaniasis: evaluation of biological activity of lectin Oreochromis niloticus

As leishmanioses constituem um grupo de doenças crônicas causadas por protozoários pertencentes ao gênero Leishmania. Tendo em vista a complexidade e ineficácia dos tratamentos atuais, o desenvolvimento de novas drogas menos tóxicas ainda é uma necessidade. Na prospecção de possíveis agentes quimioterápicos contra as leishmanioses, as lectinas apresentam-se como candidatos promissores por apresentarem um amplo espectro de atividades biológicas. No presente trabalho nós investigamos o potencial leishmanicida e imunomodulador da lectina Onil. Nossos resultados demonstraram que a Onil apresentou baixa toxidade sobre células do exsudato peritoneal (CEP) de camundongos (CC50= 317,5 ± 0,6 µg/mL), foi efetiva ao inibir o crescimento de formas promastigotas de L. braziliensis (IC50=150,58± 0,78 µg/mL), mostrou-se mais seletiva para o parasito do que para célula do hospedeiro (ISe=2,1). No entanto, não foi capaz de inibir a sobrevivência das amastigotas no interior das CEPs. A lectina Onil causou alterações ultraestruturais no flagelo, bem como mostrou um efeito sobre a divisão celular de formas promastigotas. A marcação das células tratadas com Anexina V (AV) e Iodeto de Propídio (IP) mostrou uma pequena subpopulação de células apresentava marcação para AV/IP compatíveis com o processo de morte celular por necrose/apoptose tardia. A marcação das células controles e tratadas com Onil com Rho 123 revelou que na grande maioria das células o potencial de membrana mitocondrial foi preservado. O tratamento com a lectina (75-300 µg/mL) não alterou significativamente a produção de NO e não induziu alterações na produção de citocinas em CEPs infectadas L. braziliensis. Por outro lado, uma intensa produção de citocinas associadas aos perfis Th1, Th2 e Th17 foi observada em CEPs não infectadas tratadas com 30 µg/mL da Onil. Nossos dados apontam para uma possível utilização da Onil como agente adjuvante ou como carreadora de drogas para o tratamento da leishmaniose cutânea

Leishmaniasis comprises a group of disease caused by protozoa belonging to the Leishmaniagenus. Taking in account the complexity and inefficiency of current treatments against leishmaniasis, the development of new, less toxic drugs is still needed. In a search of potential chemotherapeutic agents against leishmaniasis, lectins presented as promising candidates because for presenting a broad spectrum of biological activities. In this regard, in the present study we investigated the leishmanicidal and immunomodulatory activities of Onil in vitro. Our results demonstrated that Onil presented low toxicity to mice peritoneal exudate cells (PEC) (CC50= 317.5 ± 0.6 μg / mL), was effective to inhibit the growth of promastigotes of Leishmania braziliensis (IC50= 150.58 ± 0.78 μg / mL) and was shown to be more selective for the parasite than to the host cell, with SeI=2.1...

In vitro evaluation of new terpenoid derivatives against Leishmania infantum and Leishmania braziliensis

The activity of five (1-5) abietane phenol derivatives against Leishmania infantum and Leishmania braziliensis was studied using promastigotes and axenic and intracellular amastigotes. Infectivity and cytotoxicity tests were performed with J774.2 macrophage cells using Glucantime as a reference drug. The mechanisms of action were analysed by performing metabolite excretion and transmission electron microscopy ultrastructural studies. Compounds 1-5 were more active and less toxic than Glucantime. The infection rates and mean number of parasites per cell observed in amastigote experiments showed that derivatives 2, 4 and 5 were the most effective against both L. infantum and L. braziliensis. The ultrastructural changes observed in the treated promastigote forms confirmed that the greatest cell damage was caused by the most active compound (4). Only compound 5 caused changes in the nature and amounts of catabolites excreted by the parasites, as measured by ¹H nuclear magnetic resonance spectroscopy. All of the assayed compounds were active against the two Leishmania species in vitro and were less toxic in mammalian cells than the reference drug.