Kinetoplastid membrane protein-11 exacerbates infection with Leishmania amazonensis in murine macrophages.

In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during meta-cyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect.

Kinetoplastid membrane protein-11 exacerbates infection with Leishmania amazonensis in murine macrophages

In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during metacyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect.

Óxido nítrico sintase e proteínas quinase A como alvos de derivados amidínicos anti-Leishmania / Nitric oxide synthase and protein kinase A as the white of the amidínicos derivatives anti-Leishmania

Há muito tempo o tratamento das Leishmanioses tem feito uso de quimioterápicos utilizados em altas doses diárias que provocam diversas reações de toxicidade e o aparecimento de formas resistentes de Leishmania; tais fatores ressaltam a necessidade de estudos de drogas alternativas que possam permitir o desenvolvimento futuro de inibidores anti-leishmaniais. A abordagem moderna do desenvolvimento racional de drogas baseia-se na identificação de vias metabólicas indispensáveis à sobrevivência do microrganismo; desta forma, após a seleção de alvos biológicos, o objetivo passa a ser a caracterização detalhada de componentes de vias metabólicas envolvidas, e assim, enzimas-chave podem ser exploradas no desenvolvimento de inibidores de reações enzimáticas, sem afetar a célula hospedeira. Neste trabalho, duas importantes vias de sinalização foram avaliadas como alvos da quimioterapia experimental anti-Leishmania amazonensis utilizando uma N, NÆ-difenilbenzamidina metoxilada (metoxi-amidina): a) via do óxido nítrico (NO.) - aqui caracterizada pela detecção de nitrito em sobrenadante de culturas de promastigotas e amastigotas axênicas, imunofluorescência, cromatografia de afinidade, eletroforese de proteína, atividade de óxido nítrico sintase e inibição enzimática; e b) via da proteína quinase A (PKA), cuja atividade foi previamente caracterizada em Leishmania amazonensis e citada na literatura como participante na fosforilação da óxido nítrico sintase. Em comparação à droga de referência (Isetionato de Pentamidina), o derivado metoxilado diminuiu significativamente a produção de NO. e a atividade de PKA de promastigotas e amastigotas axênicas. Os resultados obtidos apontam a importância do NO. Como molécula sinalizadora na interação parasita-hospedeiro, e sua rota de biossíntese merece grande consideração em estudos da ação seletiva de quimioterápicos anti-Leishmania em doses toleradas pelostecidos do hospedeiro.