Vannier-Santos Marcos A, Menezes Diego, Oliveira Marcus F, de Mello Fernando G
Instituto Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia, Brazil.
Instituto de Bioquímica Médica, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, Brazil.
Microbiology (Reading). 2008 Oct;154(Pt 10):3104-3111. doi: 10.1099/mic.0.2007/013896-0.
Polyamines are important regulators of growth and differentiation in a variety of cells, including parasitic protozoa. Promastigotes of Leishmania species have high levels of putrescine and spermidine and their growth can be inhibited by polyamine biosynthesis antagonists. The putrescine analogue 1,4-diamino-2-butanone (DAB) is microbicidal against Tritrichomonas foetus and Trypanosoma cruzi, so we tested its effects on Leishmania amazonensis proliferation, viability, organization, putrescine transport and synthesis as well as in vitro infectivity. DAB impaired promastigote proliferation dose-dependently (IC(50) 144 microM) and the parasite putrescine concentration was reduced by nearly 50 %. This analogue markedly inhibited both ornithine decarboxylase activity and [H(3)]putrescine uptake by promastigotes. Pre-treatment with DAB for 24 h led to compensatory enhancement of putrescine uptake, indicating an adaptive mechanism in DAB-treated parasites. Remarkably, DAB caused mitochondrial damage, assessed by transmission electron microscopy, and 3 h treatment with 1 mM DAB enhanced lipid peroxidation, whereas incubation with 10 mM DAB or for 24 h resulted in decreased peroxidation levels in the parasites. This effect was probably due to the loss of mitochondrial function, demonstrated by the diminished reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), not observed in macrophages. Murine macrophages infected with L. amazonensis amastigotes treated with DAB had parasite loads significantly (P<0.05) lower than controls, presumably due to interference with putrescine uptake and/or synthesis. These results suggest that putrescine may be involved in leishmanial survival, possibly by maintaining the parasite's mitochondrial function. The use of analogues to interfere with polyamine/diamine synthesis and transport may shed light on its function in intracellular parasite survival and lead to identification of new targets for leishmaniasis chemotherapy.
多胺是包括寄生原生动物在内的多种细胞生长和分化的重要调节因子。利什曼原虫的前鞭毛体含有高水平的腐胺和亚精胺，其生长可被多胺生物合成拮抗剂抑制。腐胺类似物1,4-二氨基-2-丁酮（DAB）对胎儿三毛滴虫和克氏锥虫具有杀菌作用，因此我们测试了其对亚马逊利什曼原虫增殖、活力、结构、腐胺转运与合成以及体外感染性的影响。DAB剂量依赖性地损害前鞭毛体增殖（IC(50)为144微摩尔），寄生虫的腐胺浓度降低了近50%。这种类似物显著抑制前鞭毛体的鸟氨酸脱羧酶活性和[H(3)]腐胺摄取。用DAB预处理24小时导致腐胺摄取的代偿性增强，表明经DAB处理的寄生虫存在一种适应性机制。值得注意的是，通过透射电子显微镜评估发现DAB导致线粒体损伤，用1毫摩尔DAB处理3小时可增强脂质过氧化，而用10毫摩尔DAB处理或处理24小时则导致寄生虫中过氧化水平降低。这种效应可能是由于线粒体功能丧失所致，这通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）还原减少得以证明，而在巨噬细胞中未观察到这种情况。用DAB处理感染了亚马逊利什曼原虫无鞭毛体的小鼠巨噬细胞，其寄生虫负荷显著（P<0.05）低于对照组，这可能是由于对腐胺摄取和/或合成的干扰。这些结果表明，腐胺可能参与利什曼原虫的存活，可能是通过维持寄生虫的线粒体功能。使用类似物干扰多胺/二胺的合成和转运可能有助于揭示其在细胞内寄生虫存活中的功能，并有助于确定利什曼病化疗的新靶点。
