Silber Ariel M, Tonelli Renata R, Lopes Camila G, Cunha-e-Silva Narcisa, Torrecilhas Ana Cláudia T, Schumacher Robert I, Colli Walter, Alves Maria Júlia M
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof Lineu Prestes 748, São Paulo, SP, Brazil.
Mol Biochem Parasitol. 2009 Nov;168(1):102-8. doi: 10.1016/j.molbiopara.2009.07.006. Epub 2009 Jul 23.
Trypanosoma cruzi, the agent of Chagas' disease, alternates between different morphogenetic stages that face distinct physiological conditions in their invertebrate and vertebrate hosts, likely in the availability of glucose. While the glucose transport is well characterized in epimastigotes of T. cruzi, nothing is known about how the mammalian stages acquire this molecule. Herein glucose transport activity and expression were analyzed in the three developmental stages present in the vertebrate cycle of T. cruzi. The infective trypomastigotes showed the highest transport activity (V(max)=5.34+/-0.54 nmol/min per mg of protein; K(m)=0.38+/-0.01 mM) when compared to intracellular epimastigotes (V(max)=2.18+/-0.20 nmol/min per mg of protein; K(m)=0.39+/-0.01 mM). Under the conditions employed no transport activity could be detected in amastigotes. The gene of the glucose transporter is expressed at the mRNA level in trypomastigotes and in intracellular epimastigotes but not in amastigotes, as revealed by real-time PCR. In both trypomastigotes and intracellular epimastigotes protein expression could be detected by Western blot with an antibody raised against the glucose transporter correlating well with the transport activity measured experimentally. Interestingly, anti-glucose transporter antibodies showed a strong reactivity with glycosome and reservosome organelles. A comparison between proline and glucose transport among the intracellular differentiation forms is presented. The data suggest that the regulation of glucose transporter reflects different energy and carbon requirements along the intracellular life cycle of T. cruzi.
克氏锥虫是恰加斯病的病原体,它在不同形态发生阶段之间交替,在其无脊椎动物和脊椎动物宿主中面临不同的生理条件,这可能与葡萄糖的可利用性有关。虽然克氏锥虫的上鞭毛体中的葡萄糖转运已得到充分表征,但关于其哺乳动物阶段如何获取这种分子却一无所知。在此,对克氏锥虫脊椎动物周期中存在的三个发育阶段的葡萄糖转运活性和表达进行了分析。与细胞内上鞭毛体(V(max)=2.18±0.20 nmol/分钟每毫克蛋白质;K(m)=0.39±0.01 mM)相比,感染性锥鞭毛体显示出最高的转运活性(V(max)=5.34±0.54 nmol/分钟每毫克蛋白质;K(m)=0.38±0.01 mM)。在所采用的条件下,未在无鞭毛体中检测到转运活性。实时PCR显示,葡萄糖转运蛋白基因在锥鞭毛体和细胞内上鞭毛体的mRNA水平表达,但在无鞭毛体中不表达。通过用针对葡萄糖转运蛋白的抗体进行蛋白质印迹,在锥鞭毛体和细胞内上鞭毛体中均检测到蛋白质表达,这与实验测量的转运活性密切相关。有趣的是,抗葡萄糖转运蛋白抗体与糖体和储存体细胞器有强烈反应。本文还比较了细胞内分化形式之间的脯氨酸和葡萄糖转运。数据表明,葡萄糖转运蛋白的调节反映了克氏锥虫细胞内生命周期中不同的能量和碳需求。