Amino acid efflux by asexual blood-stage Plasmodium falciparum and its utility in interrogating the kinetics of hemoglobin endocytosis and catabolism in vivo.
Amino acid efflux by asexual blood-stage Plasmodium falciparum and its utility in interrogating the kinetics of hemoglobin endocytosis and catabolism in vivo.
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DOI：
10.1016/j.molbiopara.2015.07.002
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发表时间：
2015-06
影响因子：
1.5
通讯作者：
Klemba M
中科院分区：
文献类型：
--
作者：
Dalal S;Klemba M
大量宿主细胞血红蛋白的内吞和卡吞作用是人类恶性疟原虫红细胞内无性阶段的标志。众所周知，寄生虫从血红蛋白中产生的氨基酸远远超过其代谢需要。在这里，我们表明，恶性疟原虫流出大量的某些非极性（Ala，Leu，瓦尔，Pro，Phe，Gly）和极性（Ser，Thr，His）氨基酸的外部介质。这些氨基酸来源于血红蛋白催化剂，这是由个体氨基酸外排率与其在血红蛋白中的丰度之间的强相关性以及食物空泡镰状蛋白酶抑制剂E-64 d极大地抑制外排率的能力所指示的。然后，我们开发了一种快速，灵敏和精确的方法来定量通过血红蛋白的内吞分解代谢途径，是基于亮氨酸流出。该方法的优化涉及新型氨基酸限制性RPMI制剂的生成以及D-正缬氨酸作为内标物的验证。该方法的实用性被证明通过表征的影响磷脂酰肌醇-3-激酶抑制剂渥曼青霉素和双氢青蒿素的Leu流出的动力学。这两种化合物迅速抑制Leu流出，这与磷脂酰肌醇-3-磷酸生产在血红蛋白向食物泡递送中的作用一致;然而，渥曼青霉素抑制是短暂的，这可能是由于该化合物在培养基中的不稳定性。简单，方便和非侵入性的性质，Leu流出测定这里描述的，使其成为理想的特征，在体内动力学的血红蛋白内吞和catalysts，抑制剂的目标验证研究，并为中等通量筛选，以确定新的抑制剂的细胞口内吞作用。
The endocytosis and catabolism of large quantities of host cell hemoglobin is a hallmark of the intraerythrocytic asexual stage of the human malaria parasite Plasmodium falciparum. It is known that the parasite’s production of amino acids from hemoglobin far exceeds its metabolic needs. Here, we show that P. falciparum effluxes large quantities of certain non-polar (Ala, Leu, Val, Pro, Phe, Gly) and polar (Ser, Thr, His) amino acids to the external medium. That these amino acids originate from hemoglobin catabolism is indicated by the strong correlation between individual amino acid efflux rates and their abundances in hemoglobin, and the ability of the food vacuole falcipain inhibitor E-64d to greatly suppress efflux rates. We then developed a rapid, sensitive and precise method for quantifying flux through the hemoglobin endocytic-catabolic pathway that is based on leucine efflux. Optimization of the method involved the generation of a novel amino acid-restricted RPMI formulation as well as the validation of D-norvaline as an internal standard. The utility of this method was demonstrated by characterizing the effects of the phosphatidylinositol-3-kinase inhibitors wortmannin and dihydroartemisinin on the kinetics of Leu efflux. Both compounds rapidly inhibited Leu efflux, which is consistent with a role for phosphtidylinositol-3-phosphate production in the delivery of hemoglobin to the food vacuole; however, wortmannin inhibition was transient, which was likely due to the instability of this compound in culture medium. The simplicity, convenience and non-invasive nature of the Leu efflux assay described here makes it ideal for characterizing the in vivo kinetics of hemoglobin endocytosis and catabolism, for inhibitor target validation studies, and for medium-throughput screens to identify novel inhibitors of cytostomal endocytosis.