Induction of a stringent metabolic response in intracellular stages of Leishmania mexicana leads to increased dependence on mitochondrial metabolism.
Induction of a stringent metabolic response in intracellular stages of Leishmania mexicana leads to increased dependence on mitochondrial metabolism.
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DOI：
10.1371/journal.ppat.1003888
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发表时间：
2014-01
期刊：
影响因子：
6.7
通讯作者：
McConville MJ
中科院分区：
文献类型：
--
作者：
Saunders EC;Ng WW;Kloehn J;Chambers JM;Ng M;McConville MJ
利什曼原虫在昆虫载体的细胞外原鞭毛体阶段和哺乳动物宿主巨噬细胞吞噬溶酶体腔内增殖的专性细胞内无鞭毛体阶段之间交替。大多数参与利什曼原虫中心碳代谢的酶是组成性表达的，能量代谢的阶段特异性变化仍然不明确。利用13c稳定同位素分解代谢组学和2H2O标记，研究人员发现，无纺锤体分化与生长速度降低和诱导明显的严格代谢状态有关。这种状态的特点是葡萄糖和氨基酸的吸收和利用全面减少，有机酸的分泌减少，脂肪酸β-氧化增加。同位素分析表明，己糖和脂肪酸的分解代谢提供C4二羧酸（琥珀酸/苹果酸）和乙酰辅酶a，通过区隔化线粒体三羧酸（TCA）循环合成谷氨酸。体外培养和细胞内无尾线虫对线粒体乌头酶和谷氨酰胺合成酶抑制剂非常敏感，表明这些合成代谢途径对细胞内生长和毒力至关重要。损伤源性无纺丝体表现出与体外分化无纺丝体相似的代谢，表明这种严格的反应与分化信号有关，而不是与外源营养水平有关。诱导严格的代谢反应可能有助于无纺锤体在营养贫乏的细胞内生态位中存活，并使这一阶段对己糖和线粒体代谢的依赖性增加。利什曼原虫是一种由白蛉传播的寄生原生动物，可在人类中引起一系列重要疾病。虽然已对易培养昆虫（promastigote）阶段的核心代谢进行了研究，但对专性细胞内无尾虫阶段的代谢知之甚少，该阶段在哺乳动物巨噬细胞的成熟溶酶体内增殖，是抗寄生虫治疗的目标。我们利用13c示踪实验，描绘了不同promastigote阶段的碳代谢主要途径，以及培养产生和从动物病变中分离的amastigote阶段。分裂和非分裂proprotigotes均表现出较高的代谢活性，具有较高的葡萄糖和氨基酸消耗率以及代谢终产物的分泌率。相比之下，这两个阶段都表现出严格的代谢表型，其特征是葡萄糖和氨基酸摄取和分解代谢水平低，脂肪酸分解代谢增加。这种表型不是由营养限制诱导的，而是硬连线的无纺丝分化。这种反应可能导致合成代谢途径对己糖分解代谢的依赖性增加，因为化学抑制新生谷氨酸和谷氨酰胺的生物合成抑制了巨噬细胞中寄生虫的生长。这项研究强调了在巨噬细胞吞噬溶酶体中支撑其生存能力的无尾线虫代谢的关键方面。
Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using 13C-stable isotope resolved metabolomics and 2H2O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid β-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate) and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA) cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism. Leishmania are sandfly-transmitted parasitic protozoa that cause a spectrum of important diseases in humans. While the core metabolism of the readily cultivated insect (promastigote) stage has been studied, much less is known about the metabolism of the obligate intracellular amastigote stage, which proliferates within the mature lysosome of mammalian macrophages and is the target of anti-parasite therapies. We have used 13C-tracing experiments to delineate the major pathways of carbon metabolism in different promastigote stages, as well as amastigote stages generated in culture and isolated from animal lesions. Both dividing and non-dividing promastigotes exhibited high metabolic activity, with excessive rates of glucose and amino acid consumption and secretion of metabolic end-products. In contrast, both amastigote stages exhibited a stringent metabolic phenotype, characterized by low levels of glucose and amino acid uptake and catabolism and increased catabolism of fatty acids. This phenotype was not induced by nutrient limitation, but is hard-wired into amastigote differentiation. This response may lead to increased dependence on hexose catabolism for anabolic pathways, as chemical inhibition of de novo glutamate and glutamine biosynthesis inhibited parasite growth in macrophages. This study highlights key aspects of amastigote metabolism that underpin their capacity to survive in macrophage phagolysosomes.
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DOI：
10.1007/978-0-387-77570-8_12
发表时间：
2008-01-01
期刊：
DRUG TARGETS IN KINETOPLASTID PARASITES
影响因子：
--
作者：
Carter, Nicola S.;Yates, Phillip;Ullman, Buddy
通讯作者：
Ullman, Buddy
影响因子：
9
作者：
Castanys-Munoz, E.;Brown, E.;Mottram, J. C.
通讯作者：
Mottram, J. C.
影响因子：
6.7
作者：
Beste DJ;Bonde B;Hawkins N;Ward JL;Beale MH;Noack S;Nöh K;Kruger NJ;Ratcliffe RG;McFadden J
通讯作者：
McFadden J
影响因子：
3.6
作者：
Gannavaram S;Connelly PS;Daniels MP;Duncan R;Salotra P;Nakhasi HL
通讯作者：
Nakhasi HL
影响因子：
2.1
作者：
HART, DT;COOMBS, GH
通讯作者：
COOMBS, GH