Phosphofructokinase RNAi

<i>Trypanosoma brucei</i> is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of <i>T. brucei</i>.布氏锥虫（Trypanosoma brucei）是人类非洲锥虫病与动物非洲锥虫病的致病病原体。该可感染哺乳动物的寄生虫可侵袭宿主多种组织与器官，包括血液、中枢神经系统、皮肤、脂肪组织及肺部。它们依赖糖酵解产生的三磷酸腺苷（ATP），大量消耗宿主哺乳动物体内易于获取的葡萄糖。除葡萄糖外，甘油亦可作为碳源、ATP来源及糖异生（gluconeogenesis）的底物。然而，以甘油为底物的糖异生过程在感染哺乳动物的生活周期阶段中的生理相关性仍不明确。为验证该过程是否不可或缺，首先需明确该通路中的相关酶类。经典糖异生酶——果糖-1,6-二磷酸酶（fructose-1,6-bisphosphatase）的缺失并不会阻断糖异生过程，因此锥虫体内必然存在至少一种其他酶参与糖异生。本研究联合CRISPR/Cas9基因编辑技术与RNA干扰（RNA interference）技术，针对四种潜在可参与糖异生的酶类——果糖-1,6-二磷酸酶、景天庚酮糖-1,7-二磷酸酶、磷酸果糖激酶及转醛醇酶，单独或组合构建了相应的基因突变体。代谢组学分析结果显示，无论敲除上述何种基因，糖异生通路的代谢通量均得以维持。本研究数据排除了此前提出的"反向磷酸果糖激酶反应参与糖异生"的假说，同时也排除了转醛醇酶参与新型生化通路以介导糖异生的可能。在本研究构建的突变体（包括三基因敲除株）中，糖异生代谢通量仍可维持，这表明锥虫体内存在一种独特的糖异生相关酶类。此外，本研究数据为糖异生与磷酸戊糖通路（pentose phosphate pathway）提供了新的认知，并加深了当前对布氏锥虫感染哺乳动物阶段碳代谢的理解。