Sequences of DNA primers used in the study.

Trypanosoma brucei 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 T. brucei.

布氏锥虫（Trypanosoma brucei）是人类与动物非洲锥虫病（Human and Animal African Trypanosomiases）的致病原。该虫的哺乳动物宿主阶段寄生虫可侵染包括血液、中枢神经系统、皮肤、脂肪组织与肺部在内的多种组织与器官。其依赖糖酵解（glycolysis）产生的ATP，会大量消耗宿主哺乳动物体内易于获取的葡萄糖（glucose）。除葡萄糖外，甘油（glycerol）亦可作为碳源与ATP来源，同时作为糖异生（gluconeogenesis）作用的底物。然而，以甘油为底物的糖异生作用在其感染哺乳动物的生活史阶段中的生理相关性仍尚不明确。为验证该途径是否不可或缺，首先需明确该通路中的酶类组分。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，因此锥虫体内至少存在另一种酶参与糖异生途径。本研究结合CRISPR/Cas9基因编辑（CRISPR/Cas9 gene editing）与RNA干扰（RNA interference）技术，构建了4种潜在参与糖异生的酶类的单基因或多组合缺失突变株，这4种酶分别为果糖-1,6-二磷酸酶、景天庚酮糖-1,7-二磷酸酶（sedoheptulose-1,7-bisphosphatase）、磷酸果糖激酶（phosphofructokinase）与转醛醇酶（transaldolase）。代谢组学分析（metabolomic analyses）结果显示，无论缺失上述何种基因，糖异生的代谢通量均得以维持。本研究数据不支持此前提出的“反向磷酸果糖激酶反应参与糖异生”的假说，同时排除了转醛醇酶参与新型生化通路介导糖异生的可能。本研究构建的突变株（包括三基因敲除株）中糖异生代谢通量仍持续存在，这表明锥虫体内存在一种独特的酶参与糖异生过程。此外，本研究数据为糖异生与磷酸戊糖途径（pentose phosphate pathway）提供了新的见解，并加深了当前对布氏锥虫感染哺乳动物阶段碳代谢（carbon metabolism）机制的理解。