Transaldolase knock-out

<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)是人类与动物非洲锥虫病的致病原。该虫处于哺乳类宿主感染阶段的虫体可侵染血液、中枢神经系统、皮肤、脂肪组织与肺部等多种组织器官。它们依赖糖酵解途径产生的三磷酸腺苷(ATP)，并大量消耗葡萄糖，而葡萄糖在哺乳类宿主体内易于获取。除葡萄糖外，甘油也可作为碳源与ATP的来源，同时充当糖异生(gluconeogenesis)的底物。然而，以甘油为底物的糖异生过程对于布氏锥虫的哺乳感染生命周期的生理学相关性仍不明确。为验证该过程是否必需，首先需鉴定该通路中的相关酶类。经典糖异生酶——果糖-1,6-二磷酸酶的缺失并不会阻断该过程，因此布氏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基因编辑与RNA干扰(RNA interference)技术，针对四种潜在参与糖异生的酶类——果糖-1,6-二磷酸酶、景天庚酮糖-1,7-二磷酸酶、磷酸果糖激酶与转醛醇酶——分别或联合构建了敲除突变株。代谢组学分析显示，无论缺失上述何种基因，糖异生途径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的新型生化通路参与该过程的可能性。我们构建的突变株（包括三基因敲除株）中糖异生代谢通量仍可维持，这表明布氏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提供了新的研究视角，并加深了学界对布氏锥虫哺乳感染阶段碳代谢的现有认知。