Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic.
Institute of Entomology, Biology Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic.
PLoS Negl Trop Dis. 2024 Feb 23;18(2):e0012007. doi: 10.1371/journal.pntd.0012007. eCollection 2024 Feb.
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.
布氏锥虫是人类和动物非洲锥虫病的病原体。哺乳动物阶段的寄生虫感染各种组织和器官，包括血液、中枢神经系统、皮肤、脂肪组织和肺部。它们依赖糖酵解产生的 ATP，消耗大量葡萄糖，而葡萄糖在哺乳动物宿主中很容易获得。除葡萄糖外，甘油也可用作碳和 ATP 的来源，以及糖异生的底物。然而，甘油喂养的糖异生对哺乳动物感染生命周期形式的生理相关性仍然难以捉摸。为了证明其（非）必要性，我们首先必须确定该途径的酶（enzyme）。失去经典的糖异生酶果糖-1,6-二磷酸酶并不会使该过程停止，因此至少还有一种酶必须参与锥虫的糖异生。我们使用 CRISPR/Cas9 基因编辑和 RNA 干扰的组合，生成了可能有助于糖异生的四种酶（fructose-1,6-bisphosphatase、sedoheptulose-1,7-bisphosphatase、phosphofructokinase 和 transaldolase）的突变体，单独或组合使用。代谢组学分析表明，无论这些基因中的哪一个丢失，糖异生的通量都保持不变。我们的数据使先前假设的反向磷酸果糖激酶反应在糖异生中的作用不太可能，并排除了涉及 transaldolase 的新生化途径参与该过程的可能性。我们的突变体（包括三重缺失菌株）中糖异生的持续代谢通量表明存在一种独特的酶参与糖异生。此外，这些数据为糖异生和戊糖磷酸途径提供了新的见解，并提高了对 T. brucei 哺乳动物感染阶段碳代谢的现有理解。
