Leishmania stage transition using untargeted metabolomics: an emphasis on Reactive Oxygen Species (ROS) impact

Background: Leishmaniasis remains a major global health threat, requiring Leishmania parasites to undergo complex differentiation to survive contrasting environments between the sandfly vector (promastigote form) and the mammalian host (amastigote form). While temperature and pH are known triggers for this transition, the role of the macrophage oxidative burst, specifically reactive oxygen species (ROS), as signaling molecules for differentiation remains poorly understood. Methods: We employed an untargeted LC-HRMS metabolomics approach, combined with chemometric analyses to characterize the metabolic fingerprints of L. infantum procyclic promastigotes, metacyclic promastigotes, and amastigotes. Additionally, we evaluated the impact of ROS (O2•-, H2O2, and NO•) on procyclic promastigotes to determine their potency as differentiation signals. Results: Our analysis established distinct stage-specific metabolic signatures: procyclic stages are characterized by high levels of amino acids and pyrimidine nucleotides, whereas amastigotes exhibit a lipid-dominated metabolism, particularly rich in ceramides and glycerophospholipids. Crucially, we demonstrate that ROS, with nitric oxide (NO•) being the most potent, trigger an early metabolic reprogramming toward the amastigote profile. This transition, marked by decreased in promastigote biomarkers, such as glucose-6-phosphate, and an increased in amastigote-specific markers like PE 19:1_20:1, precedes morphological changes, suggesting that metabolic adaptation is an early event in the differentiation process. Conclusion: These findings confirm that ROS, specifically NO•, acts as a key environmental signal initiating coordinated transcriptional and metabolic shifts in L. infantum. This study provides the first integrative metabolomic map of L. infantum development and highlights the importance of host-derived chemical mediators in parasite adaptation.Background: 利什曼病（Leishmaniasis）仍是全球主要公共卫生威胁，利什曼原虫（Leishmania parasites）需经历复杂的分化过程，才能在白蛉媒介（sandfly vector）的前鞭毛体（promastigote form）与哺乳动物宿主的无鞭毛体（amastigote form）这两种迥异环境中存活。虽然已知温度与pH是这一转化的触发因素，但巨噬细胞氧化爆发（macrophage oxidative burst）、尤其是作为分化信号分子的活性氧（reactive oxygen species, ROS）所发挥的作用，目前仍有待深入阐明。 Methods: 本研究采用非靶向液相色谱-高分辨质谱（LC-HRMS）代谢组学方法，结合化学计量学分析（chemometric analyses），对婴儿利什曼原虫（L. infantum）的周期型前鞭毛体（procyclic promastigotes）、循环型前鞭毛体（metacyclic promastigotes）以及无鞭毛体的代谢指纹（metabolic fingerprints）特征进行了表征。此外，本研究还评估了活性氧（ROS，包括超氧阴离子O2•-、过氧化氢H2O2与一氧化氮NO•）对周期型前鞭毛体的影响，以明确其作为分化信号的效能。 Results: 本研究分析确定了阶段特异性的独特代谢特征：周期型前鞭毛体以高丰度氨基酸与嘧啶核苷酸为典型特征，而无鞭毛体则以脂质代谢为主，尤其富含神经酰胺（ceramides）与甘油磷脂（glycerophospholipids）。至关重要的是，本研究证实活性氧（ROS）中以一氧化氮（NO•）的作用最为强效，可触发早期代谢重编程，使其向无鞭毛体的代谢特征转化。这一转化以葡萄糖-6-磷酸（glucose-6-phosphate）等前鞭毛体生物标志物水平下降，以及PE 19:1_20:1等无鞭毛体特异性标志物水平上升为标志，且早于形态学变化，表明代谢适应是分化过程中的早期事件。 Conclusion: 本研究结果证实，活性氧（ROS）尤其是一氧化氮（NO•），可作为关键环境信号，启动婴儿利什曼原虫（L. infantum）的协同转录与代谢变化。本研究首次绘制了婴儿利什曼原虫发育过程的整合代谢组图谱，并凸显了宿主来源的化学介质在寄生虫适应过程中的重要性。