Leishmania infantum stage transition using untargeted metabolomics: an emphasis on reactive species 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 and nitrogen species (ROS/RNS), 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 reactive species (H 2 O 2 , 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 reactive species, with 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 reactive species, 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.