Fernández-García, MiguelGonzalez, Luis MiguelSevilla, ElenaGil, AitorSantos-Oliveira, HenriqueRevuelta, BelénBarbas, CoralRey-Stolle, Mª FernandaMontero-Clemente, EstrellaGarcía, Antonia2025-09-152025-09-152025-08-08Fernández-García, M.; Gonzalez, L.M.; Sevilla, E.; Gil, A.; Santos-Oliveira, H.; Revuelta, B.; Barbas, C.; Rey-Stolle, M.F.; Montero, E.; García, A. Application of Multiplatform Mass Spectrometry to the Study of Babesia divergens Metabolism and the Pathogenesis of Human Babesiosis. Int. J. Mol. Sci. 2025, 26, 7677. https://doi.org/10.3390/ijms261676771422-0067https://hdl.handle.net/20.500.12105/26893Babesia divergens is a tick-borne apicomplexan parasite that causes human babesiosis, a malaria-like disease. B. divergens metabolism remains poorly characterized. Here, we employed a multiplatform mass spectrometry-based metabolomics approach (using CE-TOF/MS, GC-QTOF/MS, LC-QTOF/MS, and LC-QqQ/MS) to profile intra- and extracellular metabolic changes in B. divergens-infected and uninfected red blood cells (RBCs) and their supernatants. Our results indicate alterations in the metabolome caused by B. divergens infection and proliferation within RBCs. These findings are consistent with the major metabolic dependencies of B. divergens, including extracellular glucose, glutamine, and arginine, accompanied by the accumulation of glycolytic and TCA cycle intermediates. We identified altered nucleotide metabolism, pentose phosphate pathway activity, and redox imbalance. Depletion of lysoglycerophospholipids, glucose, arginine, and glutamine, and accumulation of free heme and sphingolipids suggested pathogenic effects. Growth experiments indicate that glucose and glutamine, but not hypoxanthine, are required for parasite growth. We additionally discovered a phosphorylated HEPES derivative (PEPES) produced upon B. divergens infection of RBCs in vitro. Collectively, these findings and their global interpretation provide insights into B. divergens metabolism and metabolic dependencies and host-parasite metabolic interactions and outline potential directions for future studies on human babesiosis diagnosis, prognosis assessment, and treatment.engVoRhttps://creativecommons.org/licenses/by/4.0/Babesia divergensBabesia divergens metabolismHuman babesiosisMultiplatform metabolomicsBabesiaBabesiosisErythrocytesGlucoseGlutamineGlycolysisHumansMass SpectrometryMetabolomeMetabolomicsAttribution 4.0 International408690022616767710.3390/ijms26167677International Journal of Molecular Sciencesopen access