Profiling Temperature-Dependent Lipid Remodeling in Yersinia pestis Using Ion Mobility-Mass Spectrometry and Ozonolysis

Abstract Yersinia pestisundergoes dramatic physiological changes as it transitions from its flea vector (∼20–30 °C) to mammalian hosts (37 °C), including remodeling of its cell envelope lipids to evade innate immunity and enhance virulence. While various temperature-dependent changes in this organism have been previously documented, the broader lipidome remains poorly characterized. We applied liquid chromatography coupled with ion mobility spectrometry and tandem mass spectrometry (LC-IMS-MS/MS), in addition to ozone-induced dissociation (OzID), to comprehensively profile lipids in three attenuated Y. pestis strains (A1122, KIM D27, and KIM D1) grown at 30 and 37 °C. Untargeted lipidomic analysis identified 97 lipids across multiple classes, with principal component analysis revealing temperature-dependent clustering patterns distinct from strain-specific variations. Comparison of lipid profiles between temperatures revealed many significantly differentially expressed lipids per strain, with most strain-specific responses rather than shared across all strains. OzID fragmentation successfully identified double-bond positions for common unsaturated fatty acids and lysophospholipids, also enabling structural discrimination between monounsaturated and cyclopropyl fatty acid isomers. Notably, lipids containing cyclopropyl fatty acids were significantly upregulated at 37 °C across all three strains, suggesting a conserved temperature-responsive mechanism for enhancing membrane stability during mammalian host infection. Our study demonstrates that comprehensive, high-detailed, structurally resolved lipidomics reveals temperature-dependent lipid remodeling characteristics in Y. pestis that may support host transition and virulence, with potential applications for identifying lipid biomarkers.