Cano-Castaño, BeatrizCorral-Lugo, AndresGato, EvaTerrón-Orellana, Maria CarmenMartin-Galiano, Antonio JavierSotillo, JavierPerez Gomez, AstridMcConnell, Michael J2025-01-092025-01-092024-08-27Cano-Castaño B, Corral-Lugo A, Gato E, Terrón MC, Martín-Galiano AJ, Sotillo J, Pérez A, McConnell MJ. Loss of Lipooligosaccharide Synthesis in Acinetobacter baumannii Produces Changes in Outer Membrane Vesicle Protein Content. Int J Mol Sci. 2024 Aug 27;25(17):9272.https://hdl.handle.net/20.500.12105/25968Outer membrane vesicles (OMVs) are nanostructures derived from the outer membrane of Gram-negative bacteria. We previously demonstrated that vaccination with endotoxin-free OMVs isolated from an strain lacking lipooligosaccharide (LOS) biosynthesis, due to a mutation in , provides full protection in a murine sepsis model. The present study characterizes the protein content of highly-purified OMVs isolated from LOS-replete and LOS-deficient strains. Four purification methods were evaluated to obtain highly purified OMV preparations: ultracentrifugation, size exclusion chromatography (SEC), ultracentrifugation followed by SEC, and Optiprep™. OMVs from each method were characterized using nanoparticle tracking analysis and electron microscopy. OMVs from LOS-deficient and LOS-replete strains purified using the Optiprep™ method were subjected to LC-MS/MS analysis to determine protein content. Significant differences in protein composition between OMVs from LOS-deficient and LOS-replete strains were found. Computational analyses using Bepipred 3.0 and SEMA 2.0 indicated that the lack of LOS led to the overexpression of immunogenic proteins found in LOS-containing OMVs and the presence of immune-stimulating proteins absent in LOS-replete OMVs. These findings have important implications for developing OMV-based vaccines against , using both LOS-containing and LOS-free OMVs preparations.engVoRhttp://creativecommons.org/licenses/by/4.0/Acinetobacter baumanniiOMVsLipooligosaccharideOuter membrane vesiclesProteomicVaccineAcinetobacter InfectionsAcinetobacter baumanniiAnimalsBacterial Outer MembraneBacterial Outer Membrane ProteinsLipopolysaccharidesMiceTandem Mass SpectrometryAttribution 4.0 International392732202517927210.3390/ijms251792721422-0067International journal of molecular sciencesopen access