Loss of Lipooligosaccharide Synthesis in Produces Changes in Outer Membrane Vesicle Protein Content

Abstract

Outer 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.