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. doi: 10.3390/ijms25179272.

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 Acinetobacter baumannii strain lacking lipooligosaccharide (LOS) biosynthesis, due to a mutation in lpxD, 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 A. baumannii, using both LOS-containing and LOS-free OMVs preparations.

Keywords: Acinetobacter baumannii; OMVs; lipooligosaccharide; outer membrane vesicles; proteomic; vaccine.

MeSH terms

  • Acinetobacter Infections / microbiology
  • Acinetobacter baumannii* / genetics
  • Acinetobacter baumannii* / metabolism
  • Animals
  • Bacterial Outer Membrane / metabolism
  • Bacterial Outer Membrane Proteins* / genetics
  • Bacterial Outer Membrane Proteins* / metabolism
  • Lipopolysaccharides* / biosynthesis
  • Mice
  • Tandem Mass Spectrometry

Substances

  • Lipopolysaccharides
  • lipid-linked oligosaccharides
  • Bacterial Outer Membrane Proteins