Abstract
Outer membrane vesicles (OMVs) are 20-400 nm in size, membrane-bound, and secreted by gram-negative bacteria. OMVs play important roles in processes such as toxin delivery and immune evasion. Although many studies have revealed the critical roles played by OMVs, their heterogeneity has limited our ability to attain a comprehensive understanding of their protein compositions and functions. Therefore, studying the compositions of heterogeneous OMVs subpopulations and their biological functions is important. Herein, we used ultracentrifugation combined with density-gradient centrifugation and quantitative proteomics to systematically separate, characterize, and comprehensively analyze OMVs secreted by Escherichia coli DH5α and Pseudomonas aeruginosa PAO1. First, crude OMVs extracts from both strains were obtained by ultracentrifugation and subjected to iodixanol density-gradient centrifugation to afford six fractions each. DH5α-OMVs and PAO1-OMVs particle-size distributions were then determined via nanoparticle tracking analysis, with average particle sizes of 131.0-161.0 and 140.0-169.0 nm determined for the two subpopulation, respectively. Vesicles were observed to have classical chattel structures by transmission electron microscopy. OMVs subpopulation distributions in the density-gradated fractions were determined by silver staining and protein immunoblotting, which also identified F1a-F4a and F1b-F5b as the effective DH5α-OMVs and PAO1-OMVs subpopulation fractions, respectively. We then identified 2388 and 905 proteins from the DH5α-OMVs and PAO1-OMVs subpopulation, respectively, and used k-means clustering and gene ontology (GO) enrichment analyses to reveal the heterogeneities of the various density subpopulations in terms of biological functions, such as energy metabolism, material transport and ribosome synthesis. Comparative analysis of the E. coli DH5α-OMVs and P. aeruginosa PAO1-OMVs subpopulations finally revealed that they exhibit different functional characteristics, despite sharing commonalities in their basic OMVs functions. The F1a DH5α-OMVs subpopulation was found to be enriched for functions related to amino-acid metabolism and protein synthesis, while the F2b PAO1-OMVs subpopulation exhibited significant biomolecule synthesis functions. This study revealed that bacterial OMVs subpopulations have distinct biological functions, which in turn provides a new theoretical basis for understanding the pathogenic mechanisms of bacteria and their interactions with the host, thereby expanding their biological applications.