Proteomic analysis of outer membrane vesicle isolation methods with an Escherichia coli tolA mutant using LC MSMS

ABSTRACT: Outer membrane vesicles (OMVs) produced by Gram-negative bacteria are mediators of cell survival and pathogenesis by facilitating nutrient transport, virulence factor dissemination, and resistance to antimicrobials. Studies of OMV properties often focus on hypervesiculating Escherichia coli mutants that have increased OMV production when compared to their corresponding wild-type (WT) strains. Currently, two conventional techniques, ultracentrifugation (UC) and ultradiafiltration (UF), are used interchangeably to isolate OMVs, however, there is concern that each technique may inadvertently alter the properties of isolated OMVs during study. To address this concern, we compared two OMV isolation methods, UC and UF, with respect to final OMV quantities, size distributions, and morphologies using a hypervesiculating Escherichia coli K-12 ∆tolA mutant. Cryo-transmission electron microscopy (cryo-TEM) visualization of isolated OMVs revealed distinct morphological differences between wild-type and ∆tolA OMVs, where ∆tolA OMVs isolated by either UC or UF method possessed a greater proportion of OMVs with two or more membranes (39.6-42.4%). Proteomic OMV analysis of WT and ∆tolA OMVs confirmed that ∆tolA enhances inner plasma membrane carryover in multi-lamellar OMVs. This study demonstrates that UC and UF are useful techniques for OMV isolation, where UF may be preferable due to faster isolation, higher OMV yields and enrichment of smaller sized vesicles.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Escherichia Coli

SUBMITTER: Denice Bay  

LAB HEAD: Denice C Bay

PROVIDER: PXD022786 | Pride | 2021-09-09

REPOSITORIES: Pride