Abstract
Outer membrane vesicles (OMVs) produced by Gram-negative bacteria are mediators of cell survival and pathogenesis by facilitating 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 ΔtolA mutant. Nanoparticle tracking analysis (NTA) indicated that UC techniques result in lower vesicle yields compared to UF. However, UF permitted isolation of OMVs with smaller average sizes than UC, highlighting a potential OMV isolation size bias by each technique. Cryo-transmission electron microscopy (cryo-TEM) visualization of isolated OMVs revealed distinct morphological differences between WT and ΔtolA OMVs, where ΔtolA OMVs isolated by either UC or UF method possessed a greater proportion of OMVs with two or more membranes. Proteomic OMV analysis of WT and ΔtolA OMVs confirmed that ΔtolA 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.
Highlights
Bacterial outer membrane vesicles (OMVs) are spherical membrane structures typically ranging from 20-200 nm in diameter that are released from the outer membrane (OM) of Gram-negative bacteria into the extracellular milieu (MashburnWarren et al, 2008; Schwechheimer and Kuehn, 2015)
The goal of our study was to evaluate two of the most commonly used OMV isolation techniques, ultracentrifugation (UC) and ultradiafiltration (UF) with an E. coli K-12 BW25113 strain (WT) and JW0729, a mutant containing a singlegene deletion of TolA component in the Tol-Pal system. tolA was selected for comparison as previous studies (Bernadac et al, 1998; McBroom et al, 2006) identified that this deletion mutant confers a hypervesiculating phenotype when compared to the when compared to UF (WT) E. coli strain
Prior to UC and UF OMV isolations, we wanted to ensure that OMV formation from WT and tolA was proportional to the total quantity of cells grown in culture; this measurement was important to account for potential cell titer differences caused by growth rate differences between the mutant and WT
Summary
Bacterial outer membrane vesicles (OMVs) are spherical membrane structures typically ranging from 20-200 nm in diameter that are released from the outer membrane (OM) of Gram-negative bacteria into the extracellular milieu (MashburnWarren et al, 2008; Schwechheimer and Kuehn, 2015). OMVs have been proposed as specialized delivery vehicles, with their lipid bilayer topology ideal for transporting therapeutics to specific host cells (O’Donoghue and Krachler, 2016; Cecil et al, 2019). They have been incorporated into vaccine preparations due to their immunogenicity and ability to display antigens without the accompanying risk posed by metabolically active bacterial cells (Cecil et al, 2019). An important drawback for these applications is the low yield of vesicles that can be recovered from in vitro culture supernatants
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