Comparative Analysis of Outer Membrane Vesicle Isolation Methods With an Escherichia coli tolA Mutant Reveals a Hypervesiculating Phenotype With Outer-Inner Membrane Vesicle Content

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.

Considerations for the Analysis of Bacterial Membrane Vesicles: Methods of Vesicle Production and Quantification Can Influence Biological and Experimental Outcomes

“…We have previously shown that the overall protein composition of BMVs varies between different bacterial strains ( 7 ), throughout various stages of bacterial growth ( 4 , 45 ), and based on BMV size ( 29 ). It has also been shown that bacterial culture conditions ( 46 ) and methods of BMV isolation ( 34 , 47 ) can alter BMV protein composition. However, it is unclear whether there is variability in the overall quantity of proteins packaged into BMVs produced by different bacterial species and strains and the biological consequences of quantifying BMVs based on their variable protein cargo amounts.…”

“…Furthermore, a range of protein assays are used to quantify BMVs, including Bradford ( 28 , 29 ), bicinchoninic acid (BCA) ( 12 , 30 ), Lowry ( 31 ), or Qubit protein assays ( 32 , 33 ), with no single protein assay used as the standard method to quantify BMVs in the field. However, variables in BMV isolation ( 34 ), bacterial strains ( 35 ), culture conditions ( 36 ), growth stage ( 4 ), and BMV size ( 29 ) can all significantly alter BMV protein content, suggesting that BMV protein concentration may not directly correlate with BMV quantity.…”

Isolation and Characterization of Outer Membrane Vesicles of Pectobacterium brasiliense 1692

“…Studies also report OMVs of larger sizes, aggregates, chains, and tubes [ 19 ]. However, there are no baselines for isolation methods; hence, the types, cargo, and specific functions of OMVs will vary from one species to another, making comparisons difficult [ 20 , 21 ]. We have yet to learn more about the species and strain-specific roles of OMVs of phytopathogenic bacteria in bacteria–bacteria and bacteria–host interactions, as well as their potential to control plant diseases.…”

Exosomes: Biological Pharmaceutical Nanovectors for Theranostics

“…The easy programmability of bacteria and the many advancements in synthetic biology have enabled the development of OMVs packed with therapeutic recombinant proteins. To increase OMV yield, hypervesiculating strains can be developed by deleting or mutating genes such as the nlpI , tolR , and the mlaE gene or by overexpressing membrane proteases such as OmpT ( Premjani et al, 2014 ; Chen et al, 2016 ; Ojima et al, 2020 ; Reimer et al, 2021 ). The addition of tris-glycine to the bacterial culture media has also been shown to increase OMV production in vitro ( Hirayama and Nakao, 2020 ).…”