Differential Packaging Into Outer Membrane Vesicles Upon Oxidative Stress Reveals a General Mechanism for Cargo Selectivity

Abstract

Selective cargo packaging into bacterial extracellular vesicles has been reported and implicated in many biological processes, however, the mechanism behind the selectivity has remained largely unexplored. In this study, proteomic analysis of outer membrane (OM) and OM vesicle (OMV) fractions from enterotoxigenic E. coli revealed significant differences in protein abundance in the OMV and OM fractions for cultures shifted to oxidative stress conditions. Analysis of sequences of proteins preferentially packaged into OMVs showed that proteins with oxidizable residues were more packaged into OMVs in comparison with those retained in the membrane. In addition, the results indicated two distinct classes of OM-associated proteins were differentially packaged into OMVs as a function of peroxide treatment. Implementing a Bayesian hierarchical model, OM lipoproteins were determined to be preferentially exported during stress whereas integral OM proteins were preferentially retained in the cell. Selectivity was determined to be independent of transcriptional regulation of the proteins upon oxidative stress and was validated using randomly selected protein candidates from the different cargo classes. Based on these data, a hypothetical functional and mechanistic basis for cargo selectivity was tested using OmpA constructs. Our study reveals a basic mechanism for cargo selectivity into OMVs that may be useful for the engineering of OMVs for future biotechnological applications.

The extracellular vesicle generation paradox: a bacterial point of view

“…As the need for vesicle purification has been recognized and the purification techniques have improved (for recent reviews, see Bonnington & Kuehn, 2014Elhenawy et al, 2016;Orench-Rivera & Kuehn, 2021) does not mimic the composition of the envelope from which they are derived. Indeed, molecular "rules" have been validated that show mechanisms for specific inclusion and exclusion of soluble and membrane-associated OMV cargo based on polypeptide sequence or physical parameters (McBroom & Kuehn, 2007;Orench-Rivera & Kuehn, 2021).…”

“…As the need for vesicle purification has been recognized and the purification techniques have improved (for recent reviews, see (Prados‐Rosales et al , 2014a; Klimentová & Stulík, 2015; Tulkens et al , 2020a; Liangsupree et al , 2021; Nasukawa et al , 2021)), analysis of vesicle cargo has revealed export selectivity, a hallmark of a bona fide secretory process. Numerous quantitative proteomic and lipidomic analyses have demonstrated that the composition of OMVs (McMahon et al , 2012; Bonnington & Kuehn, 2014, 2016; Elhenawy et al , 2016; Orench‐Rivera & Kuehn, 2021) does not mimic the composition of the envelope from which they are derived. Indeed, molecular “rules” have been validated that show mechanisms for specific inclusion and exclusion of soluble and membrane‐associated OMV cargo based on polypeptide sequence or physical parameters (McBroom & Kuehn, 2007; Orench‐Rivera & Kuehn, 2021).…”

“…Numerous quantitative proteomic and lipidomic analyses have demonstrated that the composition of OMVs (McMahon et al , 2012; Bonnington & Kuehn, 2014, 2016; Elhenawy et al , 2016; Orench‐Rivera & Kuehn, 2021) does not mimic the composition of the envelope from which they are derived. Indeed, molecular “rules” have been validated that show mechanisms for specific inclusion and exclusion of soluble and membrane‐associated OMV cargo based on polypeptide sequence or physical parameters (McBroom & Kuehn, 2007; Orench‐Rivera & Kuehn, 2021). Similarly, in Gram‐positive bacteria the protein, lipid, and nucleic acid content of vesicles reveals selective packaging rather than a bulk‐flow mechanism (Resch et al , 2016; Jeon et al , 2018; Briaud & Carroll, 2020; Nagakubo et al , 2020; Tartaglia et al , 2020; Luz et al , 2021).…”

“…Subsequently, studies examined whether bacteria may also benefit from using selective vesicle export to remodel their OM (Fig 1A) (Cahill et al , 2015; Bonnington & Kuehn, 2016; Elhenawy et al , 2016; Eberlein et al , 2018; Valguarnera et al , 2018; Orench‐Rivera & Kuehn, 2021). Recently, it was determined that levels of oxidizable residues and oxidized OM‐associated proteins are increased in E. coli OMV cargo in response to oxidative stress, presumably because the oxidized proteins are detrimental to the cells (Orench‐Rivera & Kuehn, 2021).…”

“…Subsequently, studies examined whether bacteria may also benefit from using selective vesicle export to remodel their OM (Fig 1A) (Cahill et al , 2015; Bonnington & Kuehn, 2016; Elhenawy et al , 2016; Eberlein et al , 2018; Valguarnera et al , 2018; Orench‐Rivera & Kuehn, 2021). Recently, it was determined that levels of oxidizable residues and oxidized OM‐associated proteins are increased in E. coli OMV cargo in response to oxidative stress, presumably because the oxidized proteins are detrimental to the cells (Orench‐Rivera & Kuehn, 2021). Further, the OMV packaging differential between full‐length (cell‐wall bound) and truncated (unbound) OmpA increased upon oxidative stress, which may be related to the beneficial role cell‐wall associated OmpA plays in oxidative response (van der Heijden et al , 2016).…”