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

Nichole Orench-Rivera,Meta J Kuehn

doi:10.3389/fmicb.2021.561863

Nichole Orench-Rivera, Meta J Kuehn

Open Access

https://doi.org/10.3389/fmicb.2021.561863

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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.

Highlights

Outer membrane vesicles (OMVs) are produced when the outer membrane (OM) of Gramnegative bacteria bulges outwards producing spherical buds filled with periplasmic content and OM proteins
Unlike some other species examined in previous studies (Macdonald and Kuehn, 2013; van de Waterbeemd et al, 2013), a comparison of OMV yield by protein and lipid assays in treated and untreated culture supernatants revealed that the oxidative treatment did not significantly alter OMV
Production by enterotoxigenic E. coli (ETEC) (Supplementary Figure 4). These data, albeit preliminary, demonstrate that for ETEC a non-lethal shift with peroxide leads to the generation of oxidative species but not OMV induction

Summary

Introduction

Outer membrane vesicles (OMVs) are produced when the outer membrane (OM) of Gramnegative bacteria bulges outwards producing spherical buds filled with periplasmic content and OM proteins. Bacteria ubiquitously produce these vesicles and this process is known to be genetically regulated (Bodero et al, 2007; Kitagawa et al, 2010; Kulp et al, 2015; Nevermann et al, 2019; van der Westhuizen et al, 2019). Our lab has previously investigated how both vesiculation levels and specific cargo selection into and exclusion from OMVs help bacteria to quickly remodel their envelope to accommodate shifts in the environment. There are very few-to-no known mechanisms for OM lipids or OM-associated proteins to be quickly degraded or recycled (Schindler et al, 1980; Rassam et al, 2015), so these results are consistent with the notion that OMVs can serve as a remodeling and maintenance mechanism for the Gram-negative OM during environmental changes or stress

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