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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.

SUBMITTER: Orench-Rivera N

PROVIDER: S-EPMC8284480 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Smoking, which is highly prevalent in HIV-infected populations, has been shown to exacerbate HIV replication, in part via the cytochrome P450 (CYP)-induced oxidative stress pathway. Recently, we have shown that extracellular vesicles (EVs), derived from tobacco- and/or HIV-exposed macrophages, alter HIV replication in macrophages by cell-cell interactions. We hypothesize that cigarette smoke condensate (CSC) and/or HIV-exposed macrophage-derived EVs carry relatively high levels of pro-oxidant and pro-inflammatory cargos and/or low levels of antioxidant and anti-inflammatory cargos, which are key mediators for HIV pathogenesis. Therefore, in this study, we investigated differential packaging of pro- and anti-inflammatory cytokines/chemokines and pro- and anti-oxidant contents in EVs after CSC exposure to myeloid cells (uninfected U937 and HIV-infected U1 cells). Our results showed that relatively long to short exposures with CSC increased the expression of cytokines in EVs isolated from HIV-infected U1 macrophages. Importantly, pro-inflammatory cytokines, especially IL-6, were highly packaged in EVs isolated from HIV-infected U1 macrophages upon both long and short-term CSC exposures. In general, anti-inflammatory cytokines, particularly IL-10, had a lower packaging in EVs, while packaging of chemokines was mostly increased in EVs upon CSC exposure in both HIV-infected U1 and uninfected U937 macrophages. Moreover, we observed higher expression of CYPs (1A1 and 1B1) and lower expression of antioxidant enzymes (SOD-1 and catalase) in EVs from HIV-infected U1 macrophages than in uninfected U937 macrophages. Together, they are expected to increase oxidative stress factors in EVs derived from HIV-infected U1 cells. Taken together, our results suggest packaging of increased level of oxidative stress and inflammatory elements in the EVs upon exposure to tobacco constituents and/or HIV to myeloid cells, which would ultimately enhance HIV replication in macrophages via cell-cell interactions.

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

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