Proteome analysis and heterologous cargo delivery of Vibrio natriegens outer membrane vesicles

Abstract:

[Background] The fast-growing Gram-negative bacterium Vibrio natriegens is a burgeoning tool in biotechnology. Previous research has mainly focused on developing tools for in vitro and in vivo recombinant protein production using V. natriegens. However, many physiological activities that support fast growth and protein production remain largely uncharacterized. Ubiquitously produced by bacteria, outer membrane vesicles (OMVs) not only carry out important functions but also can serve as a useful delivery tool for vaccine and therapeutics development. [Objective] Characterize the proteomes of OMVs during exponential phase growth and to employ OMVs for heterologous protein delivery. [Methods] Using transmission electron microscopy, dynamic light scattering, and mass spectrometry, we characterized the morphology and size distribution of extracted OMVs and their protein composition. We used the superfolded green fluorescent protein (sfGFP) as cargo to determine OMVs protein carriers. [Results] OMVs of mid- and late-exponential phases cultures contain 288 and 317 proteins, respectively. These proteins belong to multiple functional groups including ABC transporters, flagella and two-component systems. By contrast, we identified 1 480 and 1 565 proteins in whole cell samples under these two conditions, respectively. We screened OMV proteins for candidate carriers and found an OmpA-family protein that we name OmpA24 could enrich the sfGFP as a protein-fusion cargo in OMVs. [Conclusion] We demonstrate for the first time that V. natriegens can produce OMVs throughout exponential growth and present the first proteomic snapshot of OMVs and related whole cell samples under different growth phases. OmpA24 protein is a promising carrier for delivery of heterologous protein-fusion cargo into OMVs. This study will facilitate the application of V. natriegens in protein expression and OMV-mediated secretion.