Different culture media and purification methods unveil the core proteome of Propionibacterium freudenreichii-derived extracellular vesicles

Vinícius de Rezende Rodovalho; Brenda Silva Rosa da Luz; Aurélie Nicolas; Julien Jardin; Valérie Briard-Bion; Edson Luiz Folador; Anderson Rodrigues Santos; Gwénaël Jan; Yves Le Loir; Vasco Ariston de Carvalho Azevedo; Éric Guédon

doi:10.1093/femsml/uqad029

Different culture media and purification methods unveil the core proteome of Propionibacterium freudenreichii-derived extracellular vesicles

Microlife. 2023 Jun 2:4:uqad029. doi: 10.1093/femsml/uqad029. eCollection 2023.

Affiliations

¹ INRAE, Institut Agro, STLO, 35042, Rennes, France.
² Laboratory of Cellular and Molecular Genetics, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte 31270-901, Brazil.
³ Laboratory of Immunoinflammation, Institute of Biology, University of Campinas (UNICAMP), Campinas 13000-000, Brazil.
⁴ Center of Biotechnology, Department of Biotechnology, Federal University of Paraíba, João Pessoa 58051-900, Brazil.
⁵ Faculty of Computer Science, Department of Computer Science, Federal University of Uberlândia, Uberlândia 38400902, Brazil.

Abstract

Bacterial extracellular vesicles (EVs) are natural lipidic nanoparticles implicated in intercellular communication. Although EV research focused mainly on pathogens, the interest in probiotic-derived EVs is now rising. One example is Propionibacterium freudenreichii, which produces EVs with anti-inflammatory effects on human epithelial cells. Our previous study with P. freudenreichii showed that EVs purified by size exclusion chromatography (SEC) displayed variations in protein content according to bacterial growth conditions. Considering these content variations, we hypothesized that a comparative proteomic analysis of EVs recovered in different conditions would elucidate whether a representative vesicular proteome existed, possibly providing a robust proteome dataset for further analysis. Therefore, P. freudenreichii was grown in two culture media, and EVs were purified by sucrose density gradient ultracentrifugation (UC). Microscopic and size characterization confirmed EV purification, while shotgun proteomics unveiled that they carried a diverse set of proteins. A comparative analysis of the protein content of UC- and SEC-derived EVs, isolated from cultures either in UF (cow milk ultrafiltrate medium) or YEL (laboratory yeast extract lactate medium), showed that EVs from all these conditions shared 308 proteins. This EV core proteome was notably enriched in proteins related to immunomodulation. Moreover, it showed distinctive features, including highly interacting proteins, compositional biases for some specific amino acids, and other biochemical parameters. Overall, this work broadens the toolset for the purification of P. freudenreichii-derived EVs, identifies a representative vesicular proteome, and enumerates conserved features in vesicular proteins. These results hold the potential for providing candidate biomarkers of purification quality, and insights into the mechanisms of EV biogenesis and cargo sorting.

Keywords: culture media; density gradient ultracentrifugation; membrane vesicles; probiotic; propionibacteria; proteomics; purification.