Environmental Conditions Modulate the Protein Content and Immunomodulatory Activity of Extracellular Vesicles Produced by the Probiotic Propionibacterium freudenreichii

Abstract

Propionibacterium freudenreichii is a probiotic Gram-positive bacterium with promising immunomodulatory properties. It modulates regulatory cytokines and mitigates the inflammatory response in vitro and in vivo. These properties were initially attributed to specific bacterial surface proteins. Recently, we showed that extracellular vesicles (EVs) produced by P. freudenreichii CIRM-BIA129 mimic the immunomodulatory features of parent cells in vitro (i.e., modulating NF-κB transcription factor activity and interleukin-8 release), which underlies the role of EVs as mediators of the probiotic effects of the bacterium. The modulation of EV properties, and particularly of those with potential therapeutic applications, such as the EVs produced by the probiotic P. freudenreichii, is one of the challenges in the field to achieve efficient yields with the desired optimal functionality. Here, we evaluated whether the culture medium in which the bacteria are grown could be used as a lever to modulate the protein content and, hence, the properties of P. freudenreichii CIRM-BIA129 EVs. The physical, biochemical, and functional properties of EVs produced from cells cultivated on laboratory yeast extract lactate (YEL) medium and cow milk ultrafiltrate (UF) medium were compared. UF-derived EVs were more abundant and smaller in diameter, and they displayed more intense anti-inflammatory activity than YEL-derived EVs. Furthermore, the growth media modulated EV content in terms of both the identities and abundances of their protein cargos, suggesting different patterns of interaction with the host. Proteins involved in amino acid metabolism and central carbon metabolism were modulated, as were the key surface proteins mediating host-propionibacterium interactions. IMPORTANCE Extracellular vesicles (EVs) are cellular membrane-derived nanosized particles that are produced by most cells in all three kingdoms of life. They play a pivotal role in cell-cell communication through their ability to transport bioactive molecules from donor to recipient cells. Bacterial EVs are important factors in host-microbe interactions. Recently, we have shown that EVs produced by the probiotic P. freudenreichii exhibited immunomodulatory properties. We evaluate here the impact of environmental conditions, notably culture media, on P. freudenreichii EV production and function. We show that EVs display considerable differences in protein cargo and immunomodulation depending on the culture medium used. This work offers new perspectives for the development of probiotic EV-based molecular delivery systems and reinforces the optimization of growth conditions as a tool to modulate the potential therapeutic applications of EVs.

Aerobic Adaptation and Metabolic Dynamics ofPropionibacterium freudenreichiiDSM 20271: Insights from Comparative Transcriptomics and Surfaceome Analysis

“…Interestingly, the majority of the differentially abundant ribosomal proteins identified here have previously been identified from the vesicles produced by PFR (5, 55, 56). In these studies, the proteins delivered by vesicles contributed to translation, ribosomal structure, and biogenesis.…”

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

“…Proteomic data for UC-purified EVs were achieved within this study. Proteomic data for SEC-purified EVs were retrieved from our previous publication (Rodovalho et al 2021 ). Subcellular localization and lipoprotein signals were predicted with Cello2GO (Yu et al 2014 ) and PRED-LIPO (Bagos et al 2008 ), respectively.…”

“…A diverse set of functions was attributed to bacterial EVs, including quorum sensing (Mashburn and Whiteley 2005 ), biofilm formation (Flemming et al 2016 , Caruana and Walper 2020 ), competition (Li et al 1998 ), nutrition (Elhenawy et al 2014 , Prados-Rosales et al 2014 ), defense (Manning and Kuehn 2011 , Lee et al 2013 ), pathogenesis (Pathirana and Kaparakis-Liaskos 2016 , Cecil et al 2019 ), and probiosis (Bitto and Kaparakis-Liaskos 2017 , Molina-Tijeras et al 2019 ). Among beneficial bacteria, EVs with anti-inflammatory activity were reported in several species, including Akkermansia muciniphila (Ashrafian et al 2019 , Keshavarz Azizi Raftar et al 2021 ), Bacteroides species (Shen et al 2012 , Mirjafari Tafti et al 2019 , Gul et al 2022 ), Escherichia coli Nissle 1917 (Fábrega et al 2017 ), Bifidobacterium species (López et al 2012 , Nishiyama et al 2020 ), Lactobacillus species (Seo et al 2018 , Vargoorani et al 2020 , Caruana et al 2021 , Hao et al 2021 ), and Propionibacterium freudenreichii (Rodovalho et al 2020 , 2021 ).…”

“…Bacterial EVs were reported to hold a diverse set of molecules in their internal lumen, including proteins (Lee et al 2009 , Rubio et al 2017 , Bajic et al 2020 , Nishiyama et al 2020 , Bhar et al 2021 ), DNA (Bitto et al 2017 , 2021a ; Dell'Annunziata et al 2021 ), RNA (Bitto et al 2021a , Joshi et al 2021 , Luz et al 2021 ; Pérez‐Cruz et al, 2021 ), and metabolites (Cao et al 2020 , Kim et al 2020 , 2020ba ; Sartorio et al 2022 ). This vesicular content varies in response to environmental conditions, including bacterial growth media and growth phases (Bitto et al 2021b , Briaud et al 2021 , Luz et al 2021 ; Pérez‐Cruz et al, 2021 , Rodovalho et al 2021 , da Luz et al 2022 , Mehanny et al 2022 ) and several abiotic stressors, including antibiotics, nutrient shortage, salt, and temperature (He et al 2017 , Yun et al 2018 , Godlewska et al 2019 , Lynch et al 2019 , Potter et al 2020 , Briaud et al 2021 ).…”

“…In our previous studies, we showed that the probiotic P. freudenreichii CIRM-BIA129 produces EVs with anti-inflammatory activity toward cultured human intestinal epithelial cells via NF-κB pathway modulation (Rodovalho et al 2020 ) and that bacterial growth media (UF versus YEL) impact the protein composition and the anti-inflammatory activity of EVs (Rodovalho et al 2021 ). Yeast extract lactate (YEL) is the gold-standard laboratory medium for propionibacteria, while ultrafiltrate (UF) medium was developed to mimic their growth conditions in Swiss-type cheeses after fermentation of the cheese curd by lactic acid bacteria (Malik et al 1968 , Cousin et al 2012 ).…”