Proteomic and bioinformatic analysis of Lacticaseibacillus casei BL23 extracellular vesicles

MARIANA PIURI; ANA PAULA DOMÍNGUEZ RUBIO; ANTONIO MARCILLA; CECILIA L D ANTONI; OSCAR E PÉREZ

Buenos Aires

Simposio; 6to Simposio Argentino de Jóvenes Investigadores en Bioinformática (6SAJIB); 2021

RSG Argentina

Introduction Archaea, bacteria, and eukaryotes secrete extracellular vesicles (EVs) as a mechanism for intercellular communication. We reported the isolation and characterization of EVs from the Lacticaseibacillus casei BL23, a strain that exhibits probiotic properties. Using a proteomic approach (LC-MS), we identified a total of 103 proteins; 13 exclusively present in the EVs. The EVs content included cell envelope associated and secretory proteins, heat and cold shock proteins, several metabolic enzymes, proteases, structural components of the ribosome, membrane transporters, cell wall-associated hydrolases and phage related proteins. We identified proteins described as mediators of Lacticaseibacillus probiotic effects such as P40, P75 and the product of LCABL_31160, annotated as an adhesion protein. Objectives The aim of this work was to sensitively and reliably identify abundant proteins present in EVs released by L. casei BL23 and to study their probable function and localization by bioinformatic tools. Materials and methods We conducted LC-MS/MS analysis of L. casei BL23 EVs. After gradient SDS-PAGE, only six major EV protein bands were excised to reduce the complexity of the sample and thus improve the sensitivity of the technique. Bands were in gel-digested, and tryptic peptides were separated by 1D ULTRA nanoLC and analyzed by a mass spectrometer nanoESI qQTOF. A list of peaks was generated by the ProteinPilot v4.5 software(ABSciex), where the Paragon algorithm was used to compare against Lactobacillus casei BL23 proteins database. The protein grouping was done by Pro group algorithm and similarity studies were performed using BLASTp. Functions of EV proteins were obtained from Uniprot, InterPro and Pfam. We predicted subcellular localization by PSORTb v3.0.3 and functional groups by the EggNOG database.Results We identified 69 proteins, 24 of which were putative uncharacterized proteins. The most abundant bands corresponded with membrane proteins, cell wall hydrolases and enzymes involved in metabolic pathways. Some of the latter, such as GAPDH and enolase, were previously reported to translocate to the surface of lactobacilli, where they can function as adhesins, and enhance the adhesion of EVs to the intestine. We confirmed the presence of cell wall hydrolases, such as P40, P45, P75, inulosucrase, PBPA1 and endolysin, whose occurrence could represent their importance for EV release. We have also found phage-related proteins annotated as putative uncharacterized proteins, namely a major capsid protein and endolysin, and phage receptors. Conclusion The presence of these proteins gives insight into EV biogenesis and suggests a role for the EVs in the bacteria-gastrointestinal cells interface. Studies are being carried out by our group to confirm the presence and function of these proteins, with a focus on P40 and P45, and shed light on how probiotics have beneficial effects on the host.