Proteomics of Extracellular Vesicles Produced by Granulicatella adiacens, which causes Infective Endocarditis

Granulicatella adiacens is a non-motile, non-spore-forming, facultatively anaerobic Gram-positive coccus. It is part of the normal oral flora but cause serious infections such as infective endocarditis. Many bacterial species produce extracellular vesicles (EVs) as part of normal physiology, but also use it as a virulence strategy. In this study, it was hypothesized that G. adiacens produces EVs that possibly help the species in virulence. Therefore, the objectives were to isolate and characterize EVs produced by these species and to investigate their immune-stimulatory effects. The reference strain G. adiacens CCUG 27809 was cultured on chocolate blood agar for 2 days. From subsequent broth cultures, the EVs were isolated using differential centrifugation and filtration protocol and then observed using scanning electron microscopy. Proteins in the vesicle preparations were identified by nano LC-ESI-MS/MS. The EVs proteomes were analyzed and characterized using different bioinformatics tools. The immune-stimulatory effect of the EVs was studied via ELISA quantification of IL-8, IL-1β and CCL5, major proinflammatory cytokines, produced from stimulated human PBMCs. It was revealed that both G. adiacens and G. elegans produced EVs, ranging in diameter from 30 to 250 nm. Overall, G. adiacens EVs contained 112 proteins. The proteome consist of several ribosomal proteins, DNA associated proteins, binding proteins, and metabolic enzymes. It was also shown that these EVs carry putative virulence factors including moonlighting proteins. These EVs were able to induce the production of IL-8, IL-1β and CCL5 from human PBMCs. The diversity in EVs content indicates that these vesicles may have possible roles in bacterial survival, invasion, host immune modulation as well as infection. Further characterization of the composition and immunogenicity of G. adiacens EVs may provide new insights into virulence mechanisms of this bacterial species.