Proteomics of Extracellular Vesicles Produced byGranulicatellaSpecies that cause Infective Endocarditis

Abstract

AbstractWhen oral bacteria accidentally enter the bloodstream due to transient tissue damage during dental procedures, they have the potential to attach to the endocardium or an equivalent surface of an indwelling prosthesis and cause infection. 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 thatGranulicatellaspecies produce EVs that possibly help them in virulence. Therefore, the objectives were to isolate and characterize EVs produced by these species and to investigate their immune-stimulatory effects. The reference strainsG. adiacensCCUG 27809 andG. elegansCCUG 38949 were 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 bothG. adiacensandG. elegansproduced EVs, ranging in diameter from 30 to 250 nm. Overall,G. adiacensEVs contained 160 proteins, andG. elegansEVs contained 107 proteins. Both proteomes 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 could have possible roles in bacterial survival, invasion, host immune modulation as well as infection. Further functional characterization of theGranulicatellaEVs may provide new insights into virulence mechanisms of these important but less studied oral bacterial species.