Proteomics of Extracellular Vesicles Produced byGranulicatellaSpecies that cause Infective Endocarditis

Abstract

30When oral bacteria accidentally enter the bloodstream due to transient tissue 31 damage during dental procedures, they have the potential to attach to the endocardium 32 or an equivalent surface of an indwelling prosthesis and cause infection. Many bacterial 33 species produce extracellular vesicles (EVs) as part of normal physiology, but also use 34 it as a virulence strategy. In this study, it was hypothesized that Granulicatella species 35 produce EVs that possibly help them in virulence. Therefore, the objectives were to 36 isolate and characterize EVs produced by these species and to investigate their immune-37 stimulatory effects. The reference strains G. adiacens CCUG 27809 and G. elegans 38 CCUG 38949 were cultured on chocolate blood agar for 2 days. From subsequent broth 39 cultures, the EVs were isolated using differential centrifugation and filtration protocol 40 and then observed using scanning electron microscopy. Proteins in the vesicle 41 preparations were identified by nano LC-ESI-MS/MS. The EVs proteomes were 42 analyzed and characterized using different bioinformatics tools. The immune-43 stimulatory effect of the EVs was studied via ELISA quantification of IL-8, IL-1β and 44 CCL5, major proinflammatory cytokines, produced from stimulated human PBMCs. It 45 was revealed that both G. adiacens and G. elegans produced EVs, ranging in diameter 46 from 30 to 250 nm. Overall, G. adiacens EVs contained 160 proteins, and G. elegans 47 EVs contained 107 proteins. Both proteomes consist of several ribosomal proteins, 48DNA associated proteins, binding proteins, and metabolic enzymes. It was also shown 49 that these EVs carry putative virulence factors including moonlighting proteins. These 50 EVs were able to induce the production of IL-8, IL-1β and CCL5 from human PBMCs. 51The diversity in EVs content indicates that these vesicles could have possible roles in 3 52 bacterial survival, invasion, host immune modulation as well as infection. Further 53 functional characterization of the Granulicatella EVs may provide new insights into 54 virulence mechanisms of these important but less studied oral bacterial species. 55 56 57 Granulicatella species, formerly known as nutritionally variant streptococci 58 based on their characteristic dependence on pyridoxal or cysteine supplementation for 59 their growth in standard media [1], are catalase and oxidase negative, non-motile, non-60 spore-forming, facultatively anaerobic Gram-positive cocci [2, 3]. They are part of the 61 normal oral flora [4], but cause serious infections such as infective endocarditis. The 62 genus Granulicatella consists of 3 species: Granulicatella adiacens, Granulicatella 63 elegans and Granulicatella balaenopterae [3]. The species G. balaenopterae has not 64 been isolated from human samples, whereas both G. adiacens and G. elegans have been 65 reported from IE cases [5, 6]. In addition, these oral commensal cocci have been 66 associated with endodontic infections [7, 8], dental caries [9], and periodontitis [8, 10] 67 via DNA-based s...

New frontiers of oral sciences: Focus on the source and biomedical application of extracellular vesicles

“…Given the presence of the phospholipid bilayer membrane, these contents are protected from harsh conditions (Kadurugamuwa and Beveridge, 1999). Thus, bacterial EVs resemble a destructive bullet, promoting proinflammatory signaling cascades and long-distance microbiota-host communication (Jones, et al, 2020) to cause oral disease (Peng, et al, 2020) and systemic diseases such as Alzheimer's disease (AD) (Singhrao and Olsen, 2018), neuroinflammatory diseases (Ha, et al, 2020b), infective endocarditis (Alkandari, et al, 2020) and respiratory system diseases (He, et al, 2020).…”