Proteomics of extracellular vesicles produced by Granulicatella adiacens, which causes infective endocarditis

Abstract

When 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 that Granulicatella adiacens produce EVs that possibly help it in virulence. Therefore, the objectives were to isolate and characterize EVs produced by G. adiacens and to investigate its immune-stimulatory effects. The reference strain G. adiacens CCUG 27809 was cultured on chocolate blood agar for 2 days. From subsequent broth culture, the EVs were isolated using differential centrifugation and filtration protocol and then observed using scanning electron microscopy. Proteins in the vesicle preparation were identified by nano LC-ESI-MS/MS. The EVs proteome was 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 G. adiacens produced EVs, ranging in diameter from 30 to 250 nm. Overall, G. adiacens EVs contained 112 proteins. The proteome consists 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. Further functional characterization of the G. adiacens EVs may provide new insights into virulence mechanisms of this important but less studied oral bacterial species.

Fig 1. SEM images of G. adiacens whole cells and the EVs preparation.

SEM images of bacterial whole cells (A) and the EVs preparation (B) captured at the magnification ×10000. (C) SEM images of the EVs acquired at ×40000.

Fig 2. Analysis of the proteome of G. adiacens EVs.

(A) SDS-PAGE gel showing protein bands from EVs and WCP preparations. (B) Protein sequences from LC-MS analysis of the vesicle proteome analyzed by an in silico 2-DE tool. Blue spots indicate proteins with transmembrane domains.

Fig 3. Gene Ontology analysis of the proteome of G. adiacens EVs preparations.

Gene ontology annotation was achieved using OmicsBox and an online software “WEGO”. Protein sequences were grouped into 3 categories based on their properties and functions.

Fig 4. Functional protein association networks of G. adiacens EVs proteome.

The online tool STRING was used for grouping the EVs proteins based on functional networks. Minimum interaction scores were set at a strong confidence level of 0.7. The three major network groups formed are shown in dotted circles. Seven different colors link a number of nodes and represent seven types of evidence used in predicting associations. A red line indicates the presence of fusion evidence; a green line represents neighborhood evidence; a blue line represents co-occurrence evidence; a purple line represents experimental evidence; a yellow line represents text mining evidence; a light blue line represents database evidence and a black line represents co-expression evidence.

Fig 5. KEGG pathway analysis of G. adiacens EVs proteome.

All protein sequences from G. adiacens vesicle proteome were subject to KEGG pathway analysis using the genus “streptococcus” as reference.

Fig 6

ELISA quantification of IL-8 (A), IL-1β (B), and CCL5 (C) production by human PBMCs stimulated with G. adiacens EVs and WCP (10, 25, 50, and 100 μg/ml). Cytokine induction from the EVs was considered significantly different from WCP at *p < 0.05.