Membrane Vesicles of Enterococcus faecalis: In Vitro Composition Analysis and Macrophage Inflammatory Response Under Different pH Conditions

Abstract

Enterococcus faecalis (E. faecalis) is one of the most detected bacteria in persistent apical periodontitis (PAP), with alkaline tolerance enabling post-treatment survival. In this study, we will investigate how alkaline conditions alter proteomic and metabolomic profiles of E. faecalis membrane vesicles (MVs) and preliminarily investigate the role of MVs of E. faecalis in the regulation of macrophage inflammatory response. E. faecalis MVs were characterized using transmission electron microscopy and nanoparticle tracking analysis under varying pH conditions. MVs' proteomic and metabolomic profiling across pH levels was compared. The effects of E. faecalis MVs on human dTHP-1 macrophages were evaluated using CCK-8 metabolic activity assays and ELISA-based quantitative analysis of inflammatory cytokines. In this study, the presence of E. faecalis MVs was verified, and the alkaline environment of pH 9.0 did not alter their production. Through proteomic and metabolomic analysis, we observed that ATP synthase and stress proteins, as well as lysine degradation and tryptophan metabolism pathways, were significantly enriched in the MVs at pH 9.0. Finally, we observed that both E. faecalis MVs at pH 7.0 and pH 9.0 could dose-dependently inhibit the activity of dTHP-1 cells. E. faecalis MVs promote the secretion of IL-6, TNF-α, IL-1β, IL-1ra, and TGF-β by macrophages. Compared to pH 7.0, pH 9.0 E. faecalis MVs have a reduced effect on IL-1ra and TGF-β secretion. Additionally, we observed a significant increase in the IL-1β/IL-1ra ratio after treatment with E. faecalis MVs. Our study indicated that E. faecalis can produce MVs in pH 7.0 and pH 9.0 environments. ATP synthase, stress proteins, as well as lysine degradation and tryptophan metabolism pathways, were significantly enriched in pH 9.0 MVs. Furthermore, E. faecalis MVs could promote inflammatory responses in macrophages and dose-dependently inhibit the viability of dTHP-1 cells.

Keywords: Enterococcus faecalis; immuno-inflammatory responses; macrophages; membrane vesicles; metabolomics; proteomics.

Figure 1

(A) BCA protein quantification analysis showed that the alkaline environment pH 9.0 did not alter the production of E. faecalis MVs. (B) The average particle size of MVs isolated at different pH conditions was examined by NTA, which showed that E. faecalis produces smaller sized vesicles in alkaline environment. n = 3. The experiment was repeated three times; * p < 0.05, ns: non-significant. (C–F) TEM investigation of MVs isolated at different pH conditions. (C) MVs isolated at pH 7.0, magnification ×40.0 K times. (D) MVs isolated at pH 7.0, magnification ×30.0 K times. (E,F) MVs isolated at pH 9.0, magnification ×40.0 K times.

Figure 2

(A) Venn diagram of MVs proteins at different pH conditions. (B) Subcellular structural localization of E. faecalis MVs proteins in alkaline environment by CELLO.

Figure 3

(A) Volcano plot of potential differences between MVs isolated at different pH conditions. The red dots represent proteins up-regulated in pH 9.0 MVs, and the blue dots represent proteins down-regulated, and grey represents proteins with no significant difference in expression between pH 7.0 and pH 9.0 environment. (B) Cluster analysis heatmap. Each column represents a set of samples (horizontal coordinates are sample information) and each row represents a protein (vertical coordinates are significantly different proteins), where red represents significantly up-regulated proteins, blue represents significantly down-regulated proteins, and white sections represent no protein quantification information.

Figure 4

(A) GO classification of differential proteins in pH 9.0 E. faecalis MVs. (B) GO enrichment pathway for differential proteins in pH 9.0 E. faecalis MVs. (C) KEGG pathway annotation map for differential proteins in pH 9.0 E. faecalis MVs. (D) KEGG pathway enrichment bubble map of differential proteins in pH 9.0 E. faecalis MVs.

Figure 5

(A) The quantification and proportional distribution of metabolites in E. faecalis MVs identified under the conditions of pH 7.0 and pH 9.0 across various chemical classification categories were analyzed. Metabolites lacking an identifiable chemical classification were categorized as “undefined”. Meanwhile, those chemical classification subsets harboring fewer than 10 metabolites were pooled and collectively denoted as “others”. (B,C) Heatmap of cluster analysis for DEMs of E. faecalis MVs in negative ion mode (B) and positive ion mode (C) under pH 9.0 conditions. In the graphical representation, each column is congruent to a particular group of samples, and every row symbolizes a single metabolite. Notably, within the context of the heatmap, the red hue is indicative of metabolites that have undergone significant up-regulation, whereas the blue shade denotes those metabolites with pronounced down-regulation. (D) Sankey bubble diagram of KEGG enrichment pathways for DEMs in E. faecalis MVs under pH 9.0 condition. The sankey diagram positioned on the left illustrates the specific enrichment pathways to which the DEMs pertain. Meanwhile, the bubble diagram situated on the right exhibits the Rich ratio (plotted on the X-axis), enrichment items (displayed on the Y-axis), the quantity of metabolites (indicated by the bubble size), and the p-value (depicted by the color). (E) Lollipop chart of KEGG enrichment pathways for DEMs in E. faecalis MVs under pH 9.0 condition. The Y-axis denotes the names of the pathways, while the X-axis signifies the differential abundance score (DA score). The DA score represents the comprehensive total variation of all metabolites within the pathway. When the score exceeds 0, it implies that the expression trends of all the identified metabolites in this pathway are up-regulated. Conversely, when the score is below 0, it indicates that the expression trends of all the identified metabolites in this pathway are down-regulated. The length of each line segment corresponds to the absolute value of the DA score. The size of the dot at the terminal end of the line segment reflects the quantity of metabolites in the pathway. Moreover, the darkness of the dot’s color is directly proportional to the magnitude of the DA score value. *** p < 0.001.

Figure 6

Effects of E. faecalis MVs on the secretion of IL-6 (A), TNF-α (B), IL-1β (C), IL-10 (D), IL-1ra (E), TGF-β (F), IL-6/IL-1ra ratio (G), and IL-1β/IL-1ra ratio (H) by dTHP-1 cells under different pH conditions. n = 3. The experiment was repeated three times; * p < 0.05, ** p < 0.01, *** p < 0.001, ns: non-significant.