Coaggregated E. faecalis with F. nucleatum regulated environmental stress responses and inflammatory effects

Abstract

To investigate the cell-cell interactions of intergeneric bacterial species, the study detected the survival of Enterococcus faecalis (Ef) under monospecies or coaggregation state with Fusobacterium nucleatum subsp. polymorphum (Fnp) in environmental stress. Ef and Fnp infected the human macrophages with different forms (Ef and Fnp monospecies, Ef-Fnp coaggregates, Ef + Fnp cocultures) for exploring the immunoregulatory effects and the relevant molecular mechanisms. Meanwhile, the transcriptomic profiles of coaggregated Ef and Fnp were analyzed. Ef was shown to coaggregate with Fnp strongly in CAB within 90 min by forming multiplexes clumps. Coaggregation with Fnp reinforced Ef resistance against unfavorable conditions including alkaline, hypertonic, nutrient-starvation, and antibiotic challenges. Compared with monospecies and coculture species, the coaggregation of Ef and Fnp significantly facilitates both species to invade dTHP-1 cells and aid Ef to survive within the cells. Compared with coculture species, dual-species interaction of Ef and Fnp significantly decreased the levels of pro-inflammatory cytokines IL-6, TNF-α, and chemokines MCP-1 secreted by dTHP-1 cells and lessened the phosphorylation of p38, JNK, and p65 signaling pathways. The transcriptome sequencing results showed that 111 genes were differentially expressed or Ef-Fnp coaggregated species compared to Ef monospecies; 651 genes were differentially expressed for Fnp when coaggregation with Ef. The analysis of KEGG pathway showed that Ef differentially expressed genes (DEGs) were enriched in quorum sensing and arginine biosynthesis pathway; Fnp DEGs were differentially concentrated in lipopolysaccharide (LPS) biosynthesis, biofilm formation, and lysine degradation pathway compared to monospecies. KEY POINTS: • Coaggregated with Fnp aids Ef's survival in environmental stress, especially in root canals after endodontic treatment. • The coaggregation of Ef and Fnp may weaken the pro-inflammatory response and facilitate Ef to evade killed by macrophages. • The coaggregation between Ef and Fnp altered interspecies transcriptional profiles.

Keywords: Enterococcus faecalis; Fusobacterium nucleatum; Coaggregation; Host-pathogen interaction; Immunoregulatory effects; Transcriptomics.

Fig. 1

The coaggregation index of dual-species (Ef-Fnp) or autoaggregation index of monospecies (Ef or Fnp) in CAB for 10 min to 90 min. The results shown were the coaggregation or autoaggregation index in each group. % coaggregation = [(OD600nm(Ef) +OD600nm(Fnp)-OD600nm(Ef-Fnp))/(OD600nm(Ef)+OD600nm(Fnp))]×100%. % autoaggregation = [(OD600nm(time zero value)-OD600nm(sample value))/OD600nm(time zero value)]×100%. OD600nm(Ef), OD600nm (Fnp), and OD600nm(Ef-Fnp) represents the OD600nm of Ef monoculture, Fnp monoculture, and Ef-Fnp coaggregates supernatant at the same time point. OD600nm(time zero value) represents the OD600nm of each bacterial supernatant stilled at 0 min, and OD600nm(sample value) represents the OD600nm of collected supernatant at different time points. Data represented the mean ± S.D. (standard deviation) of three independent assays. The arrow indicates the time points selected for RNA-Seq

Fig. 2

CLSM images of the coculture and coaggregation strains of HI-labeled Ef (red) and CFSE-labeled Fnp (green). (A) coculture group (Ef+Fnp) at low magnification. (B) coaggregation group (Ef-Fnp) at low magnification. (C) coaggregation group (Ef-Fnp) at high magnification. All white arrows showed the coaggregated Ef-Fnp

Fig. 3

Survival of Ef in monoculture or coaggregates under various environmental stresses. (A) Ef’s survival in different alkaline conditions. (B) Ef’s survival in hyperosmosis environment. (C) Ef’s tolerance to the low-nutrient environment. (D) Ef’s survival in culture medium supplemented with antibiotics CXM. (E) Survival of Ef in culture medium supplemented with bacteriostatic agent CHX is indicated as Log (CFU/mL)

Fig. 4

The colony counts of bacterial engulfed by dTHP-1 cells and intracellular survival. (A) The number of Ef phagocytosed by dTHP-1 cells. (B) The number of Fnp phagocytosed by dTHP-1 cells. (C) The intracellular survival rate of Ef. (D) The intracellular survival rate of Fnp. Data showed the mean ± S.D. of three independent experiments

Fig. 5

Ultrastructural changes of dTHP-1 cells under TEM. (A) dTHP-1 cells infected with Ef monoculture. (B) dTHP-1 cells infected with Fnp monoculture. (C) dTHP-1 cells infected with Ef-Fnp coaggregates. (D) dTHP-1 cells infected with Ef+Fnp cocultures. (E) Control dTHP-1 cells without bacterial infection. The yellow hollow arrow indicated the Ef; the white hollow arrow represented Fnp in cross-section; the white solid arrow showed Fnp in longitudinal section; the yellow solid hollow arrow displayed typical Ef-Fnp adhesins; and the white triangle arrow represented abundant mitochondria structures

Fig. 6

The effects of Ef-Fnp coaggregation on dTHP-1 cells proliferation and apoptosis. (A) The proliferation of dTHP-1 cells infected by bacteria in various forms after 2, 6, 24, and 48 h of infection. (B) Flow cytometric analysis of dTHP-1 cells apoptosis induced by bacteria in different forms after 2, 6, 24, and 48 h of infection. (C) The graph showed the quantitative analysis of apoptosis

Fig. 7

The expression of inflammatory factors and activation of signaling pathways in dTHP-1 cells infected by bacteria in various forms. (A) The secretion of IL-6, TNF-α, and MCP-1 by dTHP-I cells. (B) The representative western blot images of protein production in dTHP-1 cells. (C) The semi-quantitative analysis of western blotting. Data showed the mean ± S.D. of three independent experiments

Fig. 8

Volcano plots showed DEGs of Ef (A) and Fnp (B) in response to coaggregation. The x-axis displayed the log2 (Fold change) values of individual genes, and the y-axis represented the negative logarithm of the P-value to base 10. DEGs were colored red (up-regulated) and blue (down-regulated)

Fig. 9

Functional enrichment analysis of DEGs between Ef monoculture and EF-Fnp coaggregates. (A) GO functional classification. (B) GO term enrichment analysis. (C) KEGG pathways categories. (D) Bubble chart of KEGG pathway enrichment analysis. (E) PPI network analysis. Each node showed a gene. Red arrows beside the gene symbols meant gene up-regulation; blue arrows represented down-regulation of the gene

Fig. 10

Functional enrichment analysis of DEGs between Fnp monoculture and EF-Fnp coaggregates. (A) GO functional classification. (B) GO term enrichment analysis. (C) KEGG pathways categories. (D) Bubble map of KEGG pathway enrichment analysis. (E) PPI network analysis. Each node showed a gene. Red and blue arrows beside the gene symbols indicated gene up-regulation and down-regulation, respectively