Metatranscriptomic analysis of an in vitro biofilm model reveals strain-specific interactions among multiple bacterial species

Abstract

Interactions among bacteria can affect biofilm properties. Method: Here, we investigated the role of different bacteria in functional dysbiosis of an in vitro polymicrobial subgingival plaque model using both 16S rRNA and metatranscriptomic sequencing. Results: We found that high-virulence Porphyromonas gingivalis W83 had greater effects on the symbiotic species than the low-virulence P. gingivalis ATCC33277, and that Prevotella intermedia exacerbated the effects of W83. P. gingivalis significantly influenced the expression of genes related to metabolic pathways and quorum sensing of commensal oral species in a strain-specific manner. P. intermedia exerted synergistic effects with P. gingivalis W83 but antagonistic effects with strain ATCC33277, which may regulate the expression of virulence factors of P. gingivalis through the clp regulator. Discussion: The interaction networks indicated that the strongest correlation was between Fusobacterium nucleatum and Streptococcus mitis, which demonstrated their bridge and cornerstone roles in biofilm. Changes in the expression of genes relating to outer membrane proteins in F. nucleatum indicated that the addition of different bacteria can interfere with the co-adherence among F. nucleatum and other partners. Conclusion: We report here the existence of strain-specific interactions in subgingival plaque, which may enhance our understanding of periodontal micro-ecology and facilitate the development of improved plaque control strategies.

Keywords: Biofilm; bacteria interaction; periodontal microbiology; periodontal pathogen; periodontitis.

Figure 1.

Dynamics of six bacterial communities of different compositions. (a) The diagram depicts the design of six bacterial communities (C1-C6) with different compositions; (b) The relative abundance of each strain in different communities (C1-C6) at different time points (0, 1^st, 5^th and 9^th day) assessed by 16S rRNA sequencing analysis.

Figure 2.

The significantly differential expressed genes (DEGs) of S. mitis (a-f), F. nucleatum (g-l), P. gingivalis ATCC33277 (m), P. gingivalis W83 (n) and P. intermedia (o) between communities. Blue spots represent higher expression genes in a community labeled on y-axis; while red spots represent higher expression genes in a community labeled on x-axis. Differences were considered significant at a q-value ≤0.05 and fold-change ≥2.

Figure 3.

(a) The top 12 KEGG pathways of DEGs in S. mitis, F. nucleatum, P. gingivalis ATCC33277, P. gingivalis W83 and P. intermedia between communities (C2 vs C6; C3 vs C6; C4 vs C2; C5 vs C3; C5 vs C4). The y-axis shows the top 12 most abundant pathway categories. Red and blue colors represent up- and down-regulated genes, respectively. (b,c) Pathways that were specially altered in F. nucleatum by different strains. The upward arrows in red and downward arrows in blue represented up- and down-represented pathways or genes, respectively; the number of arrows represents the degree of change.

Figure 4.

The gene-level interaction network among F. nucleatum (F.n. blue spots), S. mitis (S.m. red spots) and A. naeslundii (A.n. green spots). The schematic diagram depicts the most abundant gene functional categories involved in the F. nucleatum-S. mitis interaction (grey circle).

Figure 5.

The differential expressed OMPs-related genes of F. nucleatum in each community. The color bar shows log2 fold change between two communities; * Differences were considered significant at a q-value ≤0.05 and fold-change ≥2.

Figure 6.

Model for P. intermedia regulating the expression of P. gingivalis virulence through the Clp regulator. Clp, a c-di-GMP effector that connects virulence to c-di-GMP modulation systems, i.e. RpfC/RpfG (pathway: KO 02024). The quorum sensing system may influence virulence gene expression through control of the intracellular level of Clp (crp, K10914). P. intermedia can upregulate the expression level of Clp in P. gingivalis W83, but down-regulates that in P. gingivalis ATCC33277.