Phylogenetic and functional gene structure shifts of the oral microbiomes in periodontitis patients

Abstract

Determining the composition and function of subgingival dental plaque is crucial to understanding human periodontal health and disease, but it is challenging because of the complexity of the interactions between human microbiomes and human body. Here, we examined the phylogenetic and functional gene differences between periodontal and healthy individuals using MiSeq sequencing of 16S rRNA gene amplicons and a specific functional gene array (a combination of GeoChip 4.0 for biogeochemical processes and HuMiChip 1.0 for human microbiomes). Our analyses indicated that the phylogenetic and functional gene structure of the oral microbiomes were distinctly different between periodontal and healthy groups. Also, 16S rRNA gene sequencing analysis indicated that 39 genera were significantly different between healthy and periodontitis groups, and Fusobacterium, Porphyromonas, Treponema, Filifactor, Eubacterium, Tannerella, Hallella, Parvimonas, Peptostreptococcus and Catonella showed higher relative abundances in the periodontitis group. In addition, functional gene array data showed that a lower gene number but higher signal intensity of major genes existed in periodontitis, and a variety of genes involved in virulence factors, amino acid metabolism and glycosaminoglycan and pyrimidine degradation were enriched in periodontitis, suggesting their potential importance in periodontal pathogenesis. However, the genes involved in amino acid synthesis and pyrimidine synthesis exhibited a significantly lower relative abundance compared with healthy group. Overall, this study provides new insights into our understanding of phylogenetic and functional gene structure of subgingival microbial communities of periodontal patients and their importance in pathogenesis of periodontitis.

Figure 1

Circular maximum likelihood phylogenetic tree at the genus level. The tree was constructed with MEGA 5 using neighbor-joining method with a bootstrap value of 1000 and displayed using iTOL (Letunic and Bork, 2011). The bars in the outer band represent the relative abundance of bacterial genus in the healthy (blue) and the periodontal disease (red) groups.

Figure 2

Differences between healthy (H) and periodontitis (P) subjects at the phylum, genus and OTU levels. (a) Phylum level. (b) Genus level. The genera with average relative abundance >0.4% in the periodontitis group are present. (c) Difference of average OTUs in each phylum; *P<0.05 and **P<0.001.

Figure 3

Hierarchical clustering analysis of all detected genes in all 37 samples. Results were generated by CLUSTER and visualized by TREEVIEW. Red indicates signal intensities above background, whereas black indicates signal intensities below background. Brighter red coloring indicates higher signal intensities. The groups 1–6 contained functional genes from different functional gene families, but showed different patterns between healthy and diseased groups. The y axes in the right panel indicate the sum of normalized signal intensity of each group of genes detected. H, healthy control; MP, moderate periodontitis; SP, severe periodontitis.

Figure 4

Shifts of functional genes in periodontal communities for key metabolic pathways. For each pathway, first, the relative abundance of genes between healthy controls (H) and moderate periodontitis (MP) or severe periodontitis (SP) was compared with analysis of variance (ANOVA; α=0.05). Then, the numbers of genes with statistical difference were counted. The bars represented the percentage (%) of these genes in total genes in this category and * indicated more than 40% genes altered in this category.

Figure 5

The normalized signal intensity of virulence genes. The signal intensity for each functional gene was the average of the total signal intensity from all the replicates. All data are presented as mean±s.e. (*P<0.05). Half of virulence genes in periodontitis (P) had a higher abundance than those in healthy (H) subjects.

Figure 6

The normalized signal intensity of genes involved in amino acid metabolism and synthesis. All data are presented as mean±s.e. and genes with no significant difference are not shown (P>0.05). Bars on the left of y axis represent genes with lower relative abundance in periodontitis, whereas bars on the right indicate genes with higher abundance in periodontitis. Out of 22 genes involved in amino acid metabolism, 9 showed distinct abundances between periodontitis (P) and healthy (H) subjects. As for amino acid synthesis, 19 out of 41 genes changed and 15 of these genes showed lower relative abundance in the periodontitis group.

Figure 7

The normalized signal intensity of genes involved in glycosaminoglycan degradation, pyrimidine metabolism and pyrimidine biosynthesis. The signal intensity for each functional gene was the average of the total signal intensity from all replicates. All data were presented as mean±s.e. *P<0.05. G-4-sulfohydrolase, N-acetyl-D-galactosamine-4-sulfate 4-sulfohydrolase (involved in chondroitin sulfate biosynthesis); G-6-sulfohydrolase, N-acetyl-D-glucosamine-6-sulfate 6-sulfohydrolase (involved in keratan sulfate biosynthesis); H-N-acetylhexosaminohydrolase, β-N-acetyl-D-hexosaminide N-acetylhexosaminohydrolase (involved in keratan sulfate biosynthesis).