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. 2015 Jul 28;10(7):e0134234.
doi: 10.1371/journal.pone.0134234. eCollection 2015.

Oral Administration of P. gingivalis Induces Dysbiosis of Gut Microbiota and Impaired Barrier Function Leading to Dissemination of Enterobacteria to the Liver

Mayuka Nakajima  1 , Kei Arimatsu  1 , Tamotsu Kato  2 , Yumi Matsuda  1 , Takayoshi Minagawa  1 , Naoki Takahashi  1 , Hiroshi Ohno  3 , Kazuhisa Yamazaki  4
Affiliations

Oral Administration of P. gingivalis Induces Dysbiosis of Gut Microbiota and Impaired Barrier Function Leading to Dissemination of Enterobacteria to the Liver

Mayuka Nakajima et al. PLoS One. .

Abstract

Although periodontitis has been implicated as a risk factor for various systemic diseases, the precise mechanisms by which periodontitis induces systemic disease remain to be elucidated. We have previously revealed that repeated oral administration of Porphyromonas gingivalis elicits endotoxemia via changes in the gut microbiota of the ileum, and thereby induces systemic inflammation and insulin resistance. However, it is not clear to what extent a single administration of P. gingivalis could affect gut microbiota composition, gut barrier function, and subsequent influx of gut microbiota into the liver. Therefore, in the present study, C57BL/6 mice were orally administered P. gingivalis (strain W83) once and compared to sham-inoculated mice. The phylogenetic structure and diversity of microbial communities in the gut and liver were analyzed by pyrosequencing the 16S ribosomal RNA genes. Serum endotoxin activity was determined by a Limulus amebocyte lysate test. Gene expression in the intestine and expression of 16S rRNA genes in the blood and liver were examined by quantitative polymerase chain reaction. Administration of P. gingivalis significantly altered gut microbiota, with an increased proportion of phylum Bacteroidetes, a decreased proportion of phylum Firmicutes, and increased serum endotoxin levels. In the intestinal tissues, gene expression of tjp-1 and occludin, which are involved in intestinal permeability, were downregulated. Higher amounts of bacterial DNA were detected in the liver of infected mice. Importantly, changes in gut microbiota preceded systemic inflammatory changes. These results further support the idea that disturbance of the gut microbiota composition by orally derived periodontopathic bacteria may be a causal mechanism linking periodontitis and systemic disease.

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Conflict of interest statement

Competing Interests: K. Yamazaki received a grant from SUNSTAR Inc. but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. No further competing interests exist.

Figures

Fig 1
Fig 1. Comparison of the gut microbiota between P. gingivalis-administered and sham-administered mice by 16S rRNA sequencing analysis (N = 6 in the experimental group, N = 7 in the control group).
(A) Relative abundances of each bacterial group at the phylum level at 24 hrs and 48 hrs after P. gingivalis administration are indicated by box plots. (*p < 0.05, **p < 0.01, Mann-Whitney U-test). (B) Relative abundance of each bacterial group at the genus level were significantly increased or decreased are shown. Each assigned taxonomy obtained from 16S rRNA deep sequencing was compared to Silva database using UCLUST. (*p < 0.05, **p < 0.01, Mann-Whitney U-test).
Fig 2
Fig 2. Sequencing analysis of the gut microbiota obtained from P. gingivalis-administered and sham-administered mice.
(A) Diversity of bacterial species as indicated by Chao1 rarefaction measure. (B) PCoA of fecal microbiota from P. gingivalis-administered and sham-administered mice. (Unweighted distance: R = 0.2646, p < 0.01; Weighted distance: R = -0.0251, p = 0.018, ANOSIM test).
Fig 3
Fig 3. Effect of oral administration of P. gingivalis on gut barrier function and endotoxemia.
(A) Quantitative PCR analysis of tight junction genes in the small intestine of P. gingivalis-administered mice and sham-administered mice at 24 hrs and 48 hrs after P. gingivalis administration. The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (N = 7 in each group). (B) Relative abundance of bacteria in the liver and blood of P. gingivalis-administered mice and sham-administered mice (N = 7 in each group). (C) Serum endotoxin (LPS) concentration (EU/mL) were determined after P. gingivalis administration or sham administration (N = 7 in each group). (*p < 0.05, **p < 0.01, Mann-Whitney U-test).
Fig 4
Fig 4. Effect of oral administration of P. gingivalis on the gene expression of the gut.
(A) Relative gene expression levels in the small intestine of P. gingivalis-administered and sham-administered mice (N = 7 in each group). (B) Relative gene expression levels in the large intestine of P. gingivalis-administered and sham-administered mice (N = 7 in each group). The relative mRNA expressions of the genes of interest were normalized to the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. (*p < 0.05, Mann-Whitney U-test).
Fig 5
Fig 5. Comparison of the liver microbiota in P. gingivalis-administered and sham-administered mice by 16S rRNA sequencing analysis.
(A) Relative abundances of each bacterial group at the phylum level are indicated by the box plots. (*p < 0.05, **p < 0.01, Mann-Whitney U-test). (B) Relative abundance of each bacterial group at the genus level were significantly increased or decreased are shown. Each assigned taxonomy obtained from 16S rRNA deep sequencing was compared to Silva database using UCLUST. (*p < 0.05, **p < 0.01, Mann-Whitney U-test).
Fig 6
Fig 6. Sequencing analysis of the liver microbiota obtained from P. gingivalis-administered and sham-administered mice.
(A) Diversity of bacterial species as indicated by Chao1 rarefaction measure (*p < 0.05). (B) PCoA of fecal microbiota from P. gingivalis-administered and sham-administered mice. (Unweighted distance: R = 0.3638, p < 0.01; Weighted distance: R = 0.4497, p = 0.541, ANOSIM test).
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This work was supported by the Japan Society for the Promotion of Science KAKENHI 23390476, 25670882, and 15H02578 (KY); The Japan Science and Technology Agency (HO); and Sunstar Inc. (KY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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