Potassium is a key signal in host-microbiome dysbiosis in periodontitis

Abstract

Dysbiosis, or the imbalance in the structural and/or functional properties of the microbiome, is at the origin of important infectious inflammatory diseases such as inflammatory bowel disease (IBD) and periodontal disease. Periodontitis is a polymicrobial inflammatory disease that affects a large proportion of the world's population and has been associated with a wide variety of systemic health conditions, such as diabetes, cardiovascular and respiratory diseases. Dysbiosis has been identified as a key element in the development of the disease. However, the precise mechanisms and environmental signals that lead to the initiation of dysbiosis in the human microbiome are largely unknown. In a series of previous in vivo studies using metatranscriptomic analysis of periodontitis and its progression we identified several functional signatures that were highly associated with the disease. Among them, potassium ion transport appeared to be key in the process of pathogenesis. To confirm its importance we performed a series of in vitro experiments, in which we demonstrated that potassium levels a increased the virulence of the oral community as a whole and at the same time altering the immune response of gingival epithelium, increasing the production of TNF-α and reducing the expression of IL-6 and the antimicrobial peptide human β-defensin 3 (hBD-3). These results indicate that levels of potassium in the periodontal pocket could be an important element in of dysbiosis in the oral microbiome. They are a starting point for the identification of key environmental signals that modify the behavior of the oral microbiome from a symbiotic community to a dysbiotic one.

Fig 1. Statistical differences in metatranscriptome composition after addition of ion potassium.

Phylogenetic assignment of the mRNA hits was performed using Kraken [17] and were analyzed using LEfSe with default parameters (p-value < 0.05 for Kruskal-Wallis rank sum test on classes and pairwise Wilcoxon test between subclasses of different classes) to identify significant differences in transcription activity at species level between the microbial communities compared. A) Cladogram showing the taxonomic distribution of lineages whose expression levels had a LDA value of 3.0 or higher as determined by LEfSe [18]. B) Histogram of LDA scores for differentially active taxa. In red are taxa whose activity, as determined by number of transcripts, was increased in the presence of K⁺. In green are species whose activity was higher in the absence of K⁺.

Fig 2. GO enrichment analysis comparing plaque response to the presence and absence of added ion potassium to the medium.

Enriched terms obtained using GOseq were summarized and visualized as a scatter plot using REVIGO. Only GO terms with FDR adjusted p-value < 0.05 in the 'GOseq' analysis were used. A) Summarized GO terms related to biological processes after addition of K⁺. B) Summarized GO terms related to biological processes with no K⁺ added. Circle size is proportional to the frequency of the GO terms, color indicates the log10 p-value (red higher, blue lower). Distance between circles represent GO terms' semantic similarities. Each of the circles represent a GO term, which depending on the similarity in the terms included in them they will be closer or more distant in the graph.

Fig 3. Effect of potassium concentration on hemolytic activity on dental plaque growing on blood agar plates.

Hemolysis was assayed on horse blood agar plates with different concentrations of K⁺ added and incubated under anaerobic conditions at 37°C. A) Hemolytic activity of whole dental plaque on different concentrations of K⁺ added after 6 days of incubation in 2 different biological experiments. B) Principal component analysis of HOMINGs phylogenetic profiles of the final microbial communities that grew on blood agar plates from 3 different healthy individuals, the initial inocula. Subject 1 was analyzed at 2 different times (see Fig 3a).

Fig 4. Effect of potassium concentration on hemolytic activity of different bacterial strains.

A) Hemolytic activity of supernatants from P. nigrescens ATCC 33563 and S. mitis NCTC 1226, as the percentage of lysis of horse erythrocytes with respect to a positive control (100% of activity). Results are from 4 biological replicates for each concentration. B) Hemolytic activity on agar plates with different concentrations of K⁺ added of P. nigrescens ATCC 33563 after 48 hours of incubation. C) Hemolytic activity on agar plates with different concentrations of K⁺ added of P. nigrescens ATCC 33563 after 6 days of incubation.

Fig 5. Effect of potassium concentration on gingival cytokine expression.

A three-dimensional multilayered gingival tissue model with cornified apical layers (EpiGingival, MatTek Corporation) was used to assess the effect of different concentrations of K⁺ and bacteria on the profiles of expression of different cytokines. A) Heatmap of cytokine expression measured by Luminex under different K⁺ concentrations and presence or absence of bacteria from dental plaque. B) Box plot showing the values of observed concentrations in the media of the different cytokines assayed.

Fig 6. Effect of potassium concentration on expression of human β-defensin 3 (hBD-3) in gingival tissue.

A) Immunohistochemistry examples of hBD-3 expression in the three-dimensional (3D) gingival tissue model used for the experiments. i) Negative control, using a non-immunized primary antibody, 0mM K⁺ added and no plaque, ii) 0mM K⁺ added in the absence of plaque, which gave the highest levels of hBD-3 expression, iii) 5mM K⁺ added in the presence of plaque, which gave the lowest levels of hBD-3 expression. B) Box plot showing results of the ratio of hBD-3/DAPI fluorescence measured by immunohistochemistry. The ratio represents a normalized value of hBD-3 expression (see Methods); n: number of different histological sections analyzed. Scale bars = 25 μm.