TrkA serves as a virulence modulator in Porphyromonas gingivalis by maintaining heme acquisition and pathogenesis

Abstract

Periodontitis is an inflammatory disease of the supporting tissues of the teeth, with polymicrobial infection serving as the major pathogenic factor. As a periodontitis-related keystone pathogen, Porphyromonas gingivalis can orchestrate polymicrobial biofilm skewing into dysbiosis. Some metatranscriptomic studies have suggested that modulation of potassium ion uptake might serve as a signal enhancing microbiota nososymbiocity and periodontitis progression. Although the relationship between potassium transport and virulence has been elucidated in some bacteria, less is mentioned about the periodontitis-related pathogen. Herein, we centered on the virulence modulation potential of TrkA, the potassium uptake regulatory protein of P. gingivalis, and uncovered TrkA as the modulator in the heme acquisition process and in maintaining optimal pathogenicity in an experimental murine model of periodontitis. Hemagglutination and hemolytic activities were attenuated in the case of trkA gene loss, and the entire transcriptomic profiling revealed that the trkA gene can control the expression of genes in relation to electron transport chain activity and translation, as well as some transcriptional factors, including cdhR, the regulator of the heme uptake system hmuYR. Collectively, these results link the heme acquisition process to the potassium transporter, providing new insights into the role of potassium ion in P. gingivalis pathogenesis.

Keywords: Porphyromonas gingivalis; heme acquisition; high-throughput sequencing; pathogenicity; potassium ion uptake regulatory protein.

Figure 1

Transcriptional profiling of Porphyromonas gingivalis ΔtrkA versus the wild-type (WT) strain. (A) Principal component analysis of P. gingivalis ΔtrkA versus the WT strain shows a clear divergence. (B) Volcano plot of differentially expressed genes. (C) qRT-PCR analysis of a subset of differentially expressed genes detected by RNA sequencing. Gene ontology enrichment analysis of (D) differentially expressed genes, (E) upregulated genes, and (F) downregulated genes in the P. gingivalis ΔtrkA strain. Data are presented as means ± SD, normalized to 16S rRNA and compared to WT (2^−ΔΔCT). N = 3, **p< 0.01.

Figure 2

trkA deletion attenuates hemagglutination activity of Porphyromonas gingivalis. (A) Hemagglutination activity of P. gingivalis WT, ΔtrkA, and ΔtrkA/trkA strains. (B) qRT-PCR analysis of hagA, kgp, rgpA, and hbp35 mRNA expression in P. gingivalis WT, ΔtrkA, and ΔtrkA/trkA strains. Data are presented as means ± SD, normalized to 16S rRNA and compared to WT (2^−ΔΔCT). N = 3, ****p< 0.001.

Figure 3

Loss of trkA leads to membrane potential-related hemolysis activity alteration. (A, B) Hemolytic assay, (C, D) membrane potential determination, and (E, F) Kgp and Rgp activities of Porphyromonas gingivalis WT, ΔtrkA, and ΔtrkA/trkA strains. N = 3, *p< 0.05, **p< 0.01.

Figure 4

Heme-bound capacity of Porphyromonas gingivalis is not altered except for the corresponding transcripts. (A) Heme-bound assay. qRT-PCR of (B) hmuYR and (C) ihtAB and husAB mRNA expression of P. gingivalis WT, ΔtrkA, and ΔtrkA/trkA strains. Data are presented as means ± SD, normalized to 16S rRNA and compared to WT (2^−ΔΔCT). N = 3, *p< 0.05, **p< 0.01.

Figure 5

trkA is required for the virulence of Porphyromonas gingivalis in vivo. Micro-CT analysis of alveolar bone loss in mice following infection with P. gingivalis wild-type and ΔtrkA strains. The group treated with CMC (sham) worked as a control. (A) Three-dimensional micro-CT reconstruction of the alveolar bone and (B) analysis of bone resorption. All data of the distance between ABC and CEJ in 14 sites of the first, second, and third molars were measured and presented as mean ± SD. ABC, alveolar bone crest; CEJ, cementoenamel junction. N = 10 or 11, ****p< 0.001.