Metabolic crosstalk regulates Porphyromonas gingivalis colonization and virulence during oral polymicrobial infection

Abstract

Many human infections are polymicrobial in origin, and interactions among community inhabitants shape colonization patterns and pathogenic potential ¹ . Periodontitis, which is the sixth most prevalent infectious disease worldwide ² , ensues from the action of dysbiotic polymicrobial communities ³ . The keystone pathogen Porphyromonas gingivalis and the accessory pathogen Streptococcus gordonii interact to form communities in vitro and exhibit increased fitness in vivo ^3,4 . The mechanistic basis of this polymicrobial synergy, however, has not been fully elucidated. Here we show that streptococcal 4-aminobenzoate/para-amino benzoic acid (pABA) is required for maximal accumulation of P. gingivalis in dual-species communities. Metabolomic and proteomic data showed that exogenous pABA is used for folate biosynthesis, and leads to decreased stress and elevated expression of fimbrial adhesins. Moreover, pABA increased the colonization and survival of P. gingivalis in a murine oral infection model. However, pABA also caused a reduction in virulence in vivo and suppressed extracellular polysaccharide production by P. gingivalis. Collectively, these data reveal a multidimensional aspect to P. gingivalis-S. gordonii interactions and establish pABA as a critical cue produced by a partner species that enhances the fitness of P. gingivalis while diminishing its virulence.

Figure 1

Exogenous pABA enhances P. gingivalis community formation with S. gordonii. a, P. gingivalis (green) was reacted with a substratum of S. gordonii DL-1 parental strain (WT) or Δcbe mutant (red) for 18 h without or with pABA at the concentrations indicated. A series of 20–30 μm-deep optical fluorescent x-y sections were collected by confocal microscopy to create digitally reconstructed 3D images with Imaris software. Images are representative of 6 independent experiments. Bar is 25 μm. b, Ratio of P. gingivalis:S. gordonii biovolume in images represented in (a) measured with the Imaris Isosurface function. Data are representative of 6 independent experiments and are presented with mean with standard deviation of 5 random fields from one experiment. c, Numbers of P. gingivalis in dual species communities with S. gordonii under conditions described in (a) determined by qPCR. Results are means with standard deviation from one of three independent experiments performed in triplicate. For (b) and (c) * – P < 0.05, ** – P < 0.01 compared to WT without pABA using ANOVA with Tukey’s multiple comparison test.

Figure 2

pABA increases expression of P. gingivalis effectors of community development. a, b, d–f. qRT-PCR of mRNA extracted from P. gingivalis reacted for 2 h with pABA at the concentrations indicated or with secreted components of S. gordonii WT or Δcbe in a membrane insert transwell. 16S rRNA was used for normalization. Data are expressed as fold difference (or −1/ratio for negative fold change) relative to P. gingivalis incubated with vehicle (DMSO) alone (for pABA conditions) or P. gingivalis incubated with medium only in the transwell (for S. gordonii conditions). ND: no difference. *** – P < 0.001 using ANOVA with Tukey’s multiple comparison test. Data are means with standard deviation and representative of 3 biological replicates performed in triplicate. c, Immunoblots of cell lysates of P. gingivalis reacted with pABA for 18 h at the concentrations indicated, DMSO, or PBS alone (C) and probed with antibodies to Mfa1, FimA or GppX as a control. Data are representative of 3 independent experiments.

Figure 3

Trans-omics of pABA effects of P. gingivalis on the tetrahydrofolate metabolic pathway. Red, blue and yellow short arrows represent proteins up-, down-, or non-regulated by pABA respectively (see Methods for statistical thresholds). * represents a non-detect in the proteome but down regulated at the transcriptional level. Ϯ represents a protein unchanged in the proteome but upregulated at the transcriptional level. Red and blue connecting arrows represent functionally predicted up- and down-regulation respectively. Blue font denotes PLP-dependent enzymes whose functions were suppressed as shown in the metabolome (Supplementary Table 4). Red and blue boxes represent increased and decreased metabolite production respectively.

Figure 4

Effects of pABA on P. gingivalis in vivo and expression of extracellular polysaccharide. a, Colonization of the murine oral cavity. P. gingivalis (10⁸) was reacted with pABA 1 mg/mL, or with DMSO (control) and inoculated into the oral cavity of Balb/c mice. One, 2 or 3 weeks (w) after the final inoculation, P. gingivalis was enumerated by qPCR. Each symbol represents an individual mouse and the short horizontal lines indicate the mean. One representative experiment of three is shown. b, pABA diminishes virulence of P. gingivalis in an alveolar bone loss model. P. gingivalis was inoculated orally as described in (a). Control mice were sham infected with pABA. Mice were given pABA (0.1 mg/mL) or vehicle only ad libitum in drinking water. Bone loss is expressed as mean distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) of 14 maxillary molar sites after 42 days. Each symbol represents an individual mouse and the short horizontal lines indicate the mean. One representative experiment of two is shown. c, P. gingivalis was reacted with pABA at the concentrations indicated for 18 h and extracellular polysaccharide was stained with FITC-labeled concanavalin A and wheat germ agglutinin. Bacterial cells were stained with Syto-17. The ratio of lectin binding (green) to whole cell staining (red) in confocal images was determined using Volocity software. Data are means with standard deviation of 5 random fields from one representative experiment of three. For a–c, data were analyzed with a two-tailed t-test. d, Kaplan-Meier plot showing survival of mice following subcutaneous inoculation of P. gingivalis, S. gordonii, or combinations of P. gingivalis with S. gordonii WT or Δcbe (5 x 10⁹ total bacteria). n=10 mice per group and comparison between groups was by the log-rank test. One representative experiment of two is shown. e,f, qRT-PCR of fimA (e) or mfa1 (f) expression in abscess material from groups described in (d) (except with 2.5 x 10⁹ total bacteria). Each symbol represents a single animal and the short horizontal lines indicate the mean. One representative experiment of two, performed in triplicate, is shown. Data were analyzed by a two-tailed t-test. For all panels * - P < 0.05, ** - P < 0.01, *** - P < 0.001.