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

Nature Microbiology volume 2, pages 1493–1499 (2017)Cite this article

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Abstract

Many human infections are polymicrobial in origin, and interactions among community inhabitants shape colonization patterns and pathogenic potential1. Periodontitis, which is the sixth most prevalent infectious disease worldwide2, ensues from the action of dysbiotic polymicrobial communities3. The keystone pathogen Porphyromonas gingivalis and the accessory pathogen Streptococcus gordonii interact to form communities in vitro and exhibit increased fitness in vivo3,4. 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.

Previous studies have established that both P. gingivalis and S. gordonii adapt to the community environment on a global scale10,11. Hence, we examined the impact of pABA on the proteome and metabolome of P. gingivalis. Qualitative detection of 1,153 P. gingivalis proteins (55% of the total theoretical proteome) was observed. On the basis of quantitative spectral counting, 322 proteins were significantly reduced and 207 were significantly increased, compared to the control with vehicle alone (Supplementary Table 1). The distribution of the regulated proteins in functional categories is shown in Supplementary Fig. 5. Upregulation of P. gingivalis FimA and Mfa1 structural and accessory fimbrial proteins by pABA was observed in the proteome (Supplementary Fig. 6), and this was further verified by enzyme-linked immunosorbent assay (ELISA; Supplementary Fig. 7a), and functionally through increased adherence to gingival epithelial cells (Supplementary Fig. 7b). In addition to adhesive fimbriae, proteases, haemin-uptake proteins, TonB-dependent proteins and Type IX secretion system components were upregulated (Supplementary Fig. 6), indicating that P. gingivalis also utilizes pABA as a cue to adapt to the protein- and haemin-rich subgingival environment. An increase in gingipain protease enzyme activity was observed in whole-cell lysates of pABA-treated P. gingivalis, but not on the cell surface or in the culture supernatant, indicating that secretion and/or processing had become rate-limiting (Supplementary Fig. 8). Expression of a number of stress-related proteins was decreased by pABA (Supplementary Fig. 6), suggesting that P. gingivalis has adapted for community dwelling with S. gordonii.

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Fig. 1: Exogenous pABA enhances P. gingivalis community formation with S. gordonii.
Fig. 2: pABA increases expression of P. gingivalis effectors of community development.
Fig. 3: Trans-omics of the effect of pABA on the tetrahydrofolate metabolic pathway in P. gingivalis.
Fig. 4: Effects of pABA on P. gingivalis in vivo and expression of extracellular polysaccharide.

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Acknowledgements

Supported by AMED-CREST, AMED, MEXT/JSPS KAKENHI grant numbers 15H05057, and 15K20642 (M.K.), NIH grants DE012505 and DE011111 (R.J.L.), DE023193 (M.W.) DP1 OD009572 (E.M.M.) and DE014372 (M.H.), Army Research Office Grant W911NF-12–1–0390 (E.M.M.), and the Welch Foundation grant F1515 (E.M.M.).

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Authors and Affiliations

  1. Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Masae Kuboniwa, Samar A. Alghamdi, Akito Sakanaka & Atsuo Amano

  2. AMED-CREST, Japan Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100-0004, Japan

    Masae Kuboniwa

  3. Institute for Cellular and Molecular Biology, and Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX, 78712, USA

    John R. Houser & Edward M. Marcotte

  4. Center for Microbial Proteomics and Chemical Engineering, University of Washington, Seattle, WA, 98195, USA

    Erik L. Hendrickson, Tiansong Wang, David A. C. Beck & Murray Hackett

  5. Department of Oral Immunology and Infectious Diseases, University of Louisville School of Dentistry, Louisville, KY, 40292, USA

    Qian Wang, Daniel P. Miller, Justin A. Hutcherson, Huizhi Wang & Richard J. Lamont

  6. Department of eScience, University of Washington, Seattle, WA, 98195, USA

    David A. C. Beck

  7. Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, 78712, USA

    Marvin Whiteley

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Contributions

M.K., S.A.A. and A.S. performed metabolomics and community experiments. J.R.H., E.L.H., T.W. and D.A.C.B. performed proteomics experiments. Q.W. and D.P.M. performed PCR, blots, ELISAs, protease and attachment assays. Q.W., J.A.H. and H.W. performed animal experiments. M.K., M.W., A.A., H.W., E.M.M., M.H. and R.J.L. designed the study and interpreted data. M.K., M.W., A.A., M.H. and R.J.L. wrote the manuscript.

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Correspondence to Richard J. Lamont.

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Supplementary Information

Supplementary Figures 1–10, Supplementary Tables 1 and 2, Supplementary Table 6

Supplementary Table 1

MS/MS identification of P. gingivalis proteins differentially regulated by pABA

Supplementary Table 3

Metabolomic profiles in P. gingivalis treated with pABA

Supplementary Table 4

Metabolites decreased by pABA-mediated suppression of PLP-dependent enzymes in P. gingivalis

Supplementary Table 5

Metabolite classification pathways

Supplementary Table 7

Raw protein spectral counts

Supplementary Table 8

Raw peptide spectral counts

Supplementary Table 9

Spectral counts of P. gingivalis treated with or without pABA normalized such that their averages are identical for identical biological replicates

Supplementary Table 10

Outlier analysis of proteomic data

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Kuboniwa, M., Houser, J.R., Hendrickson, E.L. et al. Metabolic crosstalk regulates Porphyromonas gingivalis colonization and virulence during oral polymicrobial infection. Nat Microbiol 2, 1493–1499 (2017). https://doi.org/10.1038/s41564-017-0021-6

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