Research ArticleHost-Microbe Interactions

Bacterial d-amino acids suppress sinonasal innate immunity through sweet taste receptors in solitary chemosensory cells

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Sci. Signal.  05 Sep 2017:
Vol. 10, Issue 495, eaam7703
DOI: 10.1126/scisignal.aam7703
  • Fig. 1 Evolutionary relationship of sweet and umami taste receptor subunits.

    (A) Condensed evolutionary tree showing mouse (m), rat (r), human (h), dog (d), frog (x), and zebra fish (z) Tas1R1 (purple), Tas1R2 (blue), and Tas1R3 (green) subunits within the class C GPCR family. This family also includes the V2R pheromone receptors, the calcium-sensing receptors (CaSRs), the metabotropic glutamate receptors, and the other GPCRs indicated in black text. The full tree is shown in fig. S1. (B) Tas1R3 (T1R3) combines with Tas1R2 (T1R2) or Tas1R1 (T1R1) to form the sweet or umami receptors, respectively. (C) Sinonasal SCCs in human primary sinonasal ALI cultures produce both T1R2 and T1R3 sweet taste receptor subunits. Images are representative of three independent experiments using ALIs from three individual patients. Scale bars, 20 μm.

  • Fig. 2 Bacteria and sweet d-amino acids in clinical sinonasal microbiologic cultures and effects on biofilm formation.

    (A) Fold increases in d-Phe, d-Leu, and d-Ile from sinonasal microorganism CM compared to starting medium. n = 5 to 12 independent culture samples for each condition tested separately for each d-amino acid. (B) Biofilm formation by P. aeruginosa strain PAO1 and MRSA strain M2 in the presence of CM from the indicated patient samples. n = 6 to 10 independent experiments for each condition and concentration; each single experiment represents an average of >8 wells of a 96-well plate. *P < 0.05 by one-way analysis of variance (ANOVA), Dunnett’s posttest comparing values to strain-specific control. (C) Dose-dependent effects of l-Phe + l-Leu + l-Tyr or d-Phe + d-Leu + d-Tyr on P. aeruginosa PAO1 and MRSA biofilm formation. Because of the poor solubility of l-Tyr and d-Tyr, the maximum concentration of l-Tyr or d-Tyr in these experiments was 0.1 mM. For amino acid concentrations indicated as greater than 0.1 mM on the graph, the indicated concentration refers to the concentration of l-Phe or d-Phe and l-Leu or d-Leu, with l-Tyr or d-Tyr at 0.1 mM. n = 6 to 10 independent experiments for each condition and concentration; each single experiment represents an average of >8 wells of a 96-well plate.

  • Fig. 3 Effects of sweet d-amino acids on P. aeruginosa pyocyanin production and swarming.

    (A) Color of PAO1 supernatant in the presence of l- or d-amino acids and representative absorption spectra of supernatant or chloroform-extracted pyocyanin. n = 3 to 10 independent experiments per strain per condition; each experiment averaged three suspension cultures. (B) Bar graph of relative pyocyanin abundance in P. aeruginosa strains. n = 3 independent experiments for each strain; each single experiment averaged three suspension cultures. Wt, wild-type. (C) Quantification of relative pyocyanin abundance in strains PAO1 and ATCC 27853, which are biofilm-competent, and PAO-JP2, which cannot form biofilms. n = 3 to 10 independent experiments per strain per condition; each single experiment averaged three suspension cultures. (D) Morphology of PAO-GFP colonies under conditions that favor swimming (0.3% agar) or swarming (0.8% agar). Scale bar, 1 cm. (E) Morphology of PAO-GFP colonies under swarming conditions and quantification of swarming in the presence of the indicated amino acids. Scale bar, 1 cm. Graphs show means ± SEM with *P < 0.05 and **P < 0.01 (one-way ANOVA, Bonferroni posttest comparing bracketed groups). n = 5 independent experiments for each condition per strain; each experiment averaged three plates.

  • Fig. 4 Effects of d-amino acids on SCC T2R Ca2+ responses.

    (A) Ca2+ responses of SCCs treated with denatonium (10 mM applied apically; abbreviated 10 D) in the presence or absence of glucose (Glc) ± lactisole (Lact) or GA2. Traces shown are the average (±SEM) of multiple experiments (n = 6 to 10); each experiment was performed on a single ALI culture from a separate individual patients (thus, cells from 6 to 10 patients were examined per condition). (B) Calcium responses in the presence of d-Leu or l-Leu (2 mM; top) and d-Phe or l-Phe (2 mM; bottom) ± lactisole. n = 5 to 8 experiments, each experiment was performed on a single ALI from a separate individual patient (thus, cells from five to eight patients were examined per condition). (C) Summary of peak Fluo-4 F/Fo values for data shown in (A) and (B). Pound symbols (#) indicate significance compared with control (denatonium only); asterisks denote significance between brackets; #P < 0.05, *P < 0.05, ##P < 0.01, and **P < 0.01 by one-way ANOVA with Bonferroni posttest. (D) β-Defensin secreted into ASL after treatment with the indicated combinations of denatonium (Denat), glucose (Glc), gymnemic acis (GA2), sucralose (S), and lactisole (Lact) in phosphate-buffered saline (PBS). n = 4 to 6 ALI cultures per condition, each from a separate individual patient; *P < 0.05 and **P < 0.01 compared with PBS (unstimulated control) by one-way ANOVA with Dunnett’s posttest.

  • Fig. 5 Effects of d-amino acids on T2R-stimulated immune responses in vitro.

    (A) Representative images showing live cells labeled with Syto9 (green) and dead cells labeled with PI (red) in sinonasal ALI cultures after apical infection with MRSA in the presence or absence of d-Phe and d-Leu, lactisole, denatonium, GA2, MRSA CM, or PAO1 CM, as indicated. n = 3 to 6 independent experiments for each condition, each experiment averaging 10 fields of view from three ALI cultures from the same patient. Scale bar, 100 μm. (B) Data from experiments as in (A) and (B) quantified as % PI-stained area. n = 3 to 6 independent experiments (each from a different individual patient) for each condition, with each experiment averaging 10 fields of view from three ALI cultures from the same patient. Asterisks denote significance compared with the first bar of each graph (control conditions for that comparison) via one-way ANOVA with Dunnett’s posttest; **P < 0.01. All graphs show means ± SEM (n = 4 to 6 patient ALI cultures for each condition).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/495/eaam7703/DC1

    Fig. S1. Phylogenetic relationship of metabotropic glutamate receptor family proteins.

    Fig. S2. Antibody validation in transfected HEK293 cells.

    Fig. S3. Dispersion of PAO-GFP microcolonies in the presence or absence of d-amino acids.

    Fig. S4. d-Amino acids did not affect salt sensitivity of P. aeruginosa strain PAO1.

    Fig. S5. Sweet d-amino acids do not affect antibiotic sensitivity of PAO-GFP or ATCC 27853.

    Fig. S6. d-Amino acid reduction of mouse nasal SCC T2R–stimulated Ca2+ signals requires T1R3.

    Table S1. Sequences used for alignment for phylogenetic tree construction.

  • Supplementary Materials for:

    Bacterial ᴅ-amino acids suppress sinonasal innate immunity through sweet taste receptors in solitary chemosensory cells

    Robert J. Lee,* Benjamin M. Hariri, Derek B. McMahon, Bei Chen, Laurel Doghramji, Nithin D. Adappa, James N. Palmer, David W. Kennedy, Peihua Jiang, Robert F. Margolskee, Noam A. Cohen*

    *Corresponding author. Email: rjl{at}mail.med.upenn.edu (R.J.L.); cohenn{at}uphs.upenn.edu (N.A.C.)

    This PDF file includes:

    • Fig. S1. Phylogenetic relationship of metabotropic glutamate receptor family proteins.
    • Fig. S2. Antibody validation in transfected HEK293 cells.
    • Fig. S3. Dispersion of PAO-GFP microcolonies in the presence or absence of ᴅ-amino acids.
    • Fig. S4. ᴅ-Amino acids did not affect salt sensitivity of P. aeruginosa strain PAO1.
    • Fig. S5. Sweet ᴅ-amino acids do not affect antibiotic sensitivity of PAO-GFP or ATCC 27853.
    • Fig. S6. ᴅ-Amino acid reduction of mouse nasal SCC T2R–stimulated Ca2+ signals requires T1R3.
    • Table S1. Sequences used for alignment for phylogenetic tree construction.

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    Citation: R. J. Lee, B. M. Hariri, D. B. McMahon, B. Chen, L. Doghramji, N. D. Adappa, J. N. Palmer, D. W. Kennedy, P. Jiang, R. F. Margolskee, N. A. Cohen, Bacterial ᴅ-amino acids suppress sinonasal innate immunity through sweet taste receptors in solitary chemosensory cells. Sci. Signal. 10, eaam7703 (2017).

    © 2017 American Association for the Advancement of Science

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