Research ArticleMicrobiome

Quorum sensing between bacterial species on the skin protects against epidermal injury in atopic dermatitis

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Science Translational Medicine  01 May 2019:
Vol. 11, Issue 490, eaat8329
DOI: 10.1126/scitranslmed.aat8329
  • Fig. 1 S. aureus PSMα induces keratinocyte protease activity and disrupts epithelial barrier homeostasis.

    NHEKs were stimulated with 5% S. aureus USA300 LAC (SA) supernatant from overnight cultures [1 × 109 colony-performing units (CFU)] of wild-type (WT) and psmα (Δpsmα) or psmβ (Δpsmβ) knockout strains for 24 hours. Both (A) trypsin activity and (B) KLK6 mRNA expression compared to the housekeeping gene GAPDH were analyzed (n = 4). (C) PSM synthetic peptides were added to NHEKs for 24 hours to analyze changes in trypsin activity (n = 4). (D) Transcript analysis by RNA-seq and (E) gene ontology (GO) analysis of genes that changed ≥2-fold 24 hours after PSMα3 treatment. The number of genes in each category shown in parentheses. (F) Eight-week-old male C57BL/6 mice were treated for 72 hours with 1 × 107 CFU SA WT (USA300 LAC) and corresponding SA Δpsmα, or SA USA300 LAC WT (AH1263) and corresponding SA Δproteases [SA WT in (F) representative of both SA WT strains]. Bacterial growth medium was used as a vehicle control. Dashed lines indicate treatment area. Scale bars, 200 μm (n = 6). (G to J) Transepidermal water loss (TEWL) or measurement of CFU/cm2 on mice after 72-hour treatment with initial 1 × 107 CFU of (G and H) SA WT (USA300 LAC) and corresponding SA Δpsmα or (I and J) SA WT [USA300 LAC (AH1263)] and corresponding SA Δproteases (n = 6). All experiments are representative of three independent experiments. Error bars are SEM. One-way ANOVA was used to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Ctl, control.

  • Fig. 2 S. epidermidis agr type I autoinducing peptide characterization and deficiency in AD skin.

    (A) SA USA300 LAC agr type I P3-YFP activity after overnight culture (1 × 109 CFU) with 25% supernatant from overnight culture of S. epidermidis (S. epi) agr types I (RP62A), II (1457), and III (8247) (n = 4). (B) Structure of S. epidermidis agr type I autoinducing peptide (AIP). (C) Analysis of agr activity as in (A) with S. epidermidis agr type I (RP62A) WT or autoinducing peptide knockout (ΔAIP) strain supernatants (n = 4). (D) NHEK trypsin activity after culture for 24 hours with 5% SA supernatant grown overnight with or without S. epidermidis agr type I (RP62A) WT or autoinducing peptide knockout (ΔAIP) (n = 4). (E) Number (#) of S. epidermidis agr types I to III strains found on AD skin from metagenomic dataset. (F and G) Ratio of S. epidermidis agr type I to SA relative abundance on the combined analysis of all sites sampled from 5 healthy individuals and 11 AD subjects during flare and ranked from “least severe” to “most severe” based on oSCORAD. (H) Analysis of agr activity as in (A) after coculture overnight with 25% supernatant of overnight cultures (1 × 109 CFU) of clinical CoNS strains (n = 3). (I) Assessment of autoinducing peptide coding regions in agrD gene of the inhibitory CoNS strains. All experiments are representative of three independent experiments. All error bars are SEM. One-way ANOVA or an (nonparametric) unpaired Kruskal-Wallis test in (G) was used to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 3 Inhibition of S. aureus agr activity by clinical S. hominis isolate correlates with prevention of skin barrier damage and inflammation.

    (A to C) SA USA300 LAC agr type I pAmi P3-Lux reporter strain (AH2759) (1 × 107 CFU) combined with or without live S. hominis C5 (1 × 107 CFU) was applied to murine back skin for 72 hours, and SA agr activity was assessed by changes in luminescence. Images are representative of n = 5; dashed boxes indicate treatment area. (D to G) Murine back skin after 72-hour bacteria treatment, assessed for changes in TEWL, trypsin activity, and Klk6 and cytokine (Il6, Il17a, and Il17f) mRNA expression normalized to housekeeping gene Gapdh (n = 5). (H) Relative abundance of SA psmα mRNA isolated from swabs of healthy (n = 6) and AD nonlesional and lesional skin (n = 5). (I) Detection of SA PSMα3 by immunofluorescence in epidermis of AD lesional skin (representative image with scale bar, 100 μm). (J) Clinical SA isolates from 11 patients were grown up to 18 hours (1 × 109 CFU) with or without 25% S. hominis C5 supernatant. psmα mRNA expression was measured at 8 hours, whereas trypsin activity was assessed from NHEKs treated 24 hours with 18-hour cultured SA (5%) supernatant (n = 3). (K) Immunoblot of culture supernatant from clinical SA isolate AD 38 grown overnight (1 × 109 CFU) with or without S. hominis C5 supernatant and probed for PSMα3. (L to N) SA AD 38 (1 × 107 CFU) was applied to murine back skin with or without S. hominis C5 (1 × 107 CFU) for 72 hours (n = 5) (dashed boxes indicate treatment area). Changes in CFU/cm2 of SA and CoNS, and TEWL are shown. All experiments are representative of two independent experiments. Error bars represent SEM. Student’s t tests, Pearson correlation coefficient (J), and an (nonparametric) unpaired Kruskal-Wallis test (H) were used to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 4 Identification of an autoinducing peptide from a clinical S. hominis isolate that inhibits S. aureus agr activity.

    (A) Determination of S. hominis C5 autoinducing peptide sequence as SYNVCGGYF. The MS-MS spectrum obtained with the synthetic peptide is shown. Bolded fragment peaks and those indicated in color were identified in the MS-MS spectra for both the synthetic peptide and the spent growth medium sample (with difference in mass of <5 parts per million). Only matching fragments above 15% abundance are shown. Key features used for identification were the y5 (blue), y6 (red), and y7 (green) fragments of the cyclic autoinducing peptide. The selected-ion chromatograms for mass/charge ratio (m/z) 991.3984 ± 5 ppm for (B) bacteria culture and (C) synthetic autoinducing peptide in growth medium confirmed the retention time (TR) and accurate m/z of the predicted autoinducing peptide sequence. (D) Analysis of S. hominis C5 synthetic autoinducing peptide on SA USA300 LAC agr type I P3-YFP activity after an overnight culture (1 × 109 CFU) and IC50 value indicated by dotted lines at midpoint of the curve. (E) Measurement of NHEK trypsin activity after 24-hour incubation with 5% supernatant from overnight culture of SA with or without S. hominis C5 autoinducing peptide (1 × 109 CFU) (n = 4). (F to H) SA USA300 LAC agr type I pAmi P3-Lux with or without S. hominis C5 synthetic autoinducing peptide was colonized (1 × 107 CFU) on murine back skin for 48 hours followed by analysis of agr activity (luminescence) and representative pictures of murine back skin after treatment (dashed lines represent treated areas) (n = 5). (I and J) SA CFU/cm2 and TEWL after treatment with S. hominis C5 synthetic autoinducing peptide. Data are representative of two experiments. Error bars are SEM. One-way ANOVA and Student’s t tests were used to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/490/eaat8329/DC1

    Materials and Methods

    Fig. S1. S. aureus PSMα changes essential barrier gene and cytokine expression in human keratinocytes.

    Fig. S2. S. aureus PSMα and proteases are responsible for barrier damage and induction of inflammation on murine skin.

    Fig. S3. Effect of AD skin disease severity on S. epidermidis agr type I to S. aureus relative abundance by body site.

    Fig. S4. Clinical CoNS isolates inhibit S. aureus agr activity without affecting growth.

    Fig. S5. S. hominis C5 live bacteria specifically inhibit S. aureus–mediated barrier damage and inflammation.

    Fig. S6. S. hominis C5 inhibits S. aureus agr types I to III but not type IV.

    Fig. S7. S. hominis C5 autoinducing peptide inhibits S. aureus–driven inflammation and barrier damage.

    Table S1. Bacteria strains and plasmids used.

    Table S2. Primers used for quantitative polymerase chain reaction and allelic replacement.

    Data file S1. Raw data (available as Excel file).

    References (4456)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. S. aureus PSMα changes essential barrier gene and cytokine expression in human keratinocytes.
    • Fig. S2. S. aureus PSMα and proteases are responsible for barrier damage and induction of inflammation on murine skin.
    • Fig. S3. Effect of AD skin disease severity on S. epidermidis agr type I to S. aureus relative abundance by body site.
    • Fig. S4. Clinical CoNS isolates inhibit S. aureus agr activity without affecting growth.
    • Fig. S5. S. hominis C5 live bacteria specifically inhibit S. aureus–mediated barrier damage and inflammation.
    • Fig. S6. S. hominis C5 inhibits S. aureus agr types I to III but not type IV.
    • Fig. S7. S. hominis C5 autoinducing peptide inhibits S. aureus–driven inflammation and barrier damage.
    • Table S1. Bacteria strains and plasmids used.
    • Table S2. Primers used for quantitative polymerase chain reaction and allelic replacement.
    • References (4456)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

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