Research ArticleHEALTH AND MEDICINE

A commensal strain of Staphylococcus epidermidis protects against skin neoplasia

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Science Advances  28 Feb 2018:
Vol. 4, no. 2, eaao4502
DOI: 10.1126/sciadv.aao4502
  • Fig. 1 S. epidermidis strains isolated from normal human skin produce 6-HAP.

    (A and B) Stability of antimicrobial molecules from S. epidermidis against GAS after heat-treatment for the indicated time (A) and incubation with indicated protease (B). The black area represents zone of growth inhibition of GAS. (C) Dose-dependent antimicrobial activity of the purified antimicrobial compound against GAS. Data are means ± SEM of three individual experiments. CFU, colony-forming unit. (D) 15N isotope incorporation into the antibiotic molecule after culturing S. epidermidis MO34 in tryptic soy broth (TSB) containing ammonium-15N chloride (12.5 mM). (E) The determined chemical structure of the active molecule, 6-HAP. (F) Capacity of 6-HAP to block in vitro DNA extension by Klenow fragment polymerase. A template that required adenosine (X = T) or cytidine (X = G) at the initial base for extension was used. (G) 5-Bromo-2′-deoxyuridine (BrdU) incorporation into tumor cell line, L5178, YAC-1 lymphoma, B16F10 melanoma, and Pam212 SCC after a 24-hour incubation in suitable media containing indicated concentrations of 6-HAP. Data are means ± SEM of four individual experiments. (H) BrdU incorporation into nontransformed human keratinocytes (NHEKs) and Pam212 cutaneous squamous cell carcinoma after 24-hour incubation in suitable media containing indicated concentrations of 6-HAP. Data are means ± SEM of four individual experiments.

  • Fig. 2 Selective antiproliferative activity of 6-HAP is mediated by mARCs.

    (A) Expression of mARC1 and mARC2 in NHEKs, squamous cell carcinoma (Pam212), melanoma (B16F10), and lymphoma cell lines (L5178). Data are means ± SEM of five individual experiments. UD, undetectable. (B) Expression of mARC1 and mARC2 in NHEKs treated with control siRNA, mARC1 siRNA, and mARC2 siRNA. Data are shown as relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and represent means ± SEM of six independent assays. (C) Effect of gene silencing with mARC1 and mARC2 siRNA on sensitivity to 6-HAP in NHEKs. Data are means ± SEM of eight individual experiments (*P < 0.05, ***P < 0.0001 by two-tailed independent t test).

  • Fig. 3 Systemic administration of 6-HAP suppresses melanoma growth in mice.

    (A) Systemic toxicity of repeated intravascular administration with 6-HAP (20 mg/kg) or with an equal volume of vehicle (2.5% DMSO in 0.9% NaCl) every 48 hours for 2 weeks (arrows) in mice. To observe toxicity of 6-HAP, we determined mouse weight at the indicated time points. Data are means ± SEM of 10 mice. (B and C) Effect of repeated intravascular administrations with 6-HAP on growth of melanoma in mice. Data are means ± SEM from 10 individual mice (*P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed independent t test versus vehicle control) (B). Representative images of tumor (yellow dashed line) in mouse treated with 6-HAP or vehicle at day 9 and day 13 are shown (C).

  • Fig. 4 Skin colonization by S. epidermidis strain producing 6-HAP protects from UV-induced neoplasia in mice.

    (A to D) Effect of colonization by S. epidermidis MO34 strain producing 6-HAP on tumor incidence (A) and number (B) in SKH-1 hairless mice treated with DMBA, followed by repeated UV-B irradiation. S. epidermidis 1457 was used as a control strain that does not produce 6-HAP. Tumor incidence and tumor number in each mouse were recorded every week. Data are means ± SEM of 19 mice (*P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed independent t test). Representative images of UV-induced tumor formation in mouse treated with S. epidermidis 1457 (C) or MO34 (D) at week 12 are shown. (E and F) A representative hematoxylin and eosin staining of UV-induced skin tumor or skin obtained from SKH-1 mice colonized by S. epidermidis 1457 (E) or MO34 (F), respectively, treated with UV-B for 12 weeks. (G and H) Immunostaining for S. epidermidis (green) and keratin-14 (red) in the UV-induced tumor or skin of SKH-1 mice treated with S. epidermidis 1457 (G) or MO34 (H), respectively. The sections were counter stained with 4′,6-diamidino-2-phenylindole (blue).

  • Fig. 5 S. epidermidis strain producing 6-HAP is commonly distributed on the human skin.

    (A to D) Productions of 6-HAP by skin isolate strains, MO34 (A) and MO38 (B), and laboratory strains of S. epidermidis, ATCC12228 (C) and 1457 (D). Production of 6-HAP was evaluated by HPLC. Arrow indicates elution time of 6-HAP. The data are representative of three independent experiments. mAU, milli absorbance units. (E) Heat map showing relative abundance of putative S. epidermidis strains producing 6-HAP in the metagenome of skin microbiome samples from 22 distinct body sites of 18 healthy subjects.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/2/eaao4502/DC1

    Supplementary data

    table S1. Mutagenic activity of 6-HAP.

    table S2. Systemic toxicity of repeated intravascular administration with 6-HAP in mice.

    table S3. List of unique marker genes that are present in S. epidermidis strains producing 6-HAP.

    fig. S1. Purification of a unique antimicrobial compound from the culture supernatant of S. epidermidis strain MO34 isolated from normal human skin.

    fig. S2. Molecular mass of purified antibiotic from the S. epidermidis MO34 strain analyzed by HR-ESI-MS.

    fig. S3. Comparison of chemical shifts of purified antibiotic with those of synthetic 6-HAP in 1H-NMR.

    fig. S4. The gHMBC spectrum (500 MHz) of 6-HAP in AcOD-D2O.

    fig. S5. Comparison of the fragmentation profile of purified antibiotic with that of synthetic 6-HAP on electron impact mass spectrometry.

    fig. S6. Capacity of 6-HAP to directly disrupt plasma membrane of human keratinocytes and sebocytes.

    fig. S7. Effect of epicutaneous application of S. epidermidis strain producing 6-HAP on the cutaneous immune system in mice.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary data
    • table S1. Mutagenic activity of 6-HAP.
    • table S2. Systemic toxicity of repeated intravascular administration with 6-HAP in mice.
    • fig. S1. Purification of a unique antimicrobial compound from the culture supernatant of S. epidermidis strain MO34 isolated from normal human skin.
    • fig. S2. Molecular mass of purified antibiotic from the S. epidermidis MO34 strain analyzed by HR-ESI-MS.
    • fig. S3. Comparison of chemical shifts of purified antibiotic with those of synthetic 6-HAP in 1H-NMR.
    • fig. S4. The gHMBC spectrum (500 MHz) of 6-HAP in AcOD-D2O.
    • fig. S5. Comparison of the fragmentation profile of purified antibiotic with that of synthetic 6-HAP on electron impact mass spectrometry.
    • fig. S6. Capacity of 6-HAP to directly disrupt plasma membrane of human keratinocytes and sebocytes.
    • fig. S7. Effect of epicutaneous application of S. epidermidis strain producing 6-HAP on the cutaneous immune system in mice.

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    Other Supplementary Material for this manuscript includes the following:

    • table S3 (Microsoft Excel format). List of unique marker genes that are present in S. epidermidis strains producing 6-HAP.

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