A commensal strain of Staphylococcus epidermidis protects against skin neoplasia

Abstract

We report the discovery that strains of Staphylococcus epidermidis produce 6-N-hydroxyaminopurine (6-HAP), a molecule that inhibits DNA polymerase activity. In culture, 6-HAP selectively inhibited proliferation of tumor lines but did not inhibit primary keratinocytes. Resistance to 6-HAP was associated with the expression of mitochondrial amidoxime reducing components, enzymes that were not observed in cells sensitive to this compound. Intravenous injection of 6-HAP in mice suppressed the growth of B16F10 melanoma without evidence of systemic toxicity. Colonization of mice with an S. epidermidis strain producing 6-HAP reduced the incidence of ultraviolet-induced tumors compared to mice colonized by a control strain that did not produce 6-HAP. S. epidermidis strains producing 6-HAP were found in the metagenome from multiple healthy human subjects, suggesting that the microbiome of some individuals may confer protection against skin cancer. These findings show a new role for skin commensal bacteria in host defense.

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.