A Derivative of Butyric Acid, the Fermentation Metabolite of Staphylococcus epidermidis, Inhibits the Growth of a Staphylococcus aureus Strain Isolated from Atopic Dermatitis Patients.

The microbiome is a rich source of metabolites for the development of novel drugs. Butyric acid, for example, is a short-chain fatty acid fermentation metabolite of the skin probiotic bacterium Staphylococcus epidermidis (S. epidermidis). Glycerol fermentation of S. epidermidis resulted in the production of butyric acid and effectively hindered the growth of a Staphylococcus aureus (S. aureus) strain isolated from skin lesions of patients with atopic dermatitis (AD) in vitro and in vivo. This approach, however, is unlikely to be therapeutically useful since butyric acid is malodorous and requires a high concentration in the mM range for growth suppression of AD S. aureus. A derivative of butyric acid, BA-NH-NH-BA, was synthesized by conjugation of two butyric acids to both ends of an -NH-O-NH- linker. BA-NH-NH-BA significantly lowered the concentration of butyric acid required to inhibit the growth of AD S. aureus. Like butyric acid, BA-NH-NH-BA functioned as a histone deacetylase (HDAC) inhibitor by inducing the acetylation of Histone H3 lysine 9 (AcH3K9) in human keratinocytes. Furthermore, BA-NH-NH-BA ameliorated AD S. aureus-induced production of pro-inflammatory interleukin (IL)-6 and remarkably reduced the colonization of AD S. aureus in mouse skin. These results describe a novel derivative of a skin microbiome fermentation metabolite that exhibits anti-inflammatory and S. aureus bactericidal activity.


The Level of IL-6 and Number of AD S. aureus in the HDAC-Depleted Mice
We next investigated if in vivo inhibition of HDAC influenced the production of IL-6 in skin and the skin colonization of AD S. aureus. HDACs in mice were depleted by administration of 3,3'diindolylmethane (DIM) according to a published protocol [1]. Skin wounds in mice treated with/without DIM were topically applied with AD S. aureus (10 8 CFU/10 µ l) bacteria for 24 h. As shown in Figure S5a, the level of IL-6 in wounded skin of mice treated with DIM (840 ± 100 pg/mL) was lower than that of wounded skin from mice without DIM treatment (2458 ± 86 pg/mL). However, there was no difference in the number of AD S. aureus colonized in skin wounds between two groups of mice treated with/without DIM ( Figure S5b,c). These results indicate that depletion of HDACs by DIM caused a decline in IL-6 production without changing the skin colonization of AD S. aureus.

Keratinocytes Treated with Bacterial Lysates in the Presence or Absence of BA-BNH-NH-BA
KERTr cells (CCD 1106 KERTr (ATCC® CRL-2309™) were grown in keratinocyte-serum-free medium (SFM) (Gibco-BRL) supplemented with bovine pituitary extract, recombinant epidermal growth factor and 1% of penicillin. Cells (5 × 10 4 cells/mL) were seeded in a culture dish and cultured for 3 d before treatments of PBS, AD S. aureus lysate (100 µ g), BA-NH-NH-BA (100 µ M), or AD S. aureus lysate with BA-NH-NH-BA) for 24 h. AD S. aureus was killed at 80 °C for 15 min. The lysate was obtained by resuspending bacterial pellets with PBS. The level of IL-6 in culture media was measured by ELISA using a human IL-6 ELISA kit (R&D systems, Minneapolis, MN, USA).

Administration of DIM into Mice
Two doses of DIM (40 mg/kg body weight, Sigma Chem-Impex, Wood Dale, IL, USA), which was dissolved by 2% dimethyl sulfoxide (DMSO) in corn oil in a total volume of 100 µ L, were intraperitoneally administered into ICR mice [1]. The second dose of DIM was given 24 h after first administration. Mice without administration of DIM serve as a control group. A 1 cm wound was made on the dorsal skin of mice and AD S. aureus (10 8 CFU/10 µ L) bacteria were topically applied onto the wound 30 min after administration of the second dose of DIM. 24 h after application of AD S. aureus, the number of bacteria and the level of IL-6 in skin wound were measured as described in Materials and Methods in the main text of manuscript.

GC Analysis
BA-NH-NH-BA (4 mM) was dissolved in PBS and stored at 4 ℃ for six months and detected by ethyl acetate liquid-liquid extraction and saturation with sodium chloride followed by GC analysis using an Agilent 5890 Series II GC [2].

Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay
To examine the cytotoxicity of BA-NH-NH-BA, dorsal skin of ICR mice were topically applied with BA-NH-NH-BA (4 mM) or PBS for 24 h. The skin was excised, immersed and fixed in 10% formalin. The tissue sections of skin were cut with a thickness of 3 µ m for TUNEL staining (R&D systems, Minneapolis, MN, USA). To quantify the TUNEL-negative (nuclear, blue staining) and -positive (nuclear, brown staining) cells, a total of at least 3 randomly selected stained images with more than 50 cells were counted.     Twenty-four h after bacterial application, the level of IL-6 was detected by ELISA (a). Bacterial CFUs in the skin wounds were enumerated by plating serial dilutions (1∶10 1 -1∶10 5 ) of the skin homogenate on TSB plates (b) and the number (log10 CFU/mL) of AD S. aureus (c) were measured. Data shown are mean ± SE. *** p < 0.001 (two-tailed t-test).