Figure 1.
Detection of porphyrins in P. acnes and human skins.
To visualize porphyrin-producing bacteria (arrows), P. acnes (a, b) or S. epidermidis (c, d) (2×108 CFU) was incubated with ALA for 4 h and observed under microscopes. Bright-field (a, c)/fluorescent (b, d) overlay of bacteria were presented. Bar = 2 µm. A Wood's lamp with an UV light source was used to image the auto-fluorescent P. acnes (red fluorescence, arrows) on the surface of nose (e). An area (square) of nose was amplified (f). Bar = 1 cm. The amounts (ng/cm2) of prophyrins on the surfaces of noses, foreheads, and arms of five volunteers (1–5) with healthy skins were compared (g). **P<0.01 or ***P<0.001 was evaluated using two-tailed t-tests. Data are the mean ± SD of three separate experiments.
Figure 2.
The production of porphyrins in P. acnes as a function of the doses of UV-B.
After radiation with or without UV-B, P. acnes was then incubated with ALA (1 mM) for 4 h at 42°C under dark conditions. The ALA induced porphyrins were monitored using the fluorescence emission spectra via a Perkin Elmer LS50B fluorescence spectrometer. The number of bacteria was determined by reading the values of OD600 as described in Materials and Methods. The production of porphyrins in individual bacteria was calculated by dividing fluorescent intensities of porphyrins by the number of bacteria. ***P<0.001 was evaluated using two-tailed t-tests. Data are the mean ± SD of three separate experiments.
Figure 3.
UV-B exposure did not alter the fluorescence emission spectra of porphyrins.
P. acnes (2×108 CFU) were incubated with 1 mM ALA for 4 h at 42°C and then exposed to UV-B (50 mJ/cm2) (UV-B). Bacteria without UV-B exposure served as a control (C). The porphyrin derivatives were detected in a spectrum between 550 and 700 nm. A spectrum of CpIII (10 µM) (Sigma, St. Louis, MO) exposed to UV-B [0 (C) or 50 mJ/cm2 (UV-B)] was presented in an inserted panel. Spectra are representative of three independent experiments.
Figure 4.
UV-B exposure decreased the production of porphyrins in bacteria isolated from human faces.
Tape trips were used to isolate facial bacteria from the nose surfaces of five volunteers (▴, •, ▾, ♦, and ▪). Bacteria were exposed to UV-B ranging from 0 to 20 mJ/cm2 and incubated with ALA (1 mM) for 4 h at 42°C. The production of porphyrins in individual bacteria was calculated as described in Materials and Methods. The mean values [fluorescence intensity/bacterium (108 CFU)] of porphyrin production in individual bacteria at a single dose of UV-B exposure were denoted ―.
Figure 5.
Simultaneous detection of in vivo responses of both P. acnes and skin cells to UV-B exposure.
Mouse ears harboring P. acnes were irradiated with (50 mJ/cm2) and without UV-B. Ears were excised and homogenized immediately (a–c; g–k) and 24 h (d–f; l–p) after radiation. P. acnes in mouse homogenates was incubated with 1 mM ALA and grown on a Brucella broth agar plate. Compared with unexposed control (C) mouse ear (a, d), the intensity of porphyrins in a single colony of P. acnes significantly decreased after UV-B exposure (UV-B) (b, e). The decrease in the porphyrin intensity is still detectable 24 h after UV-B exposure. The intensity of porphyrin fluorescence was presented as % of that in control P. acnes colonies (c, f). No CPDs were detected in an epidermal layer of UV-B-unexposed-ear skin (g, i, l, n). Immunohistochemical staining showed that the CPD formation (red; arrows) detected by a monoclonal antibody to TDM-2 was considerably increased by UV-B (h, j). Merged images showed DAPI (blue) (h) and CPD (purple) (j) staining. However, UV-B-induced CPDs faded away 24 h after UV-B exposure. The percentages of CPD-positive cells in epidermal layers were displayed (k, p). Dash lines indicate the surface of ear skin. Bar (a, b, d, e) = 200 µm; (g–j; l–o) = 50 µm. **P<0.01 or ***P<0.001 was evaluated using t-tests. Data are the mean ± SD of three separate experiments.
Figure 6.
The MS/MS spectrum of an internal peptide (THLPTGIVVSCQNER) of P. acnes RF2.
After exposure of P. acnes in PBS with and without UV-B at a dose of 50 mJ/cm2, bacterial pellets were digested with trypsin and subsequently subjected to LTQ-Orbitrap XL mass spectrometry analysis. A sequenced peptide (THLPTGIVVSCQNER) is presented and assigned as an internal peptide of P. acnes RF2 (Q6A808). The m/z value of each “y” and “b” ion in collision-induced dissociation (CID) spectra was indicated. Three independent experiments were performed. The oxidized THLPTGIVVSCQNER at H and C is reproducibly and exclusively present in the UV-B irradiated-P. acnes.
Table 1.
Mass spectrometric analysis of oxidized and de-oxidized peptides in UV-B irradiated P. acnes.