Vaccination Targeting a Surface Sialidase of P. acnes: Implication for New Treatment of Acne Vulgaris
Figure 2
Removal of Sialic Acids from Human Sebocytes by Sialidase Increases Their Susceptibility to P. acnes.
(A) Sialic acids on the cell surface of immortalized human sebocytes (SZ95) were detected by their reaction with biotinylated MAA lectin I (10 µg/ml) and streptavidin-FITC conjugate. FITC-fluorescence intensity was measured by flow cytomtery (FACSCalibur, BD Biosciences, San Jose, CA), reflecting the quantity of sialic acid. The sebocytes were pre-treated with 10 µg/ml of purified recombinant sialidase (green, a), GFP (green, b) or an equal volume of PBS (vehicle) (purple) at pH 6 for 2 h. The decrease in FITC-fluorescence intensity in sialidase-treated sebocytes validated the enzymatic activity of the purified sialidase. (B) After pre-treatment with sialidase, sebocytes were co-cultured with P. acnes (5×107 CFU/5×106 cells) for 18 h. P. acnes-induced cell death in PBS (vehicle)-, sialidase- or GFP-pretreated sebocytes was assessed by trypan blue. Cell death is presented as the % of dead cells compared to all cultured cells. (C) After washing out unbound P. acnes, the number of P. acnes adhered to sebocytes was calculated by spreading Triton-X (0.01%) lysed sebocytes on agar plates to quantify CFU/cell. (D) The CFU/cell in vehicle-pretreated sebocytes was defined as 100%. Adherence of P. acnes (arrows) to vehicle (a)-, sialidase (b)- or GFP (c)-treated sebocytes was visualized by staining with the Accustain Gram stain kit. Pre-treatment with sialidase significantly increased the adhesion of P. acnes to sebocytes. Shown are representative results of three independent experiments. Error bars are mean±SE (P<0.01*, P<0.001**, P<0.0005*** by Student's t-test). Bar: 10 µm.