Confocal microscopy study of pertussis toxin and toxoids on CHO-cells

Abstract

Pertussis toxin in its detoxified form is a major component of all current acellular pertussis vaccines. Here we report the membrane translocation and internalization activities of pertussis toxin and various pertussis toxoids using Chinese hamster ovary cells and confocal microscopy based on indirect immunofluorescence labeling. Chemically detoxified pertussis toxoids were able to translocate/internalize into cells at the concentration about 1,000 times higher than the native toxin. Pertussis toxoids detoxified with different procedures (glutaraldehyde, glutaraldehyde plus formaldehyde, hydrogen peroxide or genetic mutation) showed differences in fluorescence intensity under the same condition, indicating toxoids from different detoxification methods may have different translocation/internalization activities on cells.

Figure 1. Confocal images of CHO cells treated with 200 ng/ml of PTx for different incubation periods. (A) negative control; (B) 2 h incubation; (C) 12 h incubation, to exhibit the effect of incubation time on toxin translocation. Green fluorescence represents PTx, blue fluorescence represents nucleus, and red fluorescence represents F-actin of cytoskeleton. For figures (B and C), the images are shown (for clarity) without blue fluorescence. Scale bar = 20 μm

Figure 2.Confocal images of CHO cells treated with different PTx concentrations for 12 h. (A) Negative control; (B) PTx at 50 ng/ml; (C) PTx at 100 ng/ml; D: PTx at 200 ng/ml, to exhibit the effect of PTx concentration on toxin translocation [PTx at 20 ng/ml (data not shown)]. Green fluorescence represents PTx, blue fluorescence represents nucleus, and red fluorescence represents F-actin of cytoskeleton. For clarity, each plate is presented showing only blue/ green fluorescent channels with an insert showing red/ blue/ green fluorescence. Scale bar = 20 μm

Figure 3. Comparison of PTx and different PTds inactivated with different approaches in translocation to CHO cells shown by the fluorescence intensity of target protein in confocal images. CHO cells were treated with PTx or PTd for 12 h simultaneously. (A) Negative control; (B) Positive control, PTx (200 ng/ml); (C) PTd-I (200 ng/ml); (D) PTd-II (200 μg/ml); (E) PTd-III (200 μg/ml); (F) PTd-IV (200 μg/ml). Green fluorescence is of target protein, blue fluorescence is of nucleus, and red fluorescence representing F-actin of cytoskeleton. For clarity, each plate is presented showing only blue/ green fluorescent channels with an insert showing red/ blue/ green fluorescence. Scale bar = 20 μm.