Charakterisierung der Bindung und Aufnahme von clostridialen C3-Exoenzymen bei kultivierten makrophagenartigen Zellen

The C3 transferases from Clostridium botulinum (C3bot1) and Clostridium limosum (C3lim) ADP-ribosylate and thereby inactivate Rho A, B and C of eukaryotic cells. Cell accessibility of C3 proteins is very poor and no binding/translocation domain was identified for C3 proteins up to date. According to the current opinion, C3 proteins are taken up by unspecific pinocytosis when relatively high concentrations of C3 (> 30 µg/ml) were administered to cells for about 24 hours. Here, we show that relatively low concentrations of C3bot1 and C3lim (< 300 ng/ml) selectively intoxicated mouse J774A.1 macrophage-like cells. Both C3 transferases changed the morphology of J774A.1 cells in a concentration and time dependent manner. C3 bot1 and C3lim catalyzed the ADP-ribosylated of Rho in the cytosol, clearly indicating that the C3 proteins have been internalized into J774A.1. Enzymatic inactive C3bot1E174Q had no effect on J774A.1 cells; thus the ADP-ribosylation of Rho in the cytosol was essential to induce the observed C3-morphology. C3bot1 showed a concentration dependent binding to J774A.1 cells and competition studies with C3bot1E174Q revealed that the specific was binding. Bafilomycin A1, an inhibitor of the vacuolar ATPase proton pump, protected J774A.1 cells from intoxication with C3bot1 and C3lim. Data thus obtained imply that both C3 proteins translocate from acidified endosomes into the cytosol of cultured macrophage-like cells