RESUMEN
Clostridium botulinum C2 enterotoxin consists of two unlinked proteins designated as C2II, which recognizes a cell-surface glycoprotein and translocates an ADP-ribosyltransferase, C2I, into the cytosol of a targeted cell. Fluorescence-activated cytometry was used to study the cellular interactions of Alexa488-labeled C2I (C2I-A488) and proteolytically activated C2II (C2IIa-A488). The binding of C2IIa-A488 (4 degrees C/10 min) to Chinese hamster ovary (CHO) and African green monkey kidney (Vero) cells yielded a signal/noise ratio of 7:1 and 4:1, respectively. C2I-A488 binding required C2IIa and resulted in a 4:1 (CHO) and 10:1 (Vero) signal/noise ratio that was readily competed by unlabeled C2I. Neither C2I nor C2IIa bound to a CHO line (RK14), lacking the receptor for C2IIa. C2I-A488 did not dock with the heterologous cell-binding component (iota b) of Clostridium perfringens iota toxin, a binary toxin closely related to C2. Pretreatment of wild-type CHO or Vero cells with pronase or papain (37 degrees C/30 min) prevented a cell-associated C2IIa-specific signal. However, CHO and Vero cells pretreated with papain at 25 degrees C had a 1.5- to 2.3-fold increase in C2IIa-specific fluorescence versus untreated cells incubated with C2IIa-A488. Overall, these studies further demonstrated the utility of fluorescence-activated cytometry for studying the binding characteristics of bacterial binary toxins like C2.