Figure 5.

PA41 inhibits pore formation and the subsequent delivery of the GTD into the cytosol. A, pore formation on biological membranes. TcdB holotoxin alone (1 nm) or preincubated with PA41 mAb or Fab (10 nm) was applied to CHO-K1 cells preloaded with ⁸⁶Rb⁺, and rubidium release was compared at pH 7.5 and 4.5. Means ± S.D. (n = 6) are shown. B, Caco-2 monolayers were intoxicated with 3xFLAG-TcdB (25 nm) preincubated with equimolar (25 nm) amounts of control or PA41 mAb. Toxin cleavage assays were performed as described under “Experimental procedures.” Blots were probed with antibodies against the toxin (anti-FLAG; detects both internalized holotoxin and free GTD), unglucosylated and total Rac1, and GAPDH. C, the fraction of the internalized toxin that is cleaved and released in each condition was determined by normalizing the free GTD signal to the corresponding internalized holotoxin signal. For each time point, the fractional GTD release measurements were normalized to that of the isotype mAb controls to obtain the relative GTD cleavage. D and E, similar experiments performed with the Fab fragments. Means ± S.D. (n = 3) are shown. Error bars represent S.D.