BACTEROIDES-FRAGILIS TOXIN REARRANGES THE ACTIN CYTOSKELETON OF HT29 C1 CELLS WITHOUT DIRECT PROTEOLYSIS OF ACTIN OR DECREASE IN F-ACTIN CONTENT/

Enterotoxigenic strains of B. fragilis associated with childhood diarr hea produce a 20 kD zinc metalloprotease toxin (BFT). BFT is reported to cleave G-actin in vitro and also causes dramatic rounding and rearr angement of the F-actin cytoskeleton in human intestinal epithelial ce ll lines (HT29 and HT29/C1). To test the hypothesis that the proteolys is of cellular actin by BFT in vivo may contribute to these alteration s in morphology and cytoskeletal architecture, we assessed the F-actin content and the arrangement of the F- and G-actin cytoskeleton in BFT -treated HT29/C1 cells by spectrofluorimetry, confocal microscopy, and immunoblotting. BFT-treated cells were compared to cells treated with C. difficile toxin A (CDA) or cytochalasin D, Using spectrofluorimetr ic quantification, the F-actin content of BFT- and cytochalasin D-trea ted cells was unchanged in contrast to a significant decrease in CDA-t reated cells. By confocal microscopy, the arrangement of F- and G-acti n in all treated cells was markedly different than control cells. Ther e was no change in the immunoblotting pattern of actin in the Triton-s oluble or -insoluble cellular fractions of BFT-treated HT29/C1 cells, We conclude that BFT alters the F- and G-actin cytoskeletal architectu re of HT29/C1 cells without direct proteolysis of actin or decrease in F-actin content. (C) 1997 Wiley-Liss, Inc.