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 diarrhea produce a 20 kD zinc metalloprotease toxin (BFT). BFT is reported to cleave G-actin in vitro and also causes dramatic rounding and rearrangement of the F-actin cytoskeleton in human intestinal epithelial cell lines (HT29) and HT29/C1). To test the hypothesis that the proteolysis of cellular actin by BFT in vivo may contribute to these alterations 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 spectrofluorimetric quantification, the F-actin content of BFT- and cytochalasin D-treated cells was unchanged in contrast to a significant decrease in CDA-treated cells. By confocal microscopy, the arrangement of F- and G-actin in all treated cells was markedly different than control cells. There was no change in the immunoblotting pattern of actin in the Triton-soluble or -insoluble cellular fractions of BFT-treated HT29/C1 cells. We conclude that BFT alters the F- and G-actin cytoskeletal architecture of HT29/C1 cells without direct proteolysis of actin or decrease in F-actin content.
Saidi
R F
RF
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Jaeger
K
K
Montrose
M H
MH
Wu
S
S
Sears
C L
CL
D000199:Actins / Q000378:metabolism*; D001427:Bacterial Toxins; D001441:Bacteroides fragilis / Q000502:physiology*; D015638:Cytochalasin D / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D004768:Enterotoxins / Q000378:metabolism*; D019073:HT29 Cells; D006801:Humans; D007163:Immunosorbent Techniques; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism*; D018613:Microscopy, Confocal; D013050:Spectrometry, Fluorescence
D000199:Actins; D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; D015638:Cytochalasin D; D008666:Metalloendopeptidases; C091198:fragilysin