THE ENTEROTOXIN OF BACTEROIDES-FRAGILIS IS A METALLOPROTEASE

During the past decade, strains of Bacteroides fragilis that produce a n enterotoxin have been implicated in diarrheal disease in animals and humans, The extracellular enterotoxin has been purified and character ized as a single polypeptide (M(17) similar to 20,000). Single specifi c primer-PCR was used to clone a portion of the B. fragilis enterotoxi n gene. The recombinant protein expressed by the cloned gene fragment reacted with monospecific antibodies to B. fragilis enterotoxin by enz yme-linked immunosorbent assay and immunoblot analysis, The deduced am ino acid sequence revealed a signature zinc-binding consensus motif (H EXXHXXGXXH/Met-turn) characteristic of metalloproteases termed metzinc ins. Sequence comparisons showed close identity to matrix metalloprote ases (e.g., human fibroblast collagenase) within the zinc-binding and Met-turn region. Purified enterotoxin contained 1 g-atom of Zn2+ per m olecule and hydrolyzed gelatin, azocoll, actin, tropomyosin, and fibri nogen. The enterotoxin also underwent autodigestion. The N-terminal am ino acid sequences of two autodigestion products were identical to the deduced amino acid sequence of the recombinant enterotoxin and reveal ed cleavage at Cys-Leu and Ser-Leu peptide bonds. Gelatinase (type IV collagenase) activity comigrated with the toxin when analyzed by gel f ractionation and zymography, indicating that protease activity is due to the enterotoxin and not to a contaminating protease(s). Optimal pro teolytic activity occurred at 37 degrees C and pH 6.5. Primary proteol ytic cleavage sites in actin were identified, revealing cleavage at Gl y-Met and Thr-Leu peptide bonds. Enzymatic activity was inhibited by m etal chelators but not by inhibitors of other classes of proteases. Ad ditionally, cytotoxic activity of the enterotoxin on human carcinoma H T-29 cells was inhibited by acetoxymethyl ester EDTA. The metalloprote ase activity of the enterotoxin suggests a possible mechanism for ente rotoxicity and may have additional implications in the study of diseas e caused by B. fragilis.