The enterotoxin of Bacteroides fragilis is a metalloprotease

Abstract

During the past decade, strains of Bacteroides fragilis that produce an enterotoxin have been implicated in diarrheal disease in animals and humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (M r , ϳ20,000). Single specific primer-PCR was used to clone a portion of the B. fragilis enterotoxin gene. The recombinant protein expressed by the cloned gene fragment reacted with monospecific antibodies to B. fragilis enterotoxin by enzyme-linked immunosorbent assay and immunoblot analysis. The deduced amino acid sequence revealed a signature zinc-binding consensus motif (HEXXHXXGXXH/Met-turn) characteristic of metalloproteases termed metzincins. Sequence comparisons showed close identity to matrix metalloproteases (e.g., human fibroblast collagenase) within the zinc-binding and Met-turn region. Purified enterotoxin contained 1 g-atom of Zn 2؉ per molecule and hydrolyzed gelatin, azocoll, actin, tropomyosin, and fibrinogen. The enterotoxin also underwent autodigestion. The N-terminal amino acid sequences of two autodigestion products were identical to the deduced amino acid sequence of the recombinant enterotoxin and revealed cleavage at Cys-Leu and Ser-Leu peptide bonds. Gelatinase (type IV collagenase) activity comigrated with the toxin when analyzed by gel fractionation and zymography, indicating that protease activity is due to the enterotoxin and not to a contaminating protease(s). Optimal proteolytic activity occurred at 37؇C and pH 6.5. Primary proteolytic cleavage sites in actin were identified, revealing cleavage at Gly-Met and Thr-Leu peptide bonds. Enzymatic activity was inhibited by metal chelators but not by inhibitors of other classes of proteases. Additionally, cytotoxic activity of the enterotoxin on human carcinoma HT-29 cells was inhibited by acetoxymethyl ester EDTA. The metalloprotease activity of the enterotoxin suggests a possible mechanism for enterotoxicity and may have additional implications in the study of disease caused by B. fragilis.

Structural and functional diversity of metalloproteinases encoded by the Bacteroides fragilis pathogenicity island

“…Currently, there is valuable structural and functional information for FRAs [6,[8][9][10][11]. The crystal structure of FRA3 was recently reported [12].…”

Growth inhibition of Bacteroides fragilis by hemopexin: proteolytic degradation of hemopexin to overcome heme limitation

“…B. fragilis is known to produce several proteases [26], and more recently a metalloprotease enterotoxin [27] and a ¢brinogen-degrading serine protease [28] have been described. A trypsin-like protease is also produced by the related Bacteroides forsythus [29].…”

Purification and Characterization of a Fibrinogen‐Degrading Protease in Bacteroides fragilis Strain YCH46

“…Adherence, invasion, and production of enzymes or toxins which assist in invasion or destruction of tissue or survival of organisms are potentially significant in the understanding of pathogenesis. Different kinds of virulence factors of B. fragilis such as capsules (33,34,38), fimbriae (40,42), neuraminidase (1,35,41), iron uptake (36,37,46), superoxide dismutase (14), and enterotoxin (27,30,31,45), have been characterized. However, up to now, the critical role of proteases in infectious diseases has been unclear in B. fragilis.…”

“…Recently, enterotoxigenic strains of B. fragilis were isolated and the enterotoxin from one of them was purified (29,30,44). In addition, Moncrief et al (27) reported that the enterotoxin of B. fragilis is a zinc metalloprotease. Therefore protease production by B. fragilis has recently been the focus of intense research interest from the viewpoint of both biochemistry and pathogenicity.…”