Enterotoxigenic anaerobic Bacteroides fragilis is a significant source of inflammatory diarrheal disease and a risk factor for
colorectal cancer. Two distinct
metalloproteinase types (the homologous 1, 2, and 3
isoforms of
fragilysin (FRA1, FRA2, and FRA3, respectively) and
metalloproteinase II (MPII)) are encoded by the B. fragilis pathogenicity island. FRA was demonstrated to be important to pathogenesis, whereas MPII, also a potential virulence
protein, remained completely uncharacterized. Here, we, for the first time, extensively characterized MPII in comparison with FRA3, a representative of the FRA
isoforms. We employed a series of multiplexed
peptide cleavage assays to determine substrate specificity and proteolytic characteristics of MPII and FRA. These results enabled implementation of an efficient assay of MPII activity using a fluorescence-quenched
peptide and contributed to structural evidence for the distinct substrate cleavage preferences of MPII and FRA. Our data imply that MPII specificity mimics the dibasic Arg↓Arg cleavage motif of
furin-like
proprotein convertases, whereas the cleavage motif of FRA (Pro-X-X-
Leu-(Arg/Ala/Leu)↓) resembles that of human
matrix metalloproteinases. To the best of our knowledge, MPII is the first
zinc metalloproteinase with the dibasic cleavage preferences, suggesting a high level of versatility of
metalloproteinase proteolysis. Based on these data, we now suggest that the combined (rather than individual) activity of MPII and FRA is required for the overall B. fragilis virulence in vivo.