Functional characterization of a subtilisin-like serine protease from .

, the causative agent of the human diarrheal disease cholera, exports numerous enzymes that facilitate its adaptation to both intestinal and aquatic niches. These secreted enzymes can mediate nutrient acquisition, biofilm assembly, and interactions with its host. We recently identified a secreted serine protease, IvaP, that is active in infected rabbits and human choleric stool. IvaP alters the activity of several host and pathogen enzymes in the gut and, along with other secreted proteases, decreases binding of intelectin, an intestinal carbohydrate-binding protein, to IvaP bears homology to subtilisin-like enzymes, a large family of serine proteases primarily comprised of secreted endopeptidases. Following secretion, IvaP is cleaved at least three times to yield a truncated enzyme with serine hydrolase activity, yet little is known about the mechanism of extracellular maturation. Here, we show that IvaP maturation requires a series of sequential N- and C-terminal cleavage events congruent with the enzyme's mosaic protein domain structure. Using a catalytically inactive reporter protein, we determined that IvaP can be partially processed in , but intramolecular proteolysis is most likely required to generate the mature enzyme. Unlike many other subtilisin-like enzymes, the IvaP cleavage pattern is consistent with stepwise processing of the N-terminal propeptide, which could temporarily inhibit, and be cleaved by, the purified enzyme. Furthermore, IvaP was able to cleave purified intelectin, which inhibited intelectin binding to These results suggest that IvaP plays a role in modulating intelectin- interactions.