Defining enteric bacterial pathogenesis using organoids: uses EspC, an atypical mucinolytic protease, to penetrate mouse colonic mucus.

Enteric bacterial pathogens pose significant threats to human health; however, the mechanisms by which they infect the mammalian gut in the face of daunting host defenses remain to be fully defined. For the attaching and effacing (A/E) bacterial family member and murine pathogen , its virulence strategy appears to involve penetration of the colonic mucus barrier to reach the underlying epithelium. To better define these interactions, we grew colonoids under air-liquid interface (ALI) conditions, producing a thick mucus layer that mimicked mucus composition and glycosylation. 's penetration of ALI-derived mucus was dramatically enhanced upon exposure to sialic acid, in concert with the secretion of two serine protease autotransporter of (SPATE) proteins, Pic and EspC. Despite Pic being a class II SPATE, and already recognized as a mucinase, it was EspC, a class I SPATE family member, that degraded ALI-derived mucus, despite class I SPATEs not previously shown to possess mucinase activity. Confirming this finding, DH5α carrying a plasmid that expresses -derived EspC was able to degrade the mucus. Moreover, recombinant EspC alone also displayed mucinolytic activity in a dose-dependent manner. Collectively, our results reveal the utility of ALI-derived mucus in modeling microbe-host interactions at the intestinal mucosal surface, as well as identify EspC as an atypical class I SPATE that shows significant mucinolytic activity toward ALI-derived mucus.