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Title: Chemoproteomic Discovery of Host and Pathogen Enzymes Active in Cholera
Activity-based protein profiling (ABPP) is a powerful chemoproteomic tool for detecting active enzymes in complex biological systems. Small-molecule probes that are chemically tuned to react with the active site of a particular enzyme class facilitate selective detection, enrichment, and mass spectrometry-based identification of labeled proteins. Because these probes only react with functional enzymes, ABPP can distinguish between enzymes that are active and those that are expressed, but inactive, under a particular set of conditions. ABPP was used to identify secreted bacterial and host serine hydrolases that are active in animals infected with the cholera pathogen Vibrio cholerae. Four V. cholerae proteases were consistently active in infected rabbits, and one, VC0157 (renamed IvaP), was also active in human cholera stool. Inactivation of IvaP influenced the activity of other secreted V. cholerae and rabbit enzymes in vivo, while genetic disruption of all four proteases increased the abundance and binding of an intestinal lectin—intelectin—to V. cholerae in infected rabbits. Intelectin also bound to other enteric bacterial pathogens, suggesting it may constitute a previously unrecognized mechanism of bacterial surveillance in the intestine that is inhibited by pathogen-secreted proteases. Collectively, these findings demonstrate the power of activity-based proteomics to define host-pathogen enzymatic dialogue in an animal model of bacterial infection.