Characterizing metabolic drivers of Clostridioides difficile infection with activity-based hydrazine probes.

Many enzymes require post-translational modifications or cofactor machinery for primary function. As these catalytically essential moieties are highly regulated, they act as dual sensors and chemical handles for context-dependent metabolic activity. is a major nosocomial pathogen that infects the colon. Energy generating metabolism, particularly through amino acid Stickland fermentation, is central to colonization and persistence of this pathogen during infection. Here using activity-based protein profiling (ABPP), we revealed Stickland enzyme activity is a biomarker for infection (CDI) and annotated two such cofactor-dependent Stickland reductases. We structurally characterized the cysteine-derived pyruvoyl cofactors of D-proline and glycine reductase in cultures and showed through cofactor monitoring that their activity is regulated by their respective amino acid substrates. Proline reductase was consistently active in toxigenic , confirming the enzyme to be a major metabolic driver of CDI. Further, activity-based hydrazine probes were shown to be active site-directed inhibitors of proline reductase. As such, this enzyme activity, its druggable cofactor modality, is a promising therapeutic target that could allow for the repopulation of bacteria that compete with for proline and therefore restore colonization resistance against in the gut.