<i>In situ</i> identifying sennoside A-reducing bacteria guilds in human gut microbiota via enzymatic activity visualization

The intestinal environment determines the biochemical activity of individual microbial strains. The functions of microbial strains in their native environments often cannot be accurately predicted based solely on genomic information or the biochemical properties of cultivated isolates. In some cases, even the activity of intracellular enzymes does not correlate with the functional activity of bacteria. To address this challenge, we developed an enzymatic activity visualization platform that uses active intracellular enzymes as molecular baits to capture specific substrate probes. This platform links enzyme-mediated biochemical activity to their <i>in situ</i> localization, isolation, phylogenetic identification, and functional validation. This platform uses substrate-based probes designed to target isozymes, namely enzymes with different sequences but capable of catalyzing the same chemical reactions on a given substrate. Combined with a highly specific alkyne-azide cycloaddition reaction, these probes enable fluorescent visualization of strains harboring isozymes in human gut microbiota under anaerobic conditions. In addition, the platform can be integrated with fluorescence-activated cell sorting and 16S rRNA amplicon sequencing to isolate and taxonomically identify functional guilds containing isozymes physically. By performing metabolic capacity tests, fluorescence imaging, and proteomic analyses on four positive and three negative reference strains, we validated that our probes exhibit selectivity at the bacterial cell level. Using the platform, we successfully identified functional guilds involved in the reduction of sennoside A, a widely used laxative prodrug activated by gut bacteria. Importantly, we found that phylogenetically distinct bacteria perform similar metabolic activities toward sennoside A and discovered a novel sennoside A-reducing enzyme, StNfrA, from these functional guilds. The mechanistic study on StNfrA proved that our platform distinguished sennoside A-reducing bacteria species from microbiota by the enzymatic activity. Overall, these findings demonstrate that this enzymatic activity visualization platform is a powerful tool for the reliable localization, isolation, and identification of functional guilds in complex microbial communities.