Inducible CRISPR-targeted “knockdown” of human gut <i>Bacteroides</i> in gnotobiotic mice discloses glycan utilization strategies

Abstract

Understanding how members of the human gut microbiota prioritize nutrient resources is one component of a larger effort to decipher the mechanisms defining microbial community robustness and resiliency in health and disease. This knowledge is foundational for development of microbiota-directed therapeutics. To model how bacteria prioritize glycans in the gut, germfree mice were colonized with 13 human gut bacterial strains, including seven saccharolytic <i>Bacteroidaceae</i> species. Animals were fed a Western diet supplemented with pea fiber. After community assembly, an inducible CRISPR-based system was used to selectively and temporarily reduce the absolute abundance of <i>Bacteroides thetaiotaomicron</i> or <i>B. cellulosilyticus</i> by 10- to 60-fold. Each knockdown resulted in specific, reproducible increases in the abundances of other <i>Bacteroidaceae</i> and dynamic alterations in their expression of genes involved in glycan utilization. Emergence of these "alternate consumers" was associated with preservation of community saccharolytic activity. Using an inducible system for CRISPR base editing in vitro, we disrupted translation of transporters critical for utilizing dietary polysaccharides in <i>Phocaeicola vulgatus</i>, a <i>B. cellulosilyticus</i> knockdown-responsive taxon. In vitro and in vivo tests of the resulting <i>P. vulgatus</i> mutants allowed us to further characterize mechanisms associated with its increased fitness after knockdown. In principle, the approach described can be applied to study utilization of a range of nutrients and to preclinical efforts designed to develop therapeutic strategies for precision manipulation of microbial communities.