Inducible CRISPR-targeted “knockdown” of human gut Bacteroides 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 Bacteroidaceae 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 Bacteroides thetaiotaomicron or B. cellulosilyticus by 10- to 60-fold. Each knockdown resulted in specific, reproducible increases in the abundances of other Bacteroidaceae 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 Phocaeicola vulgatus , a B. cellulosilyticus knockdown-responsive taxon. In vitro and in vivo tests of the resulting P. vulgatus 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.

Dextran sodium sulfate-induced colitis alters the proportion and composition of replicating gut bacteria

“…These findings were further corroborated by the negative correlation between the minimal doubling time (DT) of a MAG and its median relative abundance during the symptomatic period, suggesting that taxa with the ability to replicate quickly are present at higher abundances during colitis. This is in line with previous research which suggests that faster replicating bacteria can adapt more quickly to changing environments, 33,135,136 that gut bacteria during IBD flares may be replicating more than during remission, 137 and that fast replication is one strategy gut bacteria can use to maintain or increase their biomass when cell turnover and intestinal sloughing are high 41 as in DSS-induced colitis. 22,23,138,139…”

The RNA-binding protein RbpB is a central regulator of polysaccharide utilization in gutBacteroides

“…The composition of the gut microbial community and, therefore, the pivotal functions these microbes provide to human health hinges on their ability to persist in the face of nutritional fluctuation. Understanding how gut commensals adapt their metabolism to the daily variations in feeding rhythm and types of diet has thus become an important branch of microbiota research 43,44 . The predominant bacterial genus in the healthy human gut microbiota, Bacteroides , possesses dozens of multi-protein complexes encoded on dedicated genomic loci— the PULs—to bind, clip, and import specific polysaccharides.…”

Commensal-pathogen dynamics structure disease outcomes duringClostridioides difficilecolonization

“…Stool gavage material used for animal experiments (from donors 4, 13, 15, and 16) was used to culture taxa representative of microbiomes of this patient population. For each donor, ethanol-treated stool 58 was plated on LYBHI 59 containing 0.1% sodium taurocholate, and non-ethanol-treated stool was plated on LYBHI in 5-6 10-fold dilutions. For ethanol-treated stool, stool was resuspended in an equal volume of 100% ethanol and left to sit for 4 hours.…”