Human gut bacteria produce structurally related monoglycolipids with contrasting immune functions.

Gut symbiont Bacteroides fragilis can produce α-galactosylceramides (BfaGCs), sphingolipids with immunomodulatory functions that regulate colonic natural killer T (NKT) cells. However, their synthesis pathway and whether other human gut bacteria can produce them are unclear. Here, using genetic and metabolomic approaches, we mapped the sphingolipid biosynthesis pathway of B. fragilis and determined that α-galactosyltransferase (agcT) is essential and sufficient for colonic NKT cell regulation in mice. The distribution of agcT is restricted to only a few species among Bacteroidales. However, structural homologues of AgcT, such as BgsB, are widely distributed in gut microbiota and produce α-glycosyldiacylglycerols (aGDGs), particularly in Enterococcus. Analysis of infant gut metagenomes revealed that B. fragilis predominantly accounts for agcT abundance regardless of the cohort, but bgsB-encoding bacteria were taxonomically diverse and showed dynamic changes with host age. In addition, aGDGs from bgsB-encoding species act as antagonistic ligands for BfaGC-mediated NKT cell activation in vitro and in vivo. Our findings highlight the distinct natures of immunoactive glycolipid-producing symbionts and their relevance in the human gut microbiome, particularly in early life.