Extended Data Fig. 4: Sulfonation of isoalloLCA alters its biochemical properties.

a, Flow cytometry analysis and quantification of native CD4 + T-cells cultured under T_H0 conditions (anit-CD3, anti-CD28, and IL-2) conditions in the presence of DMSO, isoalloLCA, or isoalloLCA 3-sulfate for 72 hours are shown. Cells were stained with FOXP3 as a marker for T_reg cells (n = 3 biological replicates per group, data are shown as the mean ± SEM, one-way ANOVA followed by Tukey’s multiple comparison test). b, Previously elucidated biosynthetic pathway in Bacteroidetes species for the conversion of isoLCA (1) to isoalloLCA (5). The site of biotransformation is highlighted in red for each step. c, Extracted ion chromatograms (EICs) showing that B. theta Δtdk (WT) cultures incubated with isoLCA produced isoalloLCA (5) as well as the intermediates 3-oxoLCA (2), 3-oxo-Δ4-LCA (3), and 3-oxoalloLCA (4), supporting the hypothesis that subsequent actions of a 3β-hydroxysteroid dehydrogenase (HSDH), 5β-reductase, a 5α-reductase, and a 3β-HSDH are responsible for the conversion of isoLCA to isoalloLCA by B. theta. d, The composition of bile acid metabolites was determined for cultures of P. merdae ATCC 43184, P. merdae Δsult, and P. merdae ΔPARMER_04016-18 incubated for 48 hours with isoLCA (100 µM). We observed a complete loss of the sulfonated compound in P. merdae Δsult culture, while the P. merdae ΔPARMER_04016-18 culture exhibited a loss of isoLCA-derived intermediates and a marked decrease in production of sulfonated compound compared to the WT culture. The composition of LCA derived molecules was determined by UPLC-MS (n = 3 biological replicates). e, β-sitosterol-3-sulfate levels in the cecal contents of mice monocolonized with B. theta WT were elevated when compared to levels in B. theta KO colonized mice (B. theta WT n = 8, B. theta KO n = 9, results pooled from three separate experiments, data are presented as mean ± SEM).

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