Article Figures & Data
Figures
- FIG 1
Metagenomic (16S rRNA) analysis of fecal microbiota from DEHP-exposed mice. (A) PCA on OTU counts. The percentages represent the percent variances explained by each axis. Alpha-diversity (B) and LefSe analysis (C) of fecal microbiota OTU counts. *, P < 0.05 by two-tailed t test.
- FIG 2
Metabolite analysis of fecal microbiota from DEHP-exposed mice. (A) LC-MS identification of MEHP in fecal material collected at days 7 and 14 from animals fed a low (+) or high (++) dose of DEHP. The level of MEHP in control samples (−) was below the limit of detection. (B) Scatter plot of the first two PC scores from PCA of the metabolite data.
- FIG 3
Metagenomic analysis of in vitro cecal luminal contents culture. (A) Phylum-level classification of unique OTUs in DEHP-treated cultures. (B) Relative abundance of bacterial genera on days 1 and 7 in control (GMM) and DEHP-treated cultures.
- FIG 4
Metabolite profiles from in vitro cultured cecal luminal contents. (A) Scatter plot of the first two scores from PCA representing microbial metabolites produced on day 1 and day 7. (B) Heat map of detected ion peaks with different patterns of substrate utilization and product formation. (C) Percentage distribution of detected features classified as products, substrates, or intermediates based on their time profiles. (D) Profiles of tryptophan and indole in the cecal luminal content cultures. Filled and open markers represent inoculated cultures and tubes incubated without luminal contents, respectively. Triangles and circles represent day-1 and -7 time points, respectively. The colors correspond to the classifications in the heat map and pie chart. *, P < 0.05 compared to inoculated culture at day 1 (two-tailed t test).
- FIG 5
Model of metabolic reactions in in vitro culture of cecal luminal contents. (A) Fraction of genus-level OTU counts represented by the metabolic model. (B) Hierarchical clustering of genera and metabolites in the model. (C) Correlation network showing significant Pearson correlations (absolute PCC ≥ 0.76, P < 0.05) between genera (circles) and metabolites (squares). Fold change from days 1 to 7 is indicated by red (decrease) and green (increase) colors. Solid lines between nodes indicate that the genus has at least one species capable of metabolizing the connected metabolite (per database annotation of the genome), while dotted lines indicate a purely empirical correlation.
- FIG 6
Significant microbial and metabolite changes in in vitro-cultured cecal luminal contents with DEHP. (A) LefSe analysis of genus-level microbiota changes induced by DEHP. *, P < 0.05 by two-tailed t test. (B) Scatter plot of first two PC scores from PCA of metabolite features detected in the cecal cultures (positive-mode IDA data). (C) Dose-dependent changes in p-cresol and butyric acid with DEHP on day 7. *, P < 0.05 compared to day 7 culture without DEHP addition (two-tailed t test).
Supplemental Material
FIG S1
Scatter plot of latent variable (LV) scores from PLS-DA on OTU counts for fecal samples. R2X (cumulative) = 0.145, R2Y (cumulative) = 0.989, Q2 (cumulative) = 0.714, and root-mean-square error of estimation (RMSEE) = 0.124. Download FIG S1, EPS file, 0.4 MB.
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TABLE S1
Putatively identified compounds in cecal culture. Download Table S1, DOCX file, 0.04 MB.
Copyright © 2019 Lei et al.
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TABLE S2
Confidently identified (level 2) and confirmed metabolites (level 1). Confident identification (level 2) refers to metabolites matching a standard by at least two of the following measures: accurate mass, MS/MS (external reference standards), and retention time (RT). Confirmed identification (level 1) refers to metabolites matching a standard by at least two of the following measures: accurate mass, MS/MS (internal reference standards), and retention time (RT). Download Table S2, DOCX file, 0.02 MB.
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TEXT S1
Methods for Lachnoclostridium bolteae monoculture and untargeted LC-MS experiments. Download Text S1, DOCX file, 0.01 MB.
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FIG S2
Conversion of DEHP to MEHP in cecal content culture. Error bars show one standard deviation (N = 5 independently inoculated cultures). +, 10 μM DEHP at the start of each batch cecal culture; ++, 100 μM DEHP at the start of each batch cecal culture; −, vehicle control; *, P < 0.05 compared to corresponding control samples by two-tailed t test. Download FIG S2, EPS file, 0.2 MB.
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FIG S3
Local linear regression of sample retention time against retention time of pure standard. Download FIG S3, EPS file, 0.3 MB.
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FIG S4
Production of p-hydroxyphenylacetic acid by L. bolteae. Bar graphs represent tyrosine concentration (A), p-hydroxyphenylacetic acid concentration (B), and cell density (C) after a 48-h incubation under anaerobic conditions. Data shown are means from 6 independently inoculated cultures, except vehicle controls. One vehicle control was incubated for each experimental group. Error bars represent one standard deviation. *, P < 0.05 compared to corresponding control samples by two-tailed t test. Download FIG S4, EPS file, 0.2 MB.
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FIG S5
Model of DEHP-induced increase in p-cresol production. Exposure to DEHP expands select species, e.g., L. bolteae, that synthesize p-hydroxyphenylacetate, the immediate precursor of p-cresol. The conversion of p-hydroxyphenylacetate to p-cresol likely takes place in other species, as L. bolteae lacks the required enzyme, hydroxyphenylacetate decarboxylase (Hpd). The enzyme has been detected in a number of gut bacteria, including species belonging to the genera Olsenella and Blautia, which were detected in the cecal culture. Download FIG S5, EPS file, 0.3 MB.
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FIG S6
Mirror plots of sample (red) and standard (blue) MS/MS spectra for confidently identified and confirmed metabolites. x and y axes show the m/z values and relative intensity of each peak, respectively, in the MS/MS spectrum. Metabolites are listed in order of increasing m/z. Download FIG S6, EPS file, 0.3 MB.
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FIG S7
Representative extracted ion chromatograms (±5 ppm) of metabolites with adjusted retention times based on local linear regression (see Fig. S3). Insets show MS/MS mirror plots. Metabolites are listed in order of increasing m/z. Download FIG S7, EPS file, 0.9 MB.
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