Abstract
Several enteric pathogens can gain specific metabolic advantages over other members of the microbiota by inducing host pathology and inflammation. The pathogen Clostridium difficile is responsible for a toxin-mediated colitis that causes 450,000 infections and 15,000 deaths in the United States each year1; however, the molecular mechanisms by which C. difficile benefits from this pathology remain unclear. To understand how the metabolism of C. difficile adapts to the inflammatory conditions that its toxins induce, here we use RNA sequencing to define, in a mouse model, the metabolic states of wild-type C. difficile and of an isogenic mutant that lacks toxins. By combining bacterial and mouse genetics, we demonstrate that C. difficile uses sorbitol derived from both diet and host. Host-derived sorbitol is produced by the enzyme aldose reductase, which is expressed by diverse immune cells and is upregulated during inflammation—including during toxin-mediated disease induced by C. difficile. This work highlights a mechanism by which C. difficile can use a host-derived nutrient that is generated during toxin-induced disease by an enzyme that has not previously been associated with infection.
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Data availability
Raw RNA-seq source data are available through the NCBI Sequence Read Archive. In vivo RNA-seq (Figs. 1b, 2a, Extended Data Fig. 1c, d, Supplementary Table 2) is available under accession number PRJNA666929, and in vitro RNA-seq (Extended Data Fig. 6, Supplementary Table 3) under accession number PRJNA667108. Publicly available single-cell RNA-seq data (Extended Data Fig. 7b–e) can be obtained from the Single Cell Portal (Broad Institute) under accession numbers SCP259 and SCP241. Microarray data (Fig. 3g) can be found in the Gene Expression Omnibus under accession number GSE44091. Source data are provided with this paper.
Code availability
The code used during this study is available at https://github.com/kpruss/Cdiff-AR.
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Acknowledgements
We thank S. Higginbottom for assistance with all mouse experiments. A. Chien assisted with the development of the GC–MS protocol; S. Kuehne and N. Minton provided the toxin-mutant C. difficile strains; A. Shen provided reagents for the generation of new C. difficile mutants; A. Bhatnagar and D. Mosely provided the aldose reductase knockout mice; and D. Davis shared protocols and advice regarding the development of the streptozotocin model of hyperglycaemia. This study was supported by R01-DK08502510 (with thanks to B. Karp for service and support at NIDDK) and the Chan Zuckerberg Biohub, and a Ford Foundation Pre-Doctoral Fellowship and NSF Graduate Research Fellowship to K.M.P. We thank all members of the Sonnenburg laboratory, who provided feedback throughout the development of the project.
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K.M.P. and J.L.S. conceived the project idea, designed the experiments and wrote the manuscript. K.M.P. executed the experiments and performed data analysis.
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Extended data figures and tables
Extended Data Fig. 1 C. difficile toxin production confers an advantage and alters metabolic pathways in vivo.
a, Toxin production (WT) confers an advantage in C. difficile relative abundance in the presence of a defined community (n = 5 mice per group, mean ± s.e.m., multiple unpaired t-tests with Welch’s correction, two-stage step-up procedure of Benjamini, Krieger and Yekutieli.). b, Transcriptional profiling experimental design: germ-free mice on standard diet were mono-colonized with either wild-type C. difficile 630∆erm (WT) or 630∆ermTcdA−TcdB− (Tox−). Three days post-infection, total RNA was isolated from caecal contents for RNA-seq. Created with BioRender.com. c, d, Significantly enriched Ecocyc (c) and KEGG (d) pathways based on genes differentially expressed during wild-type (positive, red bars, n = 4 mice) or Tox− C. difficile (negative, purple, n = 3 mice) infection (hypergeometric distribution followed with FDR correction).
Extended Data Fig. 2 Sorbitol impacts C. difficile growth, gene expression, and increases in the mouse gut after antibiotic treatment.
a, Schematic overview of the sorbitol utilization locus in C. difficile. The operon contains three PTS transporter subunits, a 6-phosphate dehydrogenase, an activator and an anti-terminator. b, Wild-type (red) and toxin-deficient (Tox−, purple) C. difficile grow comparably in minimal medium supplemented with various concentrations of sorbitol (mean ± s.e.m., n = 5 replicates per condition). c, The ∆srlD mutant is unable to achieve increased growth yield with 0.25% or 0.125% w/v sorbitol supplemented to minimal medium (mean ± s.e.m., n = 5 replicates per condition). d, Addition of sorbitol to minimal medium leads to upregulation of genes in the operon (srlD, annotated as sorbitol dehydrogenase; srlA, PTS transporter subunit; srlR, transcription anti-terminator) compared to base medium (mean ± s.e.m., n = 3 replicates per condition. Expression levels normalized to wild-type C. difficile in unsupplemented base medium, dotted line indicates baseline expression of 1; srlD: unpaired two-tailed t-test, srlA and srlR: one-way ANOVA with Tukey’s post hoc comparisons; srlA: F(3,8) = 31.85, srlR: F(3,8) = 27.25). e, Sorbitol administered to mice mono-colonized with wild-type C. difficile leads to induction of srlD in vivo (mean ± s.e.m., n = 4 per group, unpaired two-tailed t-test). f, Clindamycin (1 mg) treatment (n = 5) leads to increased sorbitol and mannitol in stool from conventional mice on standard diet (n = 3, mean ± s.e.m., two-tailed Mann–Whitney U-test. Sorbitol levels were below the limit of detection for two of three pre-antibiotic treatment samples and are denoted by squares at a value of 1. Samples are combined from 3 independent experiments). g, The ∆srlD C. difficile mutant is attenuated in colonization of conventional mice fed a standard diet compared to wild-type C. difficile (n = 5 mice per group, mean ± s.e.m., unpaired two-tailed t-test with Welch’s correction). h, Toxin B detected by ELISA in faecal pellets of conventional mice 24 h post-infection with wild-type or ∆srlD C. difficile; values were normalized to the absolute abundance of C. difficile from the same stool sample (n = 5 mice per group, mean ± s.e.m., unpaired two-tailed t-test with Welch’s correction).
Extended Data Fig. 3 Dietary sorbitol or mannitol availability increases C. difficile density in vivo.
a, Sorbitol (1%) (green, n = 4 mice) or mannitol (1%) (purple, n = 5 mice) were provided in drinking water to gnotobiotic mice harbouring a defined consortium of bacteria for 6 days (days 0–6). Absolute abundance of wild-type C. difficile decreases when sorbitol and mannitol are removed from drinking water (days 7–10). Replacing 1% sorbitol and mannitol in drinking water (days 11–14) restores the increase in absolute abundance (mean ± s.e.m., shaded boxes denote sorbitol or mannitol supplementation). b, Sorbitol (1%) was provided in drinking water (days 0–6, green box) to mice colonized with a defined community and subsequently infected with ∆srlD C. difficile. Supplementation of 1% mannitol in drinking water leads to an increase in abundance of the ∆srlD mutant (days 11–14, purple box) relative to sorbitol supplementation (mean ± s.e.m., n = 5 mice). c, ∆srlD C. difficile (n = 5 mice) incites a lower histopathological score than wild-type C. difficile (n = 4 mice) when 1% sorbitol is supplemented in drinking water (12 days post-infection, mean + s.e.m., unpaired two-tailed Student’s t-test). d, No significant differences in blinded histopathological scoring in the caecal blind tip of mice infected with wild-type C. difficile when sorbitol (n = 4) or mannitol (n = 5) is supplemented (as in a) or when mannitol is supplemented to ∆srlD C. difficile (as in b, n = 5, 14 days post-infection, mean ± s.e.m.).
Extended Data Fig. 4 Excess sorbitol represses toxin production in vitro and in vivo.
a, Minimal medium supplemented with 1% or 0.5% sorbitol leads to significantly lower expression of tcdC and tcdA after 8 h growth compared to un-supplemented base medium (mean ± s.e.m., n = 4 replicates per condition. Two-way ANOVA across genes: F(3,36) = 3.429, P = 0.0271; across sorbitol supplementations: F(2,36) = 11.17, P = 0.0002 with Dunnett’s multiple comparisons test using base medium as the control for sorbitol supplementation comparisons within each gene). b, Presence of sorbitol (n = 4 mice) or mannitol (n = 5 mice) in drinking water leads to relatively lower toxin production in vivo (days 4 and 12, sugar alcohol supplementation denoted by shaded boxes) compared to when sorbitol or mannitol are absent (day 7; mean ± s.e.m., mixed effects analysis with Sidak’s multiple comparisons: day is significant F(0.8915,7.132) = 18.37, P = 0.004, mannitol versus sorbitol is not). c, Addition of exogenous mannitol (day 12) leads to lower production of toxin in vivo in the ∆srlD mutant compared to sorbitol supplementation (days 1, 4) or regular water (day 7; n = 5 mice, mean ± s.e.m., one-way ANOVA F(2,8) = 45.18 with Tukey’s post hoc multiple comparisons. Day 4 was excluded from the ANOVA, as only 2 data points are present).
Extended Data Fig. 5 Streptozotocin treatment increases fasting blood glucose levels in conventional and mono-colonized mice.
a, Development of streptozotocin (STZ)-induced hyperglycaemia model in Swiss-Webster Excluded Flora mice. Mice were fasted for 4–6 h before measurement of blood glucose levels via tail vein snip. An initial injection (day 0, indicated by dashed arrow) of 4.5 mg STZ was insufficient to increase blood glucose levels. A larger dose of 9.1 mg STZ administered on day 4 (solid arrow) was sufficient to increase blood glucose (mean ± s.e.m., n = 6 mice) and was used for subsequent experiments with C. difficile infection. b, Unfasted blood glucose in germ-free mice mono-colonized with wild-type or ∆srlD at 3 days post-infection (mean ± s.e.m., one-way ANOVA F(3,20) = 36.73 with Tukey’s post hoc comparisons); STZ-treated groups (n = 7 mice per group) were treated with STZ via intraperitoneal injection 4 days before C. difficile infection and had significantly increased blood glucose compared to untreated controls (n = 5 per group). c, C. difficile gene expression of the sorbitol utilization locus in conventional (wild-type) or streptozotocin-treated (STZ) mice. An outlier (Fig. 3c, tested for with robust nonlinear regression, Q = 0.2%) from one RNA sample isolated from one mouse is indicated by the filled circle (n = 5 mice per group, bars denote median). d, Streptozotocin treatment does not alter toxin production in vivo. C. difficile toxin B quantified in the faeces of conventional mice infected with wild-type C. difficile 24 h post-infection (n = 5 mice per group, mean ± s.e.m.).
Extended Data Fig. 6 Sorbitol and mannitol lead to distinct metabolic programs in vitro.
a, Chemical structures of isomers sorbitol and mannitol. b, Sorbitol and mannitol added to minimal medium engender distinct growth kinetics (mean ± s.e.m., n = 5 replicates per condition). c, Principal component analysis of variance stabilizing-transformed RNA-seq counts from C. difficile grown for 11 h in minimal medium (control, grey), or minimal medium supplemented with 0.25% sorbitol (green), mannitol (purple) or glucose (yellow). d, Significantly differentially expressed genes between sorbitol supplementation and base medium or mannitol supplementation (n = 3 replicates per condition; colours represent row-normalized variance stabilizing-transformed counts. P < 0.01, Wald test with Bonferroni-adjusted P value.) e, Mannitol supplementation to 0.3% soft agar plates leads to significantly increased motility compared to base medium (days 3–6) and sorbitol supplementation (day 5). Sorbitol supplementation does not lead to a significant increase in motility compared to unsupplemented motility plates (n = 4 replicates per condition, mean ± s.e.m., two-way ANOVA significant by day F(4,44) = 53.50 and growth condition F(2,44) = 25.55, Tukey’s post hoc comparisons).
Extended Data Fig. 7 Aldose reductase is an immune cell-associated gene.
a, Top 10 cell types with highest Akr1b3 expression across 20 mouse organs demonstrates high prevalence of aldose reductase in immune-associated cell types24. b, Percentage of cells in mouse colonic tissue expressing isoforms of aldose reductase and sorbitol dehydrogenase. c, Percentage of different cell types in human colonic explants expressing the three isoforms of aldose reductase and sorbitol dehydrogenase. d, Akr1b1 expression (log2-TP10K+1) in cell types exhibiting significantly increased aldose reductase expression in inflammatory colonic explants from patients with ulcerative colitis (inflamed) compared to within-subject non-inflamed tissue (uninflamed) versus healthy controls that do not have ulcerative colitis (healthy; pairwise Wilcoxon-rank sum test across all immune cell types using non-zero expression levels. Means for each cell type are shown). e, Dendritic cells (DC) and plasma cells in mouse large intestine exhibited a significant increase in Akr1b3 expression (log2-TPM+1) during infection with H. polygyrus (Wilcoxon-rank sum test across all immune cell types using non-zero expression levels. Means for each cell type shown). f, Expression of Akr1b3 in the proximal colon of conventional mice infected with wild-type C. difficile. An outlier (Fig. 3e, detection method: robust nonlinear regression, Q = 0.2%) is denoted by the filled point, bars denote median.
Extended Data Fig. 8 Epalrestat inhibits C. difficile growth in vitro and in vivo.
a, Germ-free mice were mono-colonized with wild-type C. difficile and gavaged with the aldose reductase inhibitor epalrestat or vehicle control once per day. Epalrestat treatment significantly reduces C. difficile abundance (n = 5 mice per group, mean ± s.e.m., unpaired two-tailed t-tests). b, In the presence of epalrestat, wild-type C. difficile produces relatively more toxin in vivo (n = 5 mice per group, mean ± s.e.m., unpaired two-tailed t-test). c, The ∆srlD mutant colonizes germ-free mice fed standard diet equally well as does wild-type C. difficile (n = 5 mice per group, mean ± s.e.m.). d, Epalrestat inhibits growth of wild-type C. difficile 630∆erm in rich medium in a dose-dependent manner (n = 5 replicates per condition, mean ± s.e.m.). e, Absolute abundance of wild-type C. difficile after 6 h or 12 h of growth in rich medium is inhibited due to incubation with epalrestat (mean + s.e.m., n = 3 replicates per condition). f, Chemical structures of aldose reductase inhibitor epalrestat and an antibiotic with activity against cis-prenyl transferase undecaprenyl diphosphate synthase (UPPS).
Extended Data Fig. 9 A model of sorbitol utilization by C. difficile.
C. difficile can utilize diet-derived sorbitol, which spikes after disturbance to the microbiota (left). Toxin-induced tissue damage (right) leads to upregulation of host aldose reductase in the epithelium as well as recruitment of immune cells that express aldose reductase. C. difficile is able to utilize host-derived sorbitol. Created with BioRender.com.
Supplementary information
Supplementary Information
This file contains additional discussion of results for Extended Data Figs. 6 and 8.
Supplementary Table 1
Detailed results of blinded histopathological scoring for mice mono-colonized with WT or Tox- Cd.
Supplementary Table 2
DESeq2 in vivo differential gene expression analysis from mice mono-associated with WT or Tox- Cd.
Supplementary Table 3
In vitro differential gene expression analysis comparing different carbohydrate supplements to minimal medium.
Supplementary Table 4
A list of primers used in the study.
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Pruss, K.M., Sonnenburg, J.L. C. difficile exploits a host metabolite produced during toxin-mediated disease. Nature 593, 261–265 (2021). https://doi.org/10.1038/s41586-021-03502-6
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DOI: https://doi.org/10.1038/s41586-021-03502-6
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