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Diet-derived galacturonic acid regulates virulence and intestinal colonization in enterohaemorrhagic Escherichia coli and Citrobacter rodentium

Nature Microbiology volume 5, pages 368–378 (2020)Cite this article

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Abstract

Enteric pathogens sense the complex chemistry within the gastrointestinal tract to efficiently compete with the resident microbiota and establish a colonization niche. Here, we show that enterohaemorrhagic Escherichia coli and Citrobacter rodentium, its surrogate in a mouse infection model, sense galacturonic acid to initiate a multi-layered program towards successful mammalian infection. Galacturonic acid utilization as a carbon source aids the initial pathogen expansion. The main source of galacturonic acid is dietary pectin, which is converted to galacturonic acid by the prominent member of the microbiota, Bacteroides thetaiotamicron. This is regulated by the ExuR transcription factor. However, galacturonic acid is also sensed as a signal through ExuR to modulate the expression of the genes encoding a molecular syringe known as a type III secretion system, leading to infectious colitis and inflammation. Galacturonic acid acts as both a nutrient and a signal directing the exquisite microbiota–pathogen relationships within the gastrointestinal tract. This work highlights that differential dietary sugar availability influences the relationship between the microbiota and enteric pathogens, as well as disease outcomes.

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Fig. 1: ExuR activates expression of the LEE in EHEC.
Fig. 2: Galacturonic acid decreases LEE gene expression.
Fig. 3: Deletion of exuR results in decreased morbidity and colonization during mouse infection by C. rodentium.
Fig. 4: Chemically induced colitis via DSS treatment rescues colonization and pathogenesis of the exuR mutant.
Fig. 5: Regulation of the T3SS by ExuR in vivo is independent of its role in regulating galacturonic acid metabolism.
Fig. 6: Dietary pectin is the main source of galacturonic acid.

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Data availability

The data that support the findings of this study are available from the corresponding author upon request. RNA-sequencing data can be accessed at European Nucleotide Archive under accession number PRJEB30676.

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Acknowledgements

This study was supported by the National Institutes of Health grants AI053067, AI05135, AI077613 and AI114511 to V.S. A.G.J. was supported through National Institutes of Health Training Grant 5 T32 AI7520.

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Authors and Affiliations

  1. Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Angel G. Jimenez, Melissa Ellermann & Vanessa Sperandio

  2. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Angel G. Jimenez, Melissa Ellermann & Vanessa Sperandio

  3. Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada

    Wade Abbott

  4. Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada

    Wade Abbott

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  1. Angel G. Jimenez
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Contributions

A.G.J. conceived the studies, performed experiments and data analysis and wrote the paper. M.E. performed histological analysis and performed some mouse experiments. W.A. advised on experiments with Bt and pectin degradation. V.S. supervised all experiments, analysed data and wrote the paper.

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Correspondence to Vanessa Sperandio.

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Extended data

Extended Data Fig. 1 ExuR regulates genes encoding enzymes necessary for the catabolism of galacturonic-acid.

a, Schematic representation of ExuR repression of the galacturonic-acid utilization genes. b, qRT-PCR of the galacturonic utilization genes in WT (n = 3) and ΔexuR (n = 3) EHEC (n, number of biological replicates; results are representative of two independent experiments). The mean and P value for two-sided Mann-Whitney statistical analysis are shown. c, Competition EMSA of ExuR binding to the ler promoter in the absence and presence of ler cold probe. Results are representative of two independent experiments with similar results. d, qRT-PCR of the LEE espA gene in EPEC in the presence of vehicle (n = 6) or galacturonic-acid (GalA) (n = 6) (n, number of biological replicates; results are representative of two independent experiments). The mean and P value for two-sided Mann-Whitney statistical analysis are shown.

Source data

Extended Data Fig. 2 UxaC and galacturonic acid promotion of EHEC’s growth.

a, Growth curves of WT (n = 2) and ΔuxaC (n = 2) EHEC in M9 minimal medium with 5mM galacturonic-acid as a sole carbon source (n, number of biological replicates; results are representative of one independent experiments). The mean ± SD and P value for two-sided two-way ANOVA statistical analysis are shown. b, Growth curves of WT (n = 4) and ΔuxaC (n = 4) EHEC in low-glucose DMEM (n, number of biological replicates; results are representative of one independent experiments). The mean and P value for two-sided one-way ANOVA statistical analysis are shown. c, Growth of WT EHEC in low-glucose DMEM supplemented with different concentrations of galacturonic acid.

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Extended Data Fig. 3 ExuR also activates the LEE in C. rodentium.

a, Growth curve of WT (n = 3) and ΔexuR (n = 3) C. rodentium strains grown in low-glucose DMEM under microaerobic conditions (n, number of biological replicates; results are representative of one independent experiments). The mean ± SD value for two-sided one-way ANOVA statistical analysis are shown. b, RT-qPCR of the LEE-encoded genes in escC, escV, tir, and espA in WT (n = 9) and ΔexuR (n = 9) C. rodentium (n, number of biological replicates; results are representative of three independent experiments). The mean and P value for two-sided two-way ANOVA statistical analysis are shown. c, Western blot for secreted EspB in WT and ΔexuR C. rodentium. Representative blots from three independent experiments. d, Growth curves of C. rodentium with glucose (Glu) (n = 3), galacturonic acid (GalA) (n = 3) or glucuronic acid (GlcA) (n = 3) as sole carbon sources (n, number of biological replicates; results are representative of three independent experiments). The mean ± SD value for two-sided one-way ANOVA statistical analysis are shown.

Source data

Extended Data Fig. 4 Galacturonic-acid acts through ExuR to decrease LEE gene expression.

Western blot of supernatants from EHEC WT, ΔexuR, ΔuxuR and ΔexuRuxuR (ΔΔ) grown in the presence of glucose (Glu), galacturonic-acid (GalA), or glucuronic acid (GlcA) in DMEM probed with anti-EspA antiserum. BSA is used as a loading control. Representative blots from three independent experiments.

Extended Data Fig. 5 Mice infected with ΔuxaC C. rodentium have only a mild increase in inflammation.

C3H/HeJ mice under non-infected conditions as well as at post-infection day 8 with WT or the ΔexuR, ΔuxaC and ΔexuRΔuxaC mutants. a, Haematoxylin-eosin-stained cecal patch tissues of C3H/HeJ mice. Representative images from two independent experiments, scale bars = 100 µm. b, Blinded histopathology scores of non-infected mice or infected with WT (n = 4) or the ΔexuR (n = 4), ΔuxaC (n = 4), and ΔexuRΔuxaC (n = 4) C. rodentium (n, number of biological replicates; results are representative of two independent experiments). The mean and P value for two-sided one-way ANOVA statistical analysis.

Extended Data Fig. 6 Pectin engenders inflammation in C3H/HeJ mice.

C3H/HeJ mice were treated with 200µL of 2% pectin or PBS. a, Blinded histopathology scores of non-infected mice treated with 200µL of 2% pectin (n = 4) or PBS (n = 4) (n, number of biological replicates; results are representative of two independent experiments). The mean and P value for two-sided one-way ANOVA statistical analysis. b-c, RT-qPCR determined the expression of Nos2 and IL22 genes in the cecal tissue of mice treated with 200µL of 2% pectin (n = 4) or PBS (n = 4) (n, number of biological replicates; results are representative of two independent experiments). The mean and P value for two-sided Mann-Whitney statistical analysis are shown. GAPDH was used to normalize gene expression.

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Jimenez, A.G., Ellermann, M., Abbott, W. et al. Diet-derived galacturonic acid regulates virulence and intestinal colonization in enterohaemorrhagic Escherichia coli and Citrobacter rodentium. Nat Microbiol 5, 368–378 (2020). https://doi.org/10.1038/s41564-019-0641-0

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