doi: 10.1073/pnas.1904099116. Epub 2019 Jun 10.
Ruminococcus gnavus, a member of the human gut microbiome associated with Crohn's disease, produces an inflammatory polysaccharide
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- PMID:31182571
- PMCID: PMC6601261
- DOI: 10.1073/pnas.1904099116
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Ruminococcus gnavus, a member of the human gut microbiome associated with Crohn's disease, produces an inflammatory polysaccharide
Matthew T Henke et al. Proc Natl Acad Sci U S A..
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Abstract
A substantial and increasing number of human diseases are associated with changes in the gut microbiota, and discovering the molecules and mechanisms underlying these associations represents a major research goal. Multiple studies associateRuminococcus gnavus, a prevalent gut microbe, with Crohn's disease, a major type of inflammatory bowel disease. We have found thatR. gnavus synthesizes and secretes a complex glucorhamnan polysaccharide with a rhamnose backbone and glucose sidechains. Chemical and spectroscopic studies indicated that the glucorhamnan was largely a repeating unit of five sugars with a linear backbone formed from three rhamnose units and a short sidechain composed of two glucose units. The rhamnose backbone is made from 1,2- and 1,3-linked rhamnose units, and the sidechain has a terminal glucose linked to a 1,6-glucose. This glucorhamnan potently induces inflammatory cytokine (TNFα) secretion by dendritic cells, and TNFα secretion is dependent on toll-like receptor 4 (TLR4). We also identify a putative biosynthetic gene cluster for this molecule, which has the four biosynthetic genes needed to convert glucose to rhamnose and the five glycosyl transferases needed to build the repeating pentasaccharide unit of the inflammatory glucorhamnan.
Keywords: inflammatory bowel disease; microbiome; polysaccharide.
Copyright © 2019 the Author(s). Published by PNAS.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
R. gnavus produces an inflammatory glucorhamnan polysaccharide. (A) Schematic for generating fractions fromR. gnavus in culture were screened for the ability to induce the inflammatory cytokine TNFα in mBMDCs. (B) TNFα was secreted by mBMDCs that were treated withR. gnavus spent medium. This activity was resistant to proteinase-, nuclease-, lysozyme-, and heat treatment, indicating it was a polysaccharide. LPS was a positive control, and DMSO and media alone were negative controls. (C) HSQC spectrum zoom-in of anomeric region of purified polysaccharide shows the repeating unit is composed of five distinct monosaccharide types (labeled A through E). (D) The purified polysaccharide is a glucorhamnan, with a main backbone of rhamnose residues and short sidechains of glucose residues. The structure of the repeating unit is shown following three separate conventions, overlaid with the corresponding monosaccharide designation A through E.
R. gnavus glucorhamnan signals through TLR4 in a dose-dependent fashion in innate immune cells. (A)R. gnavus glucorhamnan stimulates mBMDCs to produce TNFα in dose-dependent manner roughly as potently as yeast mannan, a well-characterized inflammatory polysaccharide [error bars = SD of technical replicates (n = 4)]. (B) Secretion of TNFα by mBMDCs is lost in mice lacking TLR4 but not TLR2 (LPS) is a TLR4 ligand control; Pam3Cys is a synthetic TLR2 ligand control), n.d. = not detected; # indicates levels below 25 pg/mL detected.
Proposed biosynthetic pathway forR. gnavus glucorhamnan (A) A gene cluster (RUMGNA_03512 to RUMGNA_03534) encoding five glycosyltransferases and the biosynthetic enzyme for rhamnose is likely responsible for glucorhamnan synthesis. Five genes (designated by *) were transcribed during culture conditions (SI Appendix, Fig. S11 ). (B) A proposal for the biosynthesis of the glucorhamnan. Sequentially, monosaccharide precursors are synthesized, assembled into the repeating unit, transported across the membrane, where they are polymerized to form the full-length glucorhamnan, which is then covalently attached to the peptidoglycan.
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