Dataset Information

FluCell-SELEX Aptamers as Specific Binding Molecules for Diagnostics of the Health Relevant Gut Bacterium Akkermansia muciniphila.

ABSTRACT: Based on their unique properties, oligonucleotide aptamers have been named a gift of biological chemistry to life science. We report the development of DNA aptamers as the first high-affinity binding molecules available for fast and rapid labeling of the human gut bacterium Akkermansia muciniphila with a certain impact on Alzheimer´s disease. Fast and reliable analyses of the composition of microbiomes is an emerging field in microbiology. We describe the molecular evolution and biochemical characterization of a specific aptamer library by a FluCell-SELEX and the characterization of specific molecules from the library by bioinformatics. The aptamer AKK13.1 exerted universal applicability in different analysis techniques in modern microbiology, including fluorimetry, confocal laser scanning microscopy and flow cytometry. It was also functional as a specific binding entity hybridized to anchor primers chemically coupled via acrydite-modification to the surface of a polyacrylamide-hydrogel, which can be prototypically used for the construction of affinity surfaces in sensor chips. Together, the performance and methodological flexibility of the aptamers presented here may open new routes not only to develop novel Akkermansia-specific assays for clinical microbiology and the analyses of human stool samples but may also be an excellent starting point for the construction of novel electronic biosensors.

SUBMITTER: Raber HF

PROVIDER: S-EPMC8509069 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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FluCell-SELEX Aptamers as Specific Binding Molecules for Diagnostics of the Health Relevant Gut Bacterium <i>Akkermansia muciniphila</i>.

Raber Heinz Fabian HF Kubiczek Dennis Horst DH Bodenberger Nicholas N Kissmann Ann-Kathrin AK D'souza Deena D Xing Hu H Mayer Daniel D Xu Pengfei P Knippschild Uwe U Spellerberg Barbara B Weil Tanja T Rosenau Frank F

International journal of molecular sciences 20210927 19

Based on their unique properties, oligonucleotide aptamers have been named a gift of biological chemistry to life science. We report the development of DNA aptamers as the first high-affinity binding molecules available for fast and rapid labeling of the human gut bacterium <i>Akkermansia muciniphila</i> with a certain impact on Alzheimer´s disease. Fast and reliable analyses of the composition of microbiomes is an emerging field in microbiology. We describe the molecular evolution and biochemic ...[more]

PMID: 34638764

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Project description:The beneficial human gut bacterium Akkermansia muciniphila provides metabolites to other members of the gut microbiota by breaking down host mucin, but most of its other metabolic functions have not been investigated. A. muciniphila strain MucT is known to use cobamides, the vitamin B12 family of cofactors with structural diversity in the lower ligand. However, A. muciniphila MucT is unable to synthesize cobamides de novo, and the specific forms that can be used by A. muciniphila have not been examined. We found that the levels of growth of A. muciniphila MucT were nearly identical with each of seven cobamides tested, in contrast to nearly all bacteria that had been studied previously. Unexpectedly, this promiscuity is due to cobamide remodeling-the removal and replacement of the lower ligand-despite the absence of the canonical remodeling enzyme CbiZ in A. muciniphila We identified a novel enzyme, CbiR, that is capable of initiating the remodeling process by hydrolyzing the phosphoribosyl bond in the nucleotide loop of cobamides. CbiR does not share similarity with other cobamide remodeling enzymes or B12-binding domains and is instead a member of the apurinic/apyrimidinic (AP) endonuclease 2 enzyme superfamily. We speculate that CbiR enables bacteria to repurpose cobamides that they cannot otherwise use in order to grow under cobamide-requiring conditions; this function was confirmed by heterologous expression of cbiR in Escherichia coli Homologs of CbiR are found in over 200 microbial taxa across 22 phyla, suggesting that many bacteria may use CbiR to gain access to the diverse cobamides present in their environment.IMPORTANCE Cobamides, comprising the vitamin B12 family of cobalt-containing cofactors, are required for metabolism in all domains of life, including most bacteria. Cobamides have structural variability in the lower ligand, and selectivity for particular cobamides has been observed in most organisms studied to date. Here, we discovered that the beneficial human gut bacterium Akkermansia muciniphila can use a diverse range of cobamides due to its ability to change the cobamide structure via a process termed cobamide remodeling. We identify and characterize the novel enzyme CbiR that is necessary for initiating the cobamide remodeling process. The discovery of this enzyme has implications for understanding the ecological role of A. muciniphila in the gut and the functions of other bacteria that produce this enzyme.

Project description:Excessive mucin degradation by intestinal bacteria may contribute to inflammatory bowel diseases because access of luminal antigens to the intestinal immune system is facilitated. This study investigated how the presence of a mucin degrading commensal bacterium affects the severity of an intestinal Salmonella enterica Typhimurium-induced gut inflammation. Using a gnotobiotic C3H mouse model with a background microbiota of eight bacterial species (SIHUMI) the impact of the mucin-degrading commensal bacterium Akkermansia muciniphila (SIHUMI-A) on inflammatory and infectious symptoms caused by S. Typhimurium was investigated. Presence of A. muciniphila in S. Typhimurium-infected SIHUMI mice caused significantly increased histopathology scores and elevated mRNA levels of IFN-γ, IP-10, TNF-α, IL-12, IL-17 and IL-6 in cecal and colonic tissue. The increase in pro-inflammatory cytokines was accompanied by 10-fold higher S. Typhimurium cell numbers in mesenteric lymph nodes of SIHUMI mice associated with A. muciniphila and S. Typhimurium (SIHUMI-AS) compared to SIHUMI mice with S. Typhimurium only (SIHUMI-S). The number of mucin filled goblet cells was 2- to 3-fold lower in cecal tissue of SIHUMI-AS mice compared to SIHUMI-S, SIHUMI-A or SIHUMI mice. Reduced goblet cell numbers significantly correlated with increased IFN-γ mRNA levels (r(2) = -0.86, ***P<0.001) in all infected mice. In addition, loss of cecal mucin sulphation was observed in SIHUMI mice containing both A. muciniphila and S. Typhimurium compared to other mouse groups. Concomitant presence of A. muciniphila and S. Typhimurium resulted in a drastic change in microbiota composition of SIHUMI mice: the proportion of B. thetaiotaomicron in SIHUMI-AS mice was 0.02% of total bacteria compared to 78%-88% in the other mouse groups and the proportion of S. Typhimurium was 94% in SIHUMI-AS mice but only 2.2% in the SIHUMI-S mice. These results indicate that A. muciniphila exacerbates S. Typhimurium-induced intestinal inflammation by its ability to disturb host mucus homeostasis.

Project description:A healthy gastrointestinal tract functions as a highly selective barrier, allowing the absorption of nutrients and metabolites while preventing gut bacteria and other xenobiotic compounds from entering host circulation and tissues. The intestinal epithelium and intestinal mucus provide a physical first line of defense against resident microbes, pathogens and xenotoxic compounds. Prior studies have indicated that the gut microbe Akkermansia muciniphila, a mucin-metabolizer, can stimulate intestinal mucin thickness to improve gut barrier integrity. Grape polyphenol (GP) extracts rich in B-type proanthocyanidin (PAC) compounds have been found to increase the relative abundance of A. muciniphila, suggesting that PACs alter the gut microbiota to support a healthy gut barrier. To further investigate the effect of GPs on the gut barrier and A. muciniphila, male C57BL/6 mice were fed a high-fat diet (HFD) or low-fat diet (LFD) with or without 1% GPs (HFD-GP, LFD-GP) for 12 weeks. Compared to the mice fed unsupplemented diets, GP-supplemented mice showed increased relative abundance of fecal and cecal A. muciniphila, a reduction in total bacteria, a diminished colon mucus layer and increased fecal mucus content. GP supplementation also reduced the presence of goblet cells regardless of dietary fat. Compared to the HFD group, ileal gene expression of lipopolysaccharide (LPS)-binding protein (Lbp), an acute-phase protein that promotes pro-inflammatory cytokine expression, was reduced in the HFD-GP group, suggesting reduced LPS in circulation. Despite depletion of the colonic mucus layer, markers of inflammation (Ifng, Il1b, Tnfa, and Nos2) were similar among the four groups, with the exception that ileal Il6 mRNA levels were lower in the LFD-GP group compared to the LFD group. Our findings suggest that the GP-induced increase in A. muciniphila promotes redistribution of the intestinal mucus layer to the intestinal lumen, and that the GP-induced decrease in total bacteria results in a less inflammatory intestinal milieu.

Dataset Information

FluCell-SELEX Aptamers as Specific Binding Molecules for Diagnostics of the Health Relevant Gut Bacterium Akkermansia muciniphila.

Publications

FluCell-SELEX Aptamers as Specific Binding Molecules for Diagnostics of the Health Relevant Gut Bacterium <i>Akkermansia muciniphila</i>.

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