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RadA, a MSCRAMM Adhesin of the Dominant Symbiote Ruminococcus gnavus E1, Binds Human Immunoglobulins and Intestinal Mucins.

ABSTRACT: Adhesion to the digestive mucosa is considered a key factor for bacterial persistence within the gut. In this study, we show that Ruminococcus gnavus E1 can express the radA gene, which encodes an adhesin of the MSCRAMMs family, only when it colonizes the gut. The RadA N-terminal region contains an all-β bacterial Ig-like domain known to interact with collagens. We observed that it preferentially binds human immunoglobulins (IgA and IgG) and intestinal mucins. Using deglycosylated substrates, we also showed that the RadA N-terminal region recognizes two different types of motifs, the protein backbone of human IgG and the glycan structure of mucins. Finally, competition assays with lectins and free monosaccharides identified Galactose and N-Acetyl-Galactosamine motifs as specific targets for the binding of RadA to mucins and the surface of human epithelial cells.

SUBMITTER: Maresca M

PROVIDER: S-EPMC8615915 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Ruminococcin A (RumA) is a lanthipeptide with high activity against pathogenic clostridia and is naturally produced by the strict anaerobic bacterium Ruminococcus gnavus E1, isolated from human intestine. Cultivating R. gnavus E1 is challenging, limiting high-quality production, further biotechnological development and therapeutic exploitation of RumA. To supply an alternative production system, the gene encoding RumA-modifying enzyme (RumM) and the gene encoding the unmodified precursor peptide (preRumA) were amplified from the chromosome of R. gnavus E1 and coexpressed in Escherichia coli. Our results show that the ruminococcin-A lanthionine synthetase RumM catalyzed dehydration of threonine and serine residues and subsequently installed thioether bridges into the core structure of a mutant version of preRumA (preRumA?). These modifications were achieved when the peptide was expressed as a fusion protein together with green fluorescence protein (GFP), demonstrating that a larger attachment to the N-terminus of the leader peptide does not obstruct in vivo processivity of RumM in modifying the core peptide. The leader peptide serves as a docking sequence which the modifying enzyme recognizes and interacts with, enabling its catalytic role. We further investigated RumM catalysis in conjunction with the formation of complexes observed between RumM and the chimeric GFP fusion protein. Results obtained suggested some insights into the catalytic mechanisms of class II lanthipeptide synthetases. Our data further indicated the presence of three thioether bridges, contradicting a previous report whose findings ruled out the possibility of forming a third ring in RumA. Modified preRumA? was activated in vitro by removing the leader peptide using trypsin and biological activity was achieved against Bacillus subtilis ATCC 6633. A production yield of 6 mg of pure modified preRumA? per liter of E. coli culture was attained and considering the size ratio of the leader-to-core segments of preRumA?, this amount would generate a final yield of approximately 1-2 mg of active RumA when the leader peptide is removed. The yield of our system exceeds that attainable in the natural producer by several 1000-fold. The system developed herein supplies useful tools for product optimization and for performing in vivo peptide engineering to generate new analogs with superior anti-infective properties.

Project description:Dietary and host glycans shape the composition of the human gut microbiota with keystone carbohydrate-degrading species playing a critical role in maintaining the structure and function of gut microbial communities. Here, we focused on two major human gut symbionts, the mucin-degrader Ruminococcus gnavus ATCC 29149, and R. bromii L2-63, a keystone species for the degradation of resistant starch (RS) in human colon. Using anaerobic individual and co-cultures of R. bromii and R. gnavus grown on mucin or starch as sole carbon source, we showed that starch degradation by R. bromii supported the growth of R. gnavus whereas R. bromii did not benefit from mucin degradation by R. gnavus. Further we analyzed the growth (quantitative PCR), metabolite production (1H NMR analysis), and bacterial transcriptional response (RNA-Seq) of R. bromii cultured with RS or soluble starch (SS) in the presence or absence of R. gnavus. In co-culture fermentations on starch, 1H NMR analysis showed that R. gnavus benefits from transient glucose and malto-oligosaccharides released by R. bromii upon starch degradation, producing acetate, formate, and lactate as main fermentation end-products. Differential expression analysis (DESeq 2) on starch (SS and RS) showed that the presence of R. bromii induced changes in R. gnavus transcriptional response of genes encoding several maltose transporters and enzymes involved in its metabolism such as maltose phosphorylase, in line with the ability of R. gnavus to utilize R. bromii starch degradation products. In the RS co-culture, R. bromii showed a significant increase in the induction of tryptophan (Trp) biosynthesis genes and a decrease of vitamin B12 (VitB12)-dependent methionine biosynthesis as compared to the mono-culture, suggesting that Trp and VitB12 availability become limited in the presence of R. gnavus. Together this study showed a direct competition between R. bromii and R. gnavus on RS, suggesting that in vivo, the R. gnavus population inhabiting the mucus niche may be modulated by the supply of non-digestible carbohydrates reaching the colon such as RS.

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RadA, a MSCRAMM Adhesin of the Dominant Symbiote Ruminococcus gnavus E1, Binds Human Immunoglobulins and Intestinal Mucins.

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