Identification and characterization of novel glycan-binding bacterial adhesins encoded by the human gut microbial metagenome
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Although there have been an increasing number of scientific publications describing the adherence of gut bacteria to components of the human intestinal mucosa, very little is known about the surface molecules mediating this adhesion and their individual receptors. In the current study, we describe the identification and subsequent analysis of putative glycan-binding factors from the human gut microbiome using a combination of functional metagenomics and bioinformatics-based approaches. A fosmid library of human gut microbiota in the surrogate host Phage T-1 Resistant EPI300TM-T1R Escherichia coli was constructed and screened for enhanced adherence capability. Two out of 42,000 fosmid clones, FC3 and FC21, exhibited enhanced adherence to Caco-2 cells in functional screens. DNA segments inserted into the FC3 and FC21 clones were distinct sequences of 24.6 kb and 8.1 kb, respectively. FC21 contained three functional genes and likely originates from the dominant commensal gut species Bifidobacterium adolescentis. Sequence analysis of FC3 revealed that the 24.6 kb insert is a fragment with no current known homologs in the database, suggesting that the insert DNA is derived from a microbe with an unknown genome sequence. When carbohydrate-based and lectin microarrays were used to characterize the carbohydrate binding specificities of FC3 and FC21, the lectin microarrays revealed that the host E. coli strains carrying FC3 and FC21 had altered cell surface glycosylation, while the mucin microarrays revealed that mucin binding pattern is not altered for the two fosmid clones as compared to the control, and finally neo- glycoconjugate microarrays revealed that FC3 and FC21 exhibited binding to specific glycans in the presence of arabinose and antibiotic. Five adhesins known to mediate adhesion to specific components of the human gastrointestinal tract were identified from the literature and used as reference proteins in BLASTp searches against the genomes of 54 of the most abundant species in the gut. A homologous protein to one of the reference adhesins, MapARi, was subsequently identified in the gram-positive, butyrate-producing bacterium Roseburia intestinalis M50/1. MapARi was cloned into a Lactococcus lactis heterologous host and assessed for expression using the nisin controlled gene expression system (NICE)1 and functionally screened to detect the adherence phenotype. The results indicate that induction of the recombinant strain L. lactis NZ9000/pPTPi-MapARi with nisin does not significantly increase its adherence to 7 day-old or 3 week-old Caco-2 cells suggesting that (a) MapARi does not function as an adhesin, or (b) MapARi is not being expressed appropriately by the L. lactis NZ9000 heterologous host. The findings in this study demonstrate the power of functional screening but also raises significant questions about the usefulness of this approach and the sequence-based metagenomic approach in identifying glycan binding determinants encoded by the human gut metagenome. Identification of novel glycan binding genes which have not previously been linked to adhesion will help to broaden our understanding of host-microbe interaction and possibly lead to the identification of novel and unusual systems that play as yet undefined roles in adherence and perhaps ultimately in human gastrointestinal health.
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