[Show abstract][Hide abstract] ABSTRACT: The human intestine is one of the most competitive ecosystems on earth, housing billions of bacteria. Competition for carbohydrate substrates, production of antimicrobial compounds and adhesion to the mucosa are among the most common strategies developed by microorganisms to persist in this environment. Whereas some interactions of major Gram negative bacteria with human gut have been characterized at the molecular level, only few data exist regarding the Gram positive. We investigated the interaction of the Gram positive bacteria Ruminococcus gnavus E1 with the human intestine. R. gnavus belongs to the "core 57" species present in more than 90% individuals. The strain E1 harbors a 5.2 kb-long ORF encoding RadA, a putative adhesin of the MSCRAMMs family which is expressed in vivo, in the digestive tract of gnotobiotic animals. Biocomputerized analysis showed that the N-terminal region, residues 1 to 218 of the mature RadA devoid of SS, presents structural homologies with Immunoglobin (Ig) Binding Proteins. The corresponding fragment was overproduced as a GST-fusion protein called Rad218. We analyzed the interaction of Rad218 with human factors using a solid phase assay. We showed that Rad218 binds to human IgG, confirming its Ig binding protein ability. More important, we found that Rad218 interacts with the secreted mucins (MUC2 and MUC3) but not with the membrane-associated one (MUC1), such interaction being dependent of the glucidic moiety of the mucus. Finally, competition assays allowed us to identify particular glucides involved in the interaction of RadA with human mucins. Taken together, our data allow us to indentify the host receptors for an adhesin that probably participates in the colonization power of R. gnavus E1 and enhances the dialogue between this symbionte and the host.
[Show abstract][Hide abstract] ABSTRACT: Differential gene expression analysis was performed in monoxenic mice colonized with Ruminococcus gnavus strain E1, a major endogenous member of the gut microbiota. RNA arbitrarily primed-PCR fingerprinting assays allowed to specifically detect the in vivo expression of the aga1 gene, which was further confirmed by RT-PCR. The aga1 gene encoded a protein of 744 residues with calculated molecular mass of 85,207 Da. Aga1 exhibited significant similarity with previously characterized α-Galactosidases of the GH 36 family. Purified recombinant protein demonstrated high catalytic activity (104 ± 7 U mg(-1)) and efficient p-nitrophenyl-α-d-galactopyranoside hydrolysis [k(cat)/K(m) = 35.115 ± 8.82 s(-1) mM(-1) at 55 °C and k(cat)/K(m) = 17.48 ± 4.25 s(-1) mM(-1) at 37 °C].
Research in Microbiology 01/2012; 163(1):14-21. DOI:10.1016/j.resmic.2011.10.005 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ruminococcin C (RumC) is a trypsin-dependent bacteriocin produced by Ruminococcus gnavus E1, a gram-positive strict anaerobic strain isolated from human feces. It consists of at least three similar peptides active against Clostridium perfringens. In this article, a 15-kb region from R. gnavus E1 chromosome, containing the biosynthetic gene cluster of RumC was characterized. It harbored 17 open reading frames (called rum(c) genes) with predicted functions in bacteriocin biosynthesis and post-translational modification, signal transduction regulation, and immunity. An unusual feature of the locus is the presence of five genes encoding highly homologous, but nonidentical RumC precursors. The transcription levels of the rum(c) genes were quantified. The rumC genes were found to be highly expressed in vivo, when R. gnavus E1 colonized the digestive tract of mono-contaminated rats, whereas the amount of corresponding transcripts was below detection level when it grew in liquid culture medium. Moreover, the rumC-like genes were disseminated among 10 strains (R. gnavus or related species) previously isolated from human fecal samples and selected for their capability to produce a trypsin-dependant anti-C. perfringens compound. All harbored at least a rumC1-like copy, four exhibited rumC1-5 genes identical to those of strain E1.