Gotowa bibliografia na temat „Ruminococcus”

Les spondyloarthrites (SpA) sont desrhumatismes inflammatoires chroniques fortementassociés à l'allèle HLA-B27 du complexe majeurd'histocompatibilité de classe I. La preuve du rôlepathogène du HLA-B27 a été apportée par plusieurslignées de rats transgéniques pour le HLA-B27 et lab2 microglobuline humaine (rats B27). Ces rats B27développent spontanément des manifestationscomparables à la SpA humaine. Dans ce modèle, lescellules hématopoïétiques HLA-B27+, leslymphocytes T CD4+ et un microbiote conventionnelsont nécessaires pour développer la maladie. Le rôledu microbiote a également été étayé par la mise enévidence d'une dysbiose intestinale au cours desSpA. Ainsi, une corrélation positive entre l'activitédes SpA et l'abondance de l'espèce bactérienneanaérobie Ruminococcus gnavus dans les selles a étédémontrée.Mon premier objectif de thèse a été de déterminerles mécanismes immunologiques impliqués dans ledéclenchement de la SpA du rat B27 par l'étude despopulations productrices des cytokines clefs de laSpA : l'IL-17 et le TNF. Parallèlement, je me suisintéressée au rôle des souches de R. gnavus aucours de la SpA. Mon premier travail a démontréque les LT CD4+ conventionnels exprimant lerécepteur de chimiokines CCR6 étaient lesprincipales cellules productrices d'IL-17 et de TNFau cours de la SpA et qu'elles étaient capablesd'induire la SpA après transfert à des ratsathymiques nude B27 habituellement protégés. Lesecond axe de ma thèse m'a permis d'isoler dessouches de R. gnavus de patients atteints de SpA etde témoins sains. Des expérimentationscomplémentaires sont nécessaires pour étayer leshypothèses concernant la pathogénicité de R.gnavus<br>Spondyloarthritis (SpA) is a chronicinflammatory rheumatic disease strongly associatedwith the HLA-B27 major histocompatibility complexclass I allele. Proof of the pathogenic role of HLA-B27was provided by lines of transgenic rats for HLA-B27and human β2 microglobulin (B27 rats). These B27rats spontaneously develop manifestationscomparable to human SpA. In this model, HLA-B27+hematopoïetic cells, CD4+ T lymphocytes and aconventional microbiota are required for diseasedevelopment. The role of the microbiota is alsosupported by evidence of intestinal dysbiosis in SpA.A positive correlation between SpA activity and theabundance of the bacterial anaerobic speciesRuminococcus gnavus in stools has beendemonstrated. The first aim of my thesis was todetermine the immunological mechanismsinvolved in triggering SpA by studying thepopulations producing the key SpA cytokines IL-17and TNF in B27 rat. In parallel, I examined thepotential role of R. gnavus strains in SpA. My firstwork demonstrated that conventional CD4+LTexpressing the chemokine receptor CCR6 are themain IL-17 and TNF-producing cells during SpAand are able to induce SpA after transfer to usuallyprotected nude B27 athymic rats. In the secondpart of my thesis, I isolated R. gnavus strains fromSpA patients and healthy controls. Furtherexperiments are required to substantiate thepathogenic hypotheses of R. gnavus in SpA

O celulossomo é um complexo multienzimático extracelular utilizado por bactérias anaeróbias para a degradação de biomassa vegetal. Ele é composto por escafoldinas, estruturas alongadas que abrigam diversos módulos cohesina, às quais se ligam dockerinas, seus parceiros de interação específica de alta afinidade, fusionados às enzimas celulolíticas. Os módulos cohesina e dockerina compõem o elemento central da interação entre todos os componentes que integram o celulossomo. Esses módulos são divididos em tipos, de acordo com sua sequência primária. Essa divisão reflete efeitos funcionais distintos, sendo o tipo I responsável pela ligação de enzimas às escafoldinas, enquanto o tipo II medeia a ligação de escafoldinas à célula. O celulossomo de Ruminococcus flavefaciens é o mais complexo conhecido, e na classificação por tipos, suas sequências divergem, formando o tipo III, que foi posteriormente subdividido em 6 grupos para significância funcional. Nesse sistema, o principal responsável pela integração de enzimas ao sistema é a escafoldina primária ScaA, a qual interage com escafoldina adaptadora ScaB. A especificidade dessa ligação - dockerina de ScaA (Rf-DocA) com cohesinas de ScaB (Rf-CohB1-7) - é classificada como único membro do grupo 5, na divisão de grupos que compõem o tipo III. Assim, essa interação é de suma importância para a organização do celulossomo desse organismo, tendo sido estudada por meio de experimentos biofísicos e bioquímicos. Porém a falta de uma estrutura cristalina resolvida desses componentes limita a compreensão que podemos ter sobre a interação. 1-2 Nesse trabalho, apresentamos as estruturas cristalográficas de Rf-DocA, em complexo com a Rf-CohB4, além da estrutura dessa cohesina isolada, e ainda, a Rf-CohB1, e alguns de seus mutantes pontuais. Com isso, esclarecemos aspectos estruturais desses módulos, como a presença de dois sítios funcionais de ligação a cálcio em Rf-DocA. Também é observável pelos modelos gerados, detalhes da ligação entre eles, como os resíduos participantes da interação. Estudos de afinidade entre esses módulos foram conduzidos para a elucidar algumas propriedades da ligação entre esses módulos, de forma que descobrimos que ela ocorre de uma única maneira, e que há um loop na cohesina cuja flexibilidade afeta a afinidade da ligação. Isso sugere um mecanismo de alteração conformacional que regula a ligação à dockerina. Adicionalmente, buscamos o emprego desses módulos em uma aplicação tecnológica, desenhando redes automontáveis de proteínas, visando a construção de um nanomaterial. Essas redes são formadas por características intrínsecas das proteínas que os compõem, sendo o principal fator considerado sua simetria rotacional.3 Nesse sentido, as dockerinas e cohesinas foram utilizadas para ligação entre proteínas de diferentes simetrias. Utilizamos proteínas de simetrias C3, C4 e C6 com fusão a dockerinas, que se conectam às cohesinas fusionadas a proteínas de simetria C2, as quais formam o elemento linear da ligação entre os diferentes módulos. Esse desenho experimental permite a expressão e purificação independentes dos componentes, o que facilita a obtenção das redes, a partir da mistura dos dois componentes. Através de análises preliminares por microscopia eletrônica de transmissão, observamos a formação de filmes bidimensionais extensos e nanotubos com a construção testada.<br>The cellulosome is an intricate multienzyme extracelular complexes evolved by anaerobic bacteria for degradation of cellulosic biomass. It is composed of scaffoldins, elongated structures, which bare numerous cohesin modules, which bind to dockerin modules, their high affinity and specificity partners, borne by cellulolytic enzymes. The cohesin and dockerina modules constitute the central element of the interaction between every component of the cellulosome. These modules are categorized in types, according to their primary sequence. That distribution reflects distinct functions, in which the type I is responsible for integration of enzymes to scaffoldins, while type II mediates anchoring of scaffoldins to the cell wall. The cellulosome of Ruminococcus flavefaciens is the most intricate known to date, which is categorized into a third type of cohesins and dockerins, due to sequence diversion. The type III was further divided into 6 groups to impart functional significance. In that system, the main enzyme integrating component is the primary scaffoldin ScaA, which interacts to the adaptor scaffoldin ScaB. The specificity of this interaction - dockerina of ScaA (Rf-DocA) to ScaB cohesins (Rf-CohB1-7) - is sorted as a single member of group 5, in the subtypes of type III. Thus, this interaction is essential for cellulosome organization, having been studied by biophysical and biochemical experiments. However, the lack of a solved crystalline structure of these components narrows our understanding on this interaction. In the present study, we present the structures of Rf-DocA, complexed to Rf-CohB4, besides the structure of this isolated cohesin, and also Rf-CohB1 and its point mutants. Due to these data, we clarify structural aspects of these modules, such as the occurrence of two functioning calcium binding sites in Rf-DocA. We also identified details of their binding, such as the interacting residues. Through binding affinity studies, we concluded that the interaction between these modules occurs in a single mode, and that there is a loop in the cohesin module whose flexibility has direct effects on the binding affinity to dockerin. Additionally, we sought to utilize these modules in a downstream application, by designing self-assembling arrays of proteins, aiming for the construction of a nanomaterial. These arrays are constructed from the intrinsic properties of its constituent proteins, in which the main factor is rotational symmetry. In this context, dockerina and cohesin modules were used of binding different symmetry proteins. We utilized C3, C4 and C6 point symmetry proteins fused to dockerin modules, which bind to the cohesin modules fused to C2 point symmetry proteins, which establish the linear connection between the distinct components. This experimental design allows for the independent expression and purification of the components, which facilitates the achievement of the arrays, by simple mixture of the two components. Through preliminary analysis by transmission election microscopy, we observed the construction of two-dimensional films and nanotubes.

Here we report the enzymatic characteristics and regulation of expression for two GH36 a- galactosidases. Agu 1 and Aga,'. li'0111 R. gnavus El. Bioinforrnatics analysis of their respective genetic environment showed a different organisation. Aga 1 having a simple organisation while Aga2 is organised as part nf an ()p~wn. They were heterologously expressed in Escherichia coli. puri tied to homogeneity and their biochemical properties and substrate preferences comparatively analysed. The growth pattern (If the strain in minimum media demonstrates a preference tor complex substratcs (melibiose and raffinose) that require the expression of the a-galactosid.rscs for their utilisation and assimilation. Keywords: a-galactosidase. Ruminococcus gnavus E I. characterisation. transglycosylation. regulation. metabolism

Ruminococcus gnavus E1, une bactérie à Gram positif, anaérobie stricte, isolée du microbiote dominant d'un homme sain. Un ORF d'environ 6kb, nommé radA, a été identifié sur le chromosome de la souche E1, à proximité des clusters génétiques impliqués dans la biosynthèse des bactériocines RumA et RumC qui sont actives contre le pathogène Closthdium perfringens. RadA présente de fortes homologies avec des gènes de Staphylococcus aureus, Bacillus cereus et C. Perfingens codant pour des protéines des adhésines de la famille des MSCRAMMs. La partie du gène radA codant pour les 414 acides aminés situés à l'extrémité N-terminale de la protéine putative mature, sans, a été clonée dans le vecteur pGEXT4, exprimée chez Escherichia coli. Les tests réalisés par la méthode ELISA montrent ce fragment de RadA est impliqué dans l'adhésion au collagène de type I. Afin de localiser plus précisément la région responsable de l'adhésion, le fragment du gène radA codant pour les 218 acides aminés localisés à l'extrémité N-terminale de RadA a été clone dans le vecteur pGEXT4 et exprimé chez E. Coli. La protéine fusion GST-RadA218 présente une adhésion au collagène nettement plus forte que RadA414. Ll a été montré par RT-PCR que le gène radA est fortement exprimé in vivo, quand la souche E1 colonise le tube digestif d'animaux monoxéniques, et peu transcrit in vitro. Les expériences complémentaires montrent que radA est largement disséminé chez différentes souches isolées du microbiote dominant de l'Homme du cluster phylogénétique Clostridium coccoides qui comprend l'espèce R. Gnavus. Les résultats suggère que RadA pourrait jouer un rôle important dans la colonisation de l'écosystème digestif<br>Ruminococcus gnavus E1 is a Gram positive strict anaerobic bacterium that was isolated from the dominant faecal microbiota of a healthy adult. A 6kb-long open reading fragment called radA was identified on the E1 chromosome, next to the genetic clusters involved in the biosynthesis of the RumA and RumC bacteriocins which are active against pathogenic Clostridium perfringens. RadA shares a high sequence homology with genes of Staphylococcus aureus, Bacillus cereus and C. Perfingens encoding adhesins of the MSCRAMMs family. The gene fragment coding for the 414 amino acids located at the N-terminus of the mature protein was cloned in the pGEXT4 vector and expressed in Escherichia coli. ELISA-based tests showed that this fragment of RadA is involved in adhesion to type I collagen. To localize more precisely the region responsible for adhesion, the gene fragment coding for the 218 amino acids located at the N-terminus was cloned in the pGEXT4 vector and expressed in E. Coli. The fusion protein GST-RadA218 exhibited a stronger adhesion to collagen than RadA414. RT-PCR experiments demonstrated that the radA gene was strongly expressed in vivo, when the E1 strain colonized the digestive tract of monoxenics animals, while little transcription occured in vitro. Complementary experiences showed that radA was widely spread among various strains isolated of the human dominant microbiota that belonged to the phylogenetic duster Clostridium coccoides that includes the R. Gnavus species. Taken together, these results suggest that RadA could play an important role in the colonization of the digestive ecosystem

Ruminococcus gnavus E1 appartient au groupe des Firmicutes, l’un des deux groupes dominants du microbiote intestinal humain. Les a-galactosidases sont des glycosides hydrolase (GH) actives sur des substrats contenant des galactoses liés en a. Elles sont très largement distribuées dans tous les domaines du vivant, bactéries, champignons, plantes et animaux, mais sont absentes du tractus digestif humain. Ces travaux portent sur les caractéristiques enzymatiques et la régulation de l’expression de deux &#61537;-galactosidase, Aga1 et Aga2, de R. gnavus E1. L’analyse bioinformatique de leur environnement génétique respectif indique une organisation simple pour Aga1 tandis qu’Aga2 est organisée en opéron. Elles ont été exprimées en système hétérologue chez E. coli, purifiées et leurs propriétés biochimiques ainsi que leurs spécificités de substrat ont été analysées. Le profil de croissance de la souche indique une préférence pour des substrats complexes (raffinose et mélbiose) faisant intervenir les a-galactosidase pour leurs utilisations ainsi que leur assimilation<br>Ruminococcus gnavus E1 belongs to the Firmicutes, one of the two dominant groups in the human gut microbiota. a-galactosidases are glycoside hydrolases (GH) active on a-galactoside containing substrates. They are widely distributed through all the domains of life: bacteria, fungi, plants, and animals, but are absent from the human gastro-intestinal tract.Here we report the enzymatic characteristics and regulation of expression for two GH36 &#61537;-galactosidases, Aga1 and Aga2, from R. gnavus E1. Bioinformatics analysis of their respective genetic environment showed a different organisation, Aga1 having a simple organisation while Aga2 is organised as part of an operon. They were heterologously expressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. The growth pattern of the strain in minimum media demonstrates a preference for complex substrates (melibiose and raffinose) that require the expression of the a-galactosidases for their utilisation and assimilation

The Gram-positive, strictly anaerobic bacterium, <I>R. flavefaciens</I>, plays an important role in the degradation of plant cell wall polysaccharides in the rumen. There is a paucity of information available, however, regarding the multiplicity and organisation of <I>R. flavefaciens </I>cellulolytic and xylanolytic enzyme systems. A technique involving PCR amplification of DNA with primers designed from conserved sequences, followed by hybridisation of the PCR products to chromosomal DNA, has led to an estimate of xylanase gene multiplicity in <I>R. flavefaciens</I>. The xylanase-specific primers were also useful in the isolation and sequencing of a partial xylanase gene, <I>xynC</I>. Although <I>R. flavefaciens</I> 17 appears to produce a cellulose-binding enzyme-complex, none of the individual enzymes examined was found to bind cellulose in isolation. However, a 210 kDa protein which is present in the complex was shown to bind cellulose after isolation from a renatured SDS-gel. In order to look for genetic evidence for a cellulose-binding mechanism, sequencing of the <I>R. flavefaciens</I> 17 endoglucanase gene, <I>endA</I>, was completed from PCR products. The carboxy-terminus of the predicted <I>endA</I> product consists of a domain which is similar to dockerins found in <I>Clostridium thermocellum </I>polysaccharidases. Homologous domains are also found in the <I>R. flavefaciens </I>xylanases, XynB and XynD. As the <I>C. thermocellum</I> dockerin domains mediate binding to the 210 kDa scaffolding protein in the cellulosome complex, it is likely that the <I>R. flavefaciens</I> domains play a similar role in assembly of a cellulosome-like complex (Lamed and Bayer, 1994). A gene which maps approximately 1.5 kb downstream from <I>endA</I> on the <I>R. flavefaciens</I> 17 chromosome was sequenced and found to be homologous to <I>nifS</I> genes from nitrogen-fixing bacteria (Zheng <I>et al, </I>1993). The <I>R. flavefaciens</I> NifS product catalyses the production of sulphur from cysteine, and is suspected to partake in the assembly of iron-sulphur clusters. The precise role of NifS is not yet known, but may be related to the degradation of crystalline cellulose.

The mesophilic Ruminococcus flavefaciens FD-1 (NCDO 2215) is a Gram-positive obligate anaerobic bacterium. The aim of this thesis was to clone, sequence and analyze a cellodextrinase gene (celA) and a carboxymethylcellulase gene (celE), and study their regulation and induction at the transcriptional level. The sequence of the celA gene from FD-1 was determined and the amino acid sequence of the CelA enzyme (336 amino acid residues) deduced. It showed 40% identity with endoglucanase C of Clostridium thermocellum and 27.4% identity with endoglucanase 3 of Fibrobacter succinogenes. These three enzymes are grouped into subfamily "A3". The ATG start codon of celA is preceded by a GAGG sequence, predicted to be a ribosome binding site. The derived amino acid sequence corresponded to a protein of Mᵣ 38686. SDS-PAGE analysis of in vitro and in vivo translational products showed that CelA has a molecular mass of ca 39 kDa and was secreted into the Escherichia coli periplasmic space. Although CelA has activity on carboxymethylcellulose, further study on the enzyme showed that it degraded cellopentaose and other cellodextrins to predominantly cellobiose. Thus CelA is a cellodextrinase. It also has high activity against p-nitrophenyl-β-D-cellobioside. A gene, expressing a protein with both carboxymethyl cellulase and xylanase activity, was cloned from R. flavefaciens FD-1 using an E. coli/Bacillus subtilis shuttle vector, pEBl. The 3.6 kb DNA insert on the plasmid pWFl, which carried the gene, celE, contained an open reading frame of 963 bp encoding 320 amino acid residues with a caculated Mᵣ of 35937. Homology analysis showed 11.6% identity and 55.3% similarity with the N-terminal catalytic region of the cellulase gene of alkalophilic Bacillus sp. strain 1139. In order to obtain expression in E. coli, the gene had to be transcribed from the lambda Pᵣ promoter. To determine whether cellulase genes of R. flavefaciens FD-1 were regulated at the level of transcription, celA and celE were used as probes against RNA isolated from R. flavefaciens FD-1 grown on cellobiose, cellulose or cellotriose. Transcription of both genes was induced when cellulose was added to cells growing in cellobiose. This induction continued after cellulose depletion and after cell division had ceased. Transcription of both genes was also induced by cellotriose although not to the same extent as by cellulose. This suggests that cellotriose and possibly ether dextrins may act as key inducers to trlgger celA and eels gene expression in R. flavefaciens FD-1.