Influence du microbiote intestinal sur le métabolisme et la virulence des Escherichia coli entérohémorragiques

Les Escherichia coli entérohémorragiques (EHEC) représentent un problème majeur de santé publique dans les pays développés. Les EHEC sont régulièrement responsables de toxi-infections alimentaires graves chez l’humain et causent des colites hémorragiques et le symptôme hémolytique et urémique, mortel chez les enfants en bas âge. Les EHEC les plus virulents appartiennent au sérotype O157:H7 et le bovin constitue leur réservoir naturel. À ce jour il n’existe aucun traitement pour éviter l’apparition des symptômes liés à une infection à EHEC. Par conséquent, il est important d’augmenter nos connaissances sur les mécanismes employés par le pathogène pour réguler sa virulence et coloniser efficacement la niche intestinale. Dans un premier temps, l’adaptation de la souche EHEC O157:H7 EDL933 à l’activité métabolique du microbiote intestinal a été étudiée au niveau transcriptionnel. Pour se faire, EDL933 a été cultivée dans les contenus caecaux de rats axéniques (milieu GFC) et dans ceux provenant de rats colonisés par le microbiote intestinal humain (milieu HMC). Le HMC est un milieu cécal conditionné in vivo par le microbiote. Dans le HMC par rapport au GFC, EDL933 change drastiquement de profile métabolique en réponse à l’activité du microbiote et cela se traduit par une diminution de l’expression des voies de la glycolyse et une activation des voies de l’anaplérose (voies métaboliques dont le rôle est d’approvisionner le cycle TCA en intermédiaires métaboliques). Ces résultats, couplés avec une analyse métabolomique ciblée sur plusieurs composés, ont révélé la carence en nutriments rencontrée par le pathogène dans le HMC et les stratégies métaboliques utilisées pour s’adapter au microbiote intestinal. De plus, l’expression des gènes de virulence incluant les gènes du locus d’effacement des entérocytes (LEE) codant pour le système de sécrétion de type III sont réprimés dans le HMC par rapport au GFC indiquant la capacité du microbiote intestinal à réprimer la virulence des EHEC. L’influence de plusieurs composés intestinaux présents dans les contenus caecaux de rats sur l’expression des gènes de virulence d’EDL933 a ensuite été étudiée. Ces résultats ont démontré que deux composés, l’acide N-acétylneuraminique (Neu5Ac) et le N-acétylglucosamine (GlcNAc) répriment l’expression des gènes du LEE. La répression induite par ces composés s’effectue via NagC, le senseur du GlcNAc-6-P intracellulaire et le régulateur du catabolisme du GlcNAc et du galactose chez E. coli. NagC est un régulateur transcriptionnel inactivé en présence de GlcNAc-6-P qui dérive du catabolisme du Neu5Ac et du transport GlcNAc. Ce travail nous a permis d’identifier NagC comme un activateur des gènes du LEE et de mettre à jour un nouveau mécanisme qui permet la synchronisation de la virulence avec le métabolisme chez les EHEC O157:H7. La concentration du Neu5Ac et du GlcNAc est augmentée in vivo chez le rat par le symbiote humain Bacteroides thetaiotaomicron, indiquant la capacité de certaines espèces du microbiote intestinal à relâcher les composés répresseurs de la virulence des pathogènes. Ce travail a permis l’identification des adaptations métaboliques des EHEC O157:H7 en réponse au microbiote intestinal ainsi que la découverte d’un nouveau mécanisme de régulation de la virulence en réponse au métabolisme. Ces données peuvent contribuer à l’élaboration de nouvelles approches visant à limiter les infections à EHEC.

Caractérisation du rôle transcriptionnel et épigénétique de l’O-GlcNAcylation des histones et du facteur de transcription FOXK1

L’O-GlcNAcylation est une modification post-traductionnelle qui consiste en l’ajout covalent du N-acetylglucosamine au groupement hydroxyle des sérines et thréonines des protéines nucléaires et cytoplasmiques. Ce type de glycosylation atypique est régulé de manière très dynamique par l’action de l’O-GlcNAc transférase (OGT) et de l’O-GlcNAcase (OGA) qui catalysent et hydrolysent cette modification respectivement. Aujourd’hui, OGT émerge comme un régulateur transcriptionnel et senseur critique du métabolisme où les protéines ciblées par l’O-GlcNAcylation couvrent la presque totalité des voies de signalisation cellulaire. Récemment, des études ont aussi proposé qu’OGT soit impliquée dans la régulation épigénétique par l’O-GlcNAcylation des histones. Dans le but de caractériser le rôle fonctionnel d’OGT dans la régulation épigénétique, nous avons revisité le concept d’O-GlcNAcylation des histones et, de manière surprenante, n’avons pu confirmer cette observation. En fait, nos données indiquent que les outils disponibles pour détecter l’O-GlcNAcylation des histones génèrent des artéfacts. De ce fait, nos travaux supportent plutôt un modèle où la régulation épigénétique médiée par OGT se fait par l’O-GlcNAcylation de régulateurs transcriptionnels recrutés à la chromatine. Parmi ceux-ci, OGT s’associe au complexe suppresseur de tumeurs BAP1. En étudiant le rôle d’OGT dans ce complexe, nous avons identifié le facteur de transcription FOXK1 comme un nouveau substrat d’OGT et démontrons qu’il est régulé par O-GlcNAcylation durant la prolifération cellulaire. Enfin, nous démontrons que FOXK1 est aussi requis pour l’adipogenèse. Ensemble, nos travaux suggèrent un rôle important d’OGT dans la régulation du complexe BAP1.

Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide-protein interactions

Neoglycolipid technology is the basis of a microarray platform for assigning oligosaccharide ligands for carbohydrate-binding proteins. The strategy for generating the neoglycolipid probes by reductive amination results in ring opening of the core monosaccharides. This often limits applicability to short-chain saccharides, although the majority of recognition motifs are satisfactorily presented with neoglycolipids of longer oligosaccharides. Here, we describe neoglycolipids prepared by oxime ligation. We provide evidence from NMR studies that a significant proportion of the oxime-linked core monosaccharide is in the ring-closed form, and this form selectively interacts with a carbohydrate-binding protein. By microarray analyses we demonstrate the effective presentation with oxime-linked neoglycolipids of (1) Lewis(x) trisaccharide to antibodies to Lewisx, (2) sialyllactose analogs to the sialic acid-binding receptors, siglecs, and (3) N-glycans to a plant lectin that requires an intact N-acetylglucosamine core.

O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway

Aims Glycosylation with beta-N-acetylglucosamine (O-GlcNAcylation) is one of the most complex post-translational modifications. The cycling of O-GlcNAc is controlled by two enzymes: UDP-NAc transferase (OGT) and O-GlcNAcase (OGA). We recently reported that endothelin-1 (ET-1) augments vascular levels of O-GlcNAcylated proteins. Here we tested the hypothesis that O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway. Methods and results Incubation of vascular smooth muscle cells (VSMCs) with ET-1 (0.1 mu M) produces a time-dependent increase in O-GlcNAc levels. ET-1-induced O-GlcNAcylation is not observed when VSMCs are previously transfected with OGT siRNA, treated with ST045849 (OGT inhibitor) or atrasentan (ET(A) antagonist). ET-1 as well as PugNAc (OGA inhibitor) augmented contractions to phenylephrine in endothelium-denuded rat aortas, an effect that was abolished by the Rho kinase inhibitor Y-27632. Incubation of VSMCs with ET-1 increased expression of the phosphorylated forms of myosin phosphatase target subunit 1 (MYPT-1), protein kinase C-potentiated protein phosphatase 1 inhibitor protein (protein kinase C-potentiated phosphatase inhibitor-17), and myosin light chain (MLC) and RhoA expression and activity, and this effect was abolished by both OGT siRNA transfection or OGT inhibition and atrasentan. ET-1 also augmented expression of PDZ-Rho GEF (guanine nucleotide exchange factor) and p115-Rho GEF in VSMCs and this was prevented by OGT siRNA, ST045849, and atrasentan. Conclusion We suggest that ET-1 augments O-GlcNAcylation and this modification contributes to increased vascular contractile responses via activation of the RhoA/Rho-kinase pathway.

O-GlcNAcylation Contributes to Augmented Vascular Reactivity Induced by Endothelin 1

O-GlcNAcylation augments vascular contractile responses, and O-GlcNAc-proteins are increased in the vasculature of deoxycorticosterone-acetate salt rats. Because endothelin 1 (ET-1) plays a major role in vascular dysfunction associated with salt-sensitive forms of hypertension, we hypothesized that ET-1-induced changes in vascular contractile responses are mediated by O-GlcNAc modification of proteins. Incubation of rat aortas with ET-1 (0.1 mu mol/L) produced a time-dependent increase in O-GlcNAc levels and decreased expression of O-GlcNAc transferase and beta-N-acetylglucosaminidase, key enzymes in the O-GlcNAcylation process. Overnight treatment of aortas with ET-1 increased phenylephrine vasoconstriction (maximal effect [in moles]: 19 +/- 5 versus 11 +/- 2 vehicle). ET-1 effects were not observed when vessels were previously instilled with anti-O-GlcNAc transferase antibody or after incubation with an O-GlcNAc transferase inhibitor (3-[2-adamantanylethyl]-2-[{4-chlorophenyl}azamethylene]-4-oxo-1,3-thiazaperhyd roine-6-carboxylic acid; 100 mu mol/L). Aortas from deoxycorticosterone-acetate salt rats, which exhibit increased prepro-ET-1, displayed increased contractions to phenylephrine and augmented levels of O-GlcNAc proteins. Treatment of deoxycorticosterone-acetate salt rats with an endothelin A antagonist abrogated augmented vascular levels of O-GlcNAc and prevented increased phenylephrine vasoconstriction. Aortas from rats chronically infused with low doses of ET-1 (2 pmol/kg per minute) exhibited increased O-GlcNAc proteins and enhanced phenylephrine responses (maximal effect [in moles]: 18 +/- 2 versus 10 +/- 3 control). These changes are similar to those induced by O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate, an inhibitor of beta-N-acetylglucosaminidase. Systolic blood pressure (in millimeters of mercury) was similar between control and ET-1-infused rats (117 +/- 3 versus 123 +/- 4 mm Hg; respectively). We conclude that ET-1 indeed augments O-GlcNAc levels and that this modification contributes to the vascular changes induced by this peptide. Increased vascular O-GlcNAcylation by ET-1 may represent a mechanism for hypertension-associated vascular dysfunction or other pathological conditions associated with increased levels of ET-1. (Hypertension. 2010; 55: 180-188.)

Impaired Vasodilator Activity in Deoxycorticosterone Acetate-Salt Hypertension Is Associated With Increased Protein O-GlcNAcylation

Hyperglycemia, which increases O-linked beta-N-acetylglucosamine (O-GlcNAc) proteins, leads to changes in vascular reactivity. Because vascular dysfunction is a key feature of arterial hypertension, we hypothesized that vessels from deoxycorticosterone acetate and salt (DOCA-salt) rats exhibit increased O-GlcNAc proteins, which is associated with increased reactivity to constrictor stimuli. Aortas from DOCA rats exhibited increased contraction to phenylephrine (E(max) [mN] = 17.6 +/- 4 versus 10.7 +/- 2 control; n = 6) and decreased relaxation to acetylcholine (47.6 +/- 6% versus 73.2 +/- 10% control; n = 8) versus arteries from uninephrectomized rats. O- GlcNAc protein content was increased in aortas from DOCA rats (arbitrary units = 3.8 +/- 0.3 versus 2.3 +/- 0.3 control; n = 5). PugNAc (O- GlcNAcase inhibitor; 100 mu mol/L; 24 hours) increased vascular O- GlcNAc proteins, augmented phenylephrine vascular reactivity (18.2 +/- 2 versus 10.7 +/- 3 vehicle; n = 6), and decreased acetylcholine dilation in uninephrectomized (41.4 +/- 6 versus 73.2 +/- 3 vehicle; n = 6) but not in DOCA rats (phenylephrine, 16.5 +/- 3 versus 18.6 +/- 3 vehicle, n = 6; acetylcholine, 44.7 +/- 8 versus 47.6 +/- 7 vehicle, n = 6). PugNAc did not change total vascular endothelial nitric oxide synthase levels, but reduced endothelial nitric oxide synthase(Ser-1177) and Akt(Ser-473) phosphorylation (P < 0.05). Aortas from DOCA rats also exhibited decreased levels of endothelial nitric oxide synthase(Ser-1177) and Akt(Ser-473) (P < 0.05) but no changes in total endothelial nitric oxide synthase or Akt. Vascular O-GlcNAc-modified endothelial nitric oxide synthase was increased in DOCA rats. Blood glucose was similar in DOCA and uninephrectomized rats. Expression of O- GlcNAc transferase, glutamine: fructose-6-phosphate amidotransferase, and O- GlcNAcase, enzymes that directly modulate O-GlcNAcylation, was decreased in arteries from DOCA rats (P < 0.05). This is the first study showing that O-GlcNAcylation modulates vascular reactivity in normoglycemic conditions and that vascular O- GlcNAc proteins are increased in DOCA-salt hypertension. Modulation of increased vascular O-GlcNAcylation may represent a novel therapeutic approach in mineralocorticoid hypertension. (Hypertension. 2009; 53: 166-174.)

Direct detection of underivatized chitooligosaccharides produced through chitinase action using capillary zone electrophoresis

Capillary electrophoresis with capacitively coupled contactless conductivity detection was successfully used to quantify N-acetylglucosamine and five N-acetyl-chitooligosaccharides (C2-C6) produced after reaction with a purified chitinase (TmChi) from Tenebrio molitor (Coleoptera). No derivatization process was necessary. The separation was developed using 10 mM NaOH with 10% (v/v) acetonitrile as background electrolyte and homemade equipment with a system that avoids the harmful effect of electrolysis. The limit of detection for all oligosaccharides was ca. 3 mu M, and the results indicated that the larger the oligosaccharide, the higher the sensitivity. Analysis of the chitooligosaccharides produced revealed that TmChi has an endolytic cleavage pattern with C5 as the best substrate (higher catalytic efficiency k(cat)/K-M) releasing C2 and C3. (c) 2007 Elsevier Inc. All rights reserved.

Produção e caracterização de quitooligossacarídeos produzidos pelo fungo Metarhizium anisopliae e avaliação da citotoxicidade em células tumorais

Chitosan is a natural polymer, biodegradable, nontoxic, high molecular weight derived from marine animals, insects and microorganisms. Oligomers of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) have interesting biological activities, including antitumor effects, antimicrobial activity, antioxidant and others. The alternative proposed by this work was to study the viability of producing chitooligosaccharides using a crude enzymes extract produced by the fungus Metarhizium anisopliae. Hydrolysis of chitosan was carried out at different times, from 10 to 60 minutes to produce chitooligosaccharides with detection and quantification performed by High Performace Liquid Chromatography (HPLC). The evaluation of cytotoxicity of chitosan oligomers was carried out in tumor cells (HepG2 and HeLa) and non-tumor (3T3). The cells were treated for 72 hours with the oligomers and cell viability investigated using the method of MTT. The production of chitosan oligomers was higher for 10 minutes of hydrolysis, with pentamers concentration of 0.15 mg/mL, but the hexamers, the molecules showing greater interest in biological properties, were observed only with 30 minutes of hydrolysis with a concentration of 0.004 mg/mL. A study to evaluate the biological activities of COS including cytotoxicity in tumor and normal cells and various tests in vitro antioxidant activity of pure chitosan oligomers and the mixture of oligomers produced by the crude enzyme was performed. Moreover, the compound with the highest cytotoxicity among the oligomers was pure glucosamine, with IC50 values of 0.30; 0.49; 0.44 mg/mL for HepG2 cells, HeLa and 3T3, respectively. Superoxide anion scavenging was the mainly antioxidant activity showed by the COS and oligomers. This activity was also depending on the oligomer composition in the chitosan hydrolysates. The oligomers produced by hydrolysis for 20 minutes was analyzed for the ability to inhibit tumor cells showing inhibition of proliferation only in HeLa cells, did not show any effect in HepG2 cells and fibroblast cells (3T3)

Chitinolytic activities in the gut of Aedes aegypti (Diptera : Culicidae) larvae and their role in digestion of chitin-rich structures

Mosquito larvae are believed to be capable of digesting chitin, an insoluble polysaccharide of N-acetylglucosamine, for their nutritional benefit. Studies based on physiological and biochemical assays were conducted in order to detect the presence of chitinase activities in the gut of the detritus-feeding Aedes aegypti larvae. Larvae placed for 24 h in suspensions of chitin azure were able to digest the ingested chitin. Semi-denaturing PAGE using glycol chitin and two fluorogenic substrate analogues showed the presence of two distinct chitinase activities: an endochitinase that catalyzed the hydrolysis of chitin and an endochitinase that cleaved the short substrates [4MU(GlcNAc)(3)] and [4MU(GlcNAc)(2)] that hydrolyzed the chitobioside [4MU(GlcNAc)(2)]. The endochitinase had an extremely broad pH-activity against glycol chitin and chitin azure, pH ranging from 4.0 to 10.0. When the substrate [4MU(GlcNAc)(3)] was used, two activities were observed at pH ranges 4.0-6.0 and 8.0-10.0. Chitinase activity against [4MU(GlcNAc)(3)] was detected throughout the gut with the highest specific activity in the hindgut. The pH of the gut contents was determined by observing color changes in gut after feeding the larvae with color indicator dyes. It was observed a correlation between the pH observed in the gut of feeding larvae (pH 10-6.0) and the optimum pH for gut chitinase activities. In this work, we report that gut chitinases may be involved in the digestion of chitin-containing structures and also in the partial degradation of the chitinous peritrophic matrix in the hindgut. (C) 2003 Elsevier B.V. All rights reserved.

Lectinhistochemical staining of granuloma induced by bacillus Calmette- Guerin in Piaractus mesopotamicus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genes that encodes NAGT, MIF1 and MIF2 are not virulence factors for kala-azar caused by Leishmania infantum

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cinética de crescimento do fungo termófilo Myceliophthora thermophila M. 7.7 em cultivo no estado sólido

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Glycomic analysis of human mast cells, eosinophils and basophils

In allergic diseases such as asthma, eosinophils, basophils and mast cells, through release of preformed and newly generated mediators, granule proteins and cytokines, are recognized as key effector cells. While their surface protein phenotypes, mediator release profiles, ontogeny, cell trafficking and genomes have been generally explored and compared, there has yet to be any thorough analysis and comparison of their glycomes. Such studies are critical to understand the contribution of carbohydrates to the induction and regulation of allergic inflammatory responses and are now possible using improved technologies for detecting and characterizing cell-derived glycans. We thus report here the application of high-sensitivity mass spectrometric-based glycomics methodologies to the analysis of N-linked glycans derived from isolated populations of human mast cells, eosinophils and basophils. The samples were subjected to matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) screening analyses and MALDI-TOF/TOF sequencing studies. Results reveal substantive quantities of terminal N-acetylglucosamine containing structures in both the eosinophil and the basophil samples, whereas mast cells display greater relative quantities of sialylated terminal epitopes. For the first time, we characterize the cell surface glycan structures of principal allergic effector cells, which by interaction with glycan-binding proteins (e.g. lectins) have the possibility to dictate cellular functions, and might thus have important implications for the pathogenesis of inflammatory and allergic diseases.

Radiation metabolomics. 4. UPLC-ESI-QTOFMS-Based metabolomics for urinary biomarker discovery in gamma-irradiated rats

Radiation metabolomics has aided in the identification of a number of biomarkers in cells and mice by ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) and in rats by gas chromatography-coupled mass spectrometry (GCMS). These markers have been shown to be both dose- and time-dependent. Here UPLC-ESI-QTOFMS was used to analyze rat urine samples taken from 12 rats over 7 days; they were either sham-irradiated or γ-irradiated with 3 Gy after 4 days of metabolic cage acclimatization. Using multivariate data analysis, nine urinary biomarkers of γ radiation in rats were identified, including a novel mammalian metabolite, N-acetyltaurine. These upregulated urinary biomarkers were confirmed through tandem mass spectrometry and comparisons with authentic standards. They include thymidine, 2'-deoxyuridine, 2'deoxyxanthosine, N(1)-acetylspermidine, N-acetylglucosamine/galactosamine-6-sulfate, N-acetyltaurine, N-hexanoylglycine, taurine and, tentatively, isethionic acid. Of these metabolites, 2'-deoxyuridine and thymidine were previously identified in the rat by GCMS (observed as uridine and thymine) and in the mouse by UPLC-ESI-QTOFMS. 2'Deoxyxanthosine, taurine and N-hexanoylglycine were also seen in the mouse by UPLC-ESI-QTOFMS. These are now unequivocal cross-species biomarkers for ionizing radiation exposure. Downregulated biomarkers were shown to be related to food deprivation and starvation mechanisms. The UPLC-ESI-QTOFMS approach has aided in the advance for finding common biomarkers of ionizing radiation exposure.

Heteroresistance to Fosfomycin Is Predominant in Streptococcus pneumoniae and Depends on the murA1 Gene

Fosfomycin targets the first step of peptidoglycan biosynthesis in Streptococcus pneumoniae catalyzed by UDP-N-acetylglucosamine enolpyruvyltransferase (MurA1). We investigated whether heteroresistance to fosfomycin occurs in S. pneumoniae. We found that of 11 strains tested, all but 1 (Hungary(19A)) displayed heteroresistance and that deletion of murA1 abolished heteroresistance. Hungary(19A) differs from the other strains by a single amino acid substitution in MurA1 (Ala364Thr). To test whether this substitution is responsible for the lack of heteroresistance, it was introduced into strain D39. The heteroresistance phenotype of strain D39 was not changed. Furthermore, no relevant structural differences between the MurA1 crystal structures of heteroresistant strain D39 and nonheteroresistant strain Hungary(19A) were found. Our results reveal that heteroresistance to fosfomycin is the predominant phenotype of S. pneumoniae and that MurA1 is required for heteroresistance to fosfomycin but is not the only factor involved. The findings provide a caveat for any future use of fosfomycin in the treatment of pneumococcal infections.