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Les récepteurs nucléaires LXRs sont associés aux métabolismes des lipides, du cholestérol et des hydrates de carbone. Ses agonistes naturels seraient des dérivés oxydés du cholestérol connus sous le nom d’oxystérols. Le cholestérol et les oxystérols sont associés au développement de l’athérosclérose une maladie cardiovasculaire qui a des graves conséquences sur les sociétés modernes. L’un des facteurs de risque majeurs de l’athérosclérose est le diabète de type II. Dans cette étude, qui essaye de faire un lien entre athérosclérose et diabètes, nous avons étudié les effets des LXRs sur le transport du glucose en conditions d’hyperglycémie et d’hyperglycémie/hyperinsulinémie qui sont des caractéristiques du diabète de type II. Nous avons mis en évidence que les transporteurs des hydrates de carbone Glut1, Glut3, Glut5 et Glut9 sont exprimés dans le macrophage humain THP1. Glut5 est connu comme étant un transporteur de fructose et Glut9 comme un transporteur d’urée. Nous avons démontré que Glut1 est impliqué dans le transport glucose des macrophages humain THP-1 et que ce transport est sensible à l’insuline. Tant l’activation spécifique que l’activation non spécifique de LXRs augmentent le transport de glucose dans les conditions étudiées, mais d’une manière différente. Cette étude nous a permis de démontrer qu’il pouvait y avoir un lien entre l’activation des récepteurs nucléaires LXRs sensibles aux oxystérols et une amélioration du transport du glucose dans les macrophages humains. / LXRs are nuclear receptors involved in lipids, cholesterol and carbohydrates metabolism. Their natural agonists are believed to be oxidized derivatives of cholesterol known as oxysterols. Cholesterol and oxysterols are associated with the development of atherosclerosis a cardiovascular disease having devastating consequences in modern societies. One of the major risk factors for atherosclerosis is type II diabetes. In this report, trying to make a link between atherosclerosis and diabetes, we attempt to demonstrate that LXRs may have an effect on glucose transport in conditions of hyperglycemia or hyperglycemia/hyperinsulinemia, two specific characteristics of type II diabetes. We found that in the THP1 human macrophage Glut1, Glut3, Glut5 and Glut9 are expressed. Glut5 is known as a fructose transporter and Glut9 as a urea transporter. We unveil that Glut1 was involved in glucose transport in human THP1 macrophages, and it was sensible to induction with insulin. Both specific and less specific activation of LXR increased glucose transport in hyperglycemic and hyperglycemic/hyperinsulinemic conditions but in a different way. This study demonstrated that activation of LXRs nuclear receptors that are sensible to oxysterols have benefic effects on glucose transport in human macrophages.

The clearance of circulating glucose and long-chain fatty acids (FA) into skeletal muscle involves the translocation of glucose transporter GLUT4, fatty acid translocase (FAT/CD36), plasma membrane associated fatty acid binding protein (FABPpm) and fatty acid transport protein (FATP) 1 and 4 to the plasma membrane (PM). FAT/CD36 also appears to participate in the regulation of mitochondrial FA oxidation. Metabolic challenges are known to increase FA transport and/or oxidation, but whether this is solely attributable to the translocation of FAT/CD36 to the sarcolemma and/or mitochondria is unknown. Moreover, the signaling and trafficking pathways involved in the translocation of FA transporters are largely unexplored. In this thesis it was found that FA transport was markedly increased following insulin (+2.9-fold) or contraction (+1.7-fold) stimulation of skeletal muscle, along with the PM contents of FAT/CD36 (+78%, +55%,), FABPpm (+61%, +62%), FATP1 (+84%, +61%) and FATP4 (+60%, +66%) (p<0.05). Upon combining the two stimuli, only the translocation of FAT/CD36 (+179%) and FATP1 (+125%) to the PM was additive, suggesting that these transporters may reside in distinct insulin-sensitive and contraction-sensitive intracellular compartments. The translocation of FA transporters may involve the insulin-signaling protein Akt2. It was found that insulin-stimulated FA transport and PM translocation of FA transporters was essentially prevented in Akt2 knockout mice. Following contraction, FA transport was also markedly blunted, along with an impaired translocation of both FAT/CD36 and FATP1, but not FABPpm or FATP4. FA oxidation and mitochondrial FAT/CD36 appearance were also inhibited following muscle contraction in knockout mice (p<0.05). Whether the GLUT4 trafficking protein Munc18c is important for the vesicular re-distribution of FA transporters to the PM or mitochondria was also investigated. FA uptake was comparably increased 1.4 fold with insulin and contraction in both wildtype and heterozygous Munc18c-/+ mice, as were PM FA transporters FAT/CD36 (+82%, +84%), FABPpm (+39%, +43%), FATP1 (+40%, +38%) and FATP4 (+33%, +32%) (p<0.05). Contraction-stimulated mitochondrial FA oxidation was also increased similarly in wildtype (+39%) and Munc18c-/+ mice (+33%). These studies demonstrate that a number of FA transporters are involved in upregulating skeletal muscle FA transport, although their signaling and trafficking pathways may differ from that of GLUT4.

The facilitative glucose transporter GLUT1 catalyzes the passive translocation of glucose across plasma membranes. Previous studies have demonstrated that regulation of GLUT1 activity is complex and subject to modulation by a variety of environmental and allosteric effectors. Mathematical models of GLUT1 kinetics have been derived which successfully account for subsets of these factors, though efforts to develop a single kinetic theory has not yet been achieved. Limitations in conventional experimental methodologies cannot provide kinetic data with the precision and continuity needed for further refinement of said models. We utilized a glucose-sensitive fluorescent protein (GlcSnFR) to develop novel experimental methodologies to facilitate elucidation of the GLUT1 kinetic mechanism. Characterization of the kinetic and optic properties of GlcSnFR and glucose binding (kon: 389.5 ± 12.1 M-1s-1, koff: 0.593 ± 0.103 s-1, kD: 1.52 ± 0.31 mM) allowed us to construct analyses which continuously estimate glucose flux through GLUT1 with subsecond temporal resolution and spacial resolution of a single human erythrocyte. We additionally revised the analytic methodology for interpreting the collected kinetic data sets to reduce the a priori assumptions regarding equilibrium states of the GLUT1 enzyme ensemble, which provided more flexible, model-independent characterizations of transporter activity. Using resealed human erythrocyte ghosts containing GlcSnFR, we applied techniques in confocal microscopy and fluorometry to achieve new insights into the kinetics of GLUT1. By microscopy, we observed transport in single cells to achieve conclusive evidence that populations of resealed ghosts are homogeneous in their composition, a previously unachieved result which retroactively validates to a great many previous studies. Further, we confirmed, unambiguously, that ATP induces directional asymmetry in GLUT1 through a reduction in rates of sugar efflux. By fluorometry, we demonstrated that previous measurements of the endofacial KM of GLUT1 for glucose are underestimated (~4 mM) and closer to ~20 mM. Finally, these new methods provide access to a new paradigm for measuring glucose transport in general and will serve to advance the field significantly in years to come.

Aujourd’hui, face à l’épidémie d’obésité, de plus en plus de personnes ont recours à la chirurgie bariatrique, qui permet une perte de poids importante et une amélioration des conditions métaboliques associées à l’obésité. L’adaptation gastro-intestinale après la chirurgie et ses conséquences métaboliques sont cependant peu connues. Nous avons développé des modèles murins de bypass gastriques et de gastrectomie longitudinale (sleeve) et nous avons caractérisé l’adaptation morphologique et fonctionnelle de l’épithélium gastro-intestinal après ces chirurgies afin de comprendre l’origine des améliorations métaboliques. Nous avons montré que l’estomac était remodelé après les deux chirurgies, suggérant une augmentation de la production acide par les cellules pariétales et une altération de la production de gastrine et de ghréline. Après le bypass, l’anse alimentaire était hyperplasique et la consommation intestinale de glucose était augmentée chez le rat et l’homme; après la sleeve, l’absorption de glucose lors du repas était diminuée. De plus, l’augmentation du nombre de cellules entéroendocrines observée après le bypass, et l’augmentation de leur densité après la sleeve pourraient participer à l’hypersécrétion des hormones incrétines. L’ensemble de ces mécanismes pourrait contribuer à améliorer le contrôle de la glycémie. Enfin, le mini-bypass chez le rat a induit une malabsorption protéique et des fuites énergétiques majeures qui n’étaient pas compensées par l’hyperplasie intestinale ou l’augmentation de l’expression des transporteurs de peptides. Cette thèse montre l’importance du tractus gastro-intestinal dans les conséquences métaboliques de la chirurgie bariatrique / In today’s global epidemic of obesity, more and more people are undergoing bariatric surgery, which is the best known treatment available to lose weight and treat obesity-associated diseases. Yet, little is known about gastrointestinal (GI) adaptation and its metabolic consequences after surgery. We developed original models of Roux-en-Y gastric bypass (RYGB), mini-bypass (MGB) and vertical sleeve gastrectomy (VSG) in rats, and we characterized the morphological and functional adaptations of the GI epithelium after these surgeries in order to understand the origin of the observed metabolic improvements. We reported profound changes in the remaining gastric mucosa of rats having undergone RYGB and VSG, suggesting an increase in acid production by parietal cells and an impaired production of gastrin and ghrelin. In RYGB rats and patients, the alimentary limb was hyperplasic and intestinal glucose consumption was increased. After VSG, the absorption of glucose during meals appeared diminished. These adaptations could participate in the lowering of blood glucose after surgery. In addition, the increased number of enteroendocrine cells observed in RYGB rats and patients, and their increased density in VSG rats, could contribute to the higher secretion of incretin hormone and improved glycemic control in operated individuals. Finally, we demonstrated in rats that the MGB was more malabsorptive than the RYGB. Indeed, we observed an increased fecal loss of nitrogen and energy despite intestinal overgrowth and higher expression of peptide transporters. This thesis brings new insight to the role of the GI tract in the metabolic outcomes of bariatric surgeries

Chez les mammifères, le muscle squelettique constitue un tissu d'importance majeure dans la régulation du transport et du métabolisme du glucose durant l'exercice physique ou en période postprandiale. La captation musculaire de glucose induite par l'insuline et la contraction musculaire s'effectue grâce à la translocation des transporteurs de glucose GLUT4 à la membrane plasmique et aux tubules transversaux à partir d'un réservoir interne. La première partie des travaux constituant cette thèse a été effectuée dans le but de clarifier si l'insuline et la contraction musculaire activent la translocation de GLUT4 à partir de réservoirs internes distincts. Par fractionnement et immunoadsorption membranaire, nous avons démontré que la contraction musculaire recrutait GLUT4 à partir de deux compartiments distincts : un compartiment associé au récepteur de la transferrine sélectivement mobilisé à la membrane plasmique et insensible à l'insuline, et un second compartiment non associé à cette protéine recruté au niveau des tubules transversaux. / Cette étude a permis de déterminer que l'insuline et la contraction musculaire recrutaient GLUT4 à partir de réservoirs distincts et que la contraction musculaire induisait la translocation de GLUT4 à la surface cellulaire à partir d'au moins deux différentes populations de vésicules GLUT4.Les deux dernières parties des travaux faisant l'objet de cette thèse ont permis de déterminer l'implication de certains médiateurs intracellulaires dans la stimulation du transport du glucose induite par la contraction musculaire ou par le AICAR, un agent mimétique de la contraction. Tout d'abord, nos travaux ont révélé que l'infusion de AICAR induit sélectivement la translocation de GLUT4 à la membrane plasmique à partir d'un compartiment enrichi en récepteur de la transferrine. De plus, nous avons démontré que la stimulation du transport du glucose par le AICAR était dépendante de l'activation de la p38 MAPK, une kinase proposée comme agent régulateur de l'activité intrinsèque de GLUT4. / D'autre part, une dernière étude nous a permis de déterminer que le AICAR active spécifiquement le transport du glucose au niveau des muscles glycolytiques isolés et que le monoxyde d'azote n'est pas impliqué dans l'effet stimulateur du AICAR sur la captation du glucose au niveau de ce type de muscle. Toutefois, l'infusion de AICAR in vivo stimule le transport de glucose dans tous les types de fibres musculaires ainsi que la production de monoxyde d'azote. De plus, l'injection de AICAR stimule la phosphorylation et l'activation de eNOS, suggérant que l'activation du transport du glucose induite par le AICAR in vivo est dépendante de l'augmentation du flot sanguin via la production de monoxyde d'azote. Globalement, ces études nous ont permis de mieux comprendre les mécanismes intracellulaires par lesquels la contraction musculaire active le transport du glucose in vitro et in vivo dans le muscle squelettique.

No description available.

GLUT4 trafficking in muscle cells has been studied to determine how distinct signalling pathways induce GLUT4 translocation. Two different cell models were adopted for these investigations; cardiomyocytes isolated from a transgenic mouse line expressing HA-GLUT4-GFP in muscle and L6 myotubes retrovirally expressing HA-GLUT4. The GLUT4 constructs were largely excluded from the external membrane under basal conditions in both cell models. GLUT4 was trafficked to the external membrane in to response all stimuli studied in cardiomyocytes (insulin, contraction and hypoxia) and L6 myotubes (insulin, AICAR and A-769662). By comparing the anti-HA and GFP signals at the sarcolemma and transverse tubules in cardiomyocytes, it has also be possible to observe an enhancement of GSV fusion with the sarcolemma following stimulation with insulin and contraction. This effect was specific to these stimuli and to the sarcolemma. Insulin-stimulation of GLUT4 exocytosis was not detected under steady-state conditions in L6 myotubes. Here, the major effect of insulin-stimulation and AMPK-activation was on GLUT4 internalisation. The rate constant for GLUT4 internalisation was very rapid in basal cells and was decreased during the steady-state responses to insulin and the AMPK-activators AICAR and A-769662. In cardiomyocytes, internalising GLUT4 colocalised with clathrin at puncta at the sarcolemma. This indicates that GLUT4 is internalised via a clathrin-mediated route. Investigations into the amount of GLUT4 recycling in L6 cells under steady-state conditions revealed that a large proportion of cellular GLUT4 recycles with the cell surface under basal conditions. Insulin-stimulation and AMPK-activation additively mobilised GLUT4 in L6 cells. This implies a non-convergent mobilisation of GLUT4 in response to activation of the PKB/Akt and AMPK signalling pathways. Data obtained from an in vitro kinase assay confirmed that serine 237 of TBC1D1 is a bone fide AMPK phosphorylation site. Furthermore, phosphorylation of this site in L6 myotubes incubated with AMPK activators has been confirmed using a novel antibody specific to TBC1D1 phosphorylated at serine 237. This thesis discusses the consequences and importance of multiple controls impinging on GLUT4 traffic and highlights the advantages and limitations of kinetic studies of these processes.

Introdução: O diabetes mellitus (DM) está associado a complicações que comprometem a qualidade de vida e a sobrevida dos indivíduos. Além disso, acarreta elevados custos para o controle metabólico e o tratamento de suas complicações, sendo assim caracterizado como um problema de saúde pública. A regulação da digestão e da absorção intestinal dos carboidratos, com vista a manter a homeostase da glicose plasmática, constituem importantes estratégias de proteção em condições clínicas como o diabetes tipo 2 (DM2), obesidade e síndrome metabólica. Os compostos fenólicos compreendem um grupo complexo de fitoquímicos bioativos presentes nos vegetais. Estudos in vitro e in vivo têm demonstrado que os compostos fenólicos inibem a atividade de carbohidrases (&#945;-amilase e &#945;-glicosidase) e o transporte intestinal de glicose mediado pelos transportadores SGLT1 e GLUT2. O cerrado brasileiro compreende uma larga biodiversidade, porém, apesar de muitas espécies terem sido identificadas, o seu potencial nutritivo e funcional ainda é pouco conhecido. Dentre estas espécies nativas é destacado o jatobá-do-cerrado. O jatobá-do-cerrado é uma leguminosa nativa brasileira, cuja a polpa farinácea que envolve suas sementes apresenta quantidades significativas de compostos fenólicos, podendo ter um potencial efeito sobre o metabolismo da glicose. Objetivos: Verificar os efeitos dos compostos fenólicos da farinha de jatobá-do-cerrado na digestão de carboidratos e na captação de glicose em células intestinais Caco-2. Metodologia: Os compostos fenólicos da farinha de jatobá foram obtidos por extração sequencial com as soluções de etanol (60%) e acetona (70%). Em seguida, o extrato foi digerido utilizando enzimas (&#945;-amilase, pepsina e pancreatina) em pH fisiológico. Os compostos fenólicos presentes no extrato antes e após a digestão foram identificados por cromatografia líquida de ultra performance - espectrômetro de massas (UPLC-MS/MS). Foi avaliada a capacidade de inibição dos extratos de jatobá digeridos em relação à atividade das enzimas &#945;-amilase e &#945;-glicosidase. Células intestinais Caco-2 foram incubadas com diferentes concentrações (0,05 mg/mL - 0,1 mg/mL) de extratos de farinha de jatobá digeridos em diferentes tempos (30 min, 2h e 12 h) para a avaliação da captação de glicose e da expressão gênica dos transportadores de glicose SGLT1 e GLUT2. Resultados: 44 compostos fenólicos foram identificados, dentre eles, a principal classe presente são os flavonoides. Compostos como o ácido cafeico, o kaempferol, quercetina-3- rutinosideo e a quercetrina estavam presentes no extrato antes da digestão. O conteúdo de compostos fenólicos do extrato foi reduzido após a digestão, entretanto o mesmo ainda apresentou compostos de relevância biológica como o ácido p-cumárico, ácido 3-o-feruloilquinico, theaflavina, crisina e grandinina que já apresentaram efeito positivo sobre o metabolismo da glicose in vitro em outros trabalhos. Os extratos fenólicos de jatobá após a digestão in vitro inibiram significativamente a atividade das enzimas &#945;-amilase (76 e 91%) e &#945;- glicosidase (53 e 77%). Os extratos também demonstraram inibir significativamente tanto a captação de glicose independente de sódio quanto a expressão gênica dos transportadores de glicose SGLT1 e GLUT2 de maneira dose-dependente. Conclusão: Este é o primeiro trabalho que identificou os compostos fenólicos presentes na farinha de jatobá. A partir do exposto, podemos concluir que a farinha de jatobá apresenta potencial benefício a saúde devido ao seu conteúdo de compostos fenólicos e a capacidade destes compostos de regular a digestão e a absorção de carboidratos in vitro. / Introduction: Diabetes mellitus (DM) is associated with complications that decrease the quality of life and survival of individuals. In addition, it entails high costs for metabolic control and treatment of its complications, thus being characterized as a public health problem. The regulation of digestion and intestinal absorption of carbohydrates to maintain plasma glucose homeostasis are important strategies for protection in chronic diseases such as type 2 diabetes (DM2), obesity and metabolic syndrome. Phenolic compounds are a complex group of chemical substances present in plants. In vitro and in vivo studies have shown that phenolic compounds are able to inhibit the activity of carbohydrases (&#945;-amylase and &#945;-glycosidase) and the intestinal transport of glucose mediated by SGLT1 and GLUT2 transporters. Brazilian Cerrado present a large biodiversity, but although many species have been identified, its nutritional and functional potential is still little known. Among these native species is the jatobá-docerrado. Jatobá-do-cerrado is a brazilian native legume, whose farinaceous pulp that surrounds its seeds presents significant amounts of phenolic compounds and may have a potential effect on glucose metabolism. Objectives: To verify the effects of phenolic compounds from jatobá-do-cerrado flour in the digestion of carbohydrates and uptake of glucose in Caco-2 intestinal cells. Methods: Phenolic compounds of jatobá flour were obtained by sequential extraction with solutions of ethanol (60%) and acetone (70%). The extract was digested using enzymes (&#945;-amylase, pepsin and pancreatin) at physiological pH. The phenolic compounds present in the extract before and after the digestion were identified by liquid chromatography of ultra-performance - mass spectrometer (UPLCMS / MS). The ability of inhibition of the extracts of jatobá digested in relation to the activity of &#945;-amylase and &#945;-glycosidase enzymes was evaluated. Caco-2 intestinal cells were incubated with different concentrations of jatobá flour extracts (0.1 mg / mL - 0.05 mg / mL) for different time (30 min, 2 h and 12 h) to the evaluation of facilitated uptake (sodium-free buffer) and gene expression of SGLT1 and GLUT2 glucose transporters. Results: 44 phenolic compounds have been identified, among them a major class present are flavonoids. Compounds such as caffeic acid, quercetin-3-rutinoside and quercetrine were present in the extract before in vitro digestion. The content of phenolic compounds of the extract after digestion was reduced. However, the extract presents compounds with biological activity such as p-coumaric acid, 3-o-feruloylquinic acid , theaflavin, chrysin and grandinine, which already presented positive effects on glucose metabolism in vitro in other studies. Phenolic extracts of jatobá after in vitro digestion inhibited the activity of &#945;-amylase (76 and 91%) and &#945;-glycosidase (53 and 77%). The extracts also shown to inhibit both glucose uptake and gene expression of glucose transporters SGLT1 and GLUT2 in a dose-dependent manner. Conclusion: This is the first work that identified the phenolic compounds present in jatobá flour. Thus, we can conclude that the jatobá flour presents potential health benefit by modulate digestion and the absorption of carbohydrates in vitro.

Listeria monocytogenes est une bactérie à Gram+, ubiquiste, pathogène intracellulaire d’origine alimentaire, responsable chez l’homme, de nombreuses infections telles que les infections foeto-maternelles, des méningo-encéphalites et des septicémies. La bactérie utilise préférentiellement le glucose qui est transporté via le système phosphoenolpyruvate:sucre phsosphotransferase (PTS) et des perméases non-PTS. Les deux principaux transporteurs de glucose chez L. monocytogenes seraient des PTS de la classe mannose. Le premier est codé par l’opéron manLMN (man) et le deuxième, par l’opéron mpoABCD (mpo). Nous avons, dans un premier temps, mis en évidence le transport de glucose par ces PTS chez L. monocytogenes et aussi identifier d’autres transporteurs non-PTS de glucose. Des tests de croissance en milieu minimum (MM) additionné de glucose et des tests de consommation de glucose ont permis de montrer que les mutants ΔmanL (manL code pour l’EIIABMan) et ΔmanM (manM code pour l’EIICMan) utilisent moins vite le glucose que la souche sauvage AML73 ou EGDe (3 à 4 fois moins vite). Le mutant ΔmpoA (mpoA code pour l’EIIAMpo) montre un phénotype similaire à la souche sauvage tandis que le mutant ΔmpoB (mpoB code pour l’EIIBMpo) utilise 4 à 5 fois moins vite le glucose que la souche sauvage. Des tests de qRT-PCR ont par ailleurs permis de montrer que la délétion du gène mpoA permet une expression constitutive de l’opéron man tandis que la délétion du gène mpoB entraîne une inhibition de l’expression de cet opéron. Nous avons aussi montré que l’opéron man est induit par le glucose et l’opéron mpo est exprimé constitutivement. Le PTSMan est le principal système de transport de glucose chez L. monocytogenes et le PTSMpo pourrait fonctionner comme un senseur de glucose qui en présence de ce sucre stimule l’expression de l’opéron man en régulant l’activité de ManR. Le mutant ΔptsI (ptsI code pour la protéine générale EI du PTS) utilise 8 à 10 fois moins vite le glucose que la souche sauvage et présente une très faible expression de l’opéron man. L’utilisation du glucose (bien que faible) par le mutant ΔptsI permet d’affirmer qu’il existerait des transporteurs non-PTS qui permettraient à ce mutant d’utiliser le glucose. Des tests de complémentation hétérologue dans la souche E. coli LJ140 (incapable de transporter le glucose) ont permis de montrer que les trois protéines GlcU (GlcU1, GlcU2 et GlcU3, identifiées par homologie de séquences aux GlcU d’autres firmicutes) permettent le transport de glucose chez L. monocytogenes mais avec une très faible affinité. Un rôle potentiel du PTS et des transporteurs non-PTS dans la régulation de PrfA a également été mis en évidence par des tests de dosage β-D-glucuronidase à partir de cultures bactériennes réalisées en milieux liquides ou sur géloses et aussi par des tests de qRT-PCR (pour l’expression des gènes actA et hly). Ces tests ont été réalisés à partir de la souche L. monocytogenes AML73 (portant la fusion Phly-gus) et des mutants ΔmanL, ΔmanM, ΔmpoB, ΔmpoA, ΔptsI et glcU (construits dans cette souche). Les mutations manL, manM, mpoB, ptsI entraînent une augmentation de l’activité de PrfA (de 2 à 14 fois) et une augmentation de l’expression des gènes de virulence PrfA-dépendants (hly et actA) est également observée dans les mutants ΔmanL, ΔmanM et ΔmpoB. Les mutations glcU et mpoA ne montrent aucun effet sur l’activité de PrfA. Les mutants montrant une forte activité de PrfA contiennent peu ou pas de protéine EIIABMan qui est supposée jouer un rôle dans la régulation de l’activité de PrfA par le glucose. L’effet des mutations PTS observé sur l’expression des gènes de virulence dépend de PrfA car cet effet disparaît quand le gène prfA est délété dans les mutants ΔmanL, ΔmanM et ΔmpoB. Les mutations montrant un effet sur l’activité de PrfA ont également été étudiées in vitro par des infections des cellules épithéliales (Caco-2 et Jeg-3) avec les différents mutants et également in vivo dans la souris. La délétion du gène ptsI montre un effet dans l’infection plus particulièrement dans l’entrée des bactéries dans les cellules / L. monocytogenes is a ubiquitous foodborne pathogenic Gram-positive bacterium, which can multiply in host cells and infect humans causing septicemia, spontaneous abortion and méningoencephalitis. This bacterium transports glucose via phosphoenolpyruvate:sugar phosphotransferase systems (PTS) and non-PTS permeases. Two major glucose-transporting PTSs belong to the mannose class. One is encoded by the manLMN (man) operon and the second by the mpoABCD (mpo) operon. One goal was to study the transport of glucose by the proteins encoded by these operons and to identify non-PTS glucose transporters. Growth studies in MM supplemented with glucose and glucose consumption assays with several mutants revealed that deletion of manL (encodes EIIABMan) or manM (encodes EIICMan) significantly slowed glucose utilization (3- to 4-fold) compared to the WT AML73 or EGDe strain. Deletion of mpoA (encodes EIIAMpo) had no significant effect on glucose utilization (same phenotype as the WT) whereas deletion of mpoB (encodes EIIBMpo) significantly slowed glucose utilization (4- to -5 fold). By using qRT-PCR, we show that expression of the man operon is induced by glucose, whereas the mpo operon is expressed constitutively. Nevertheless, deletion of mpoA causes constitutive man operon expression whereas deletion of mpoB inhibits it. The PTSMpo therefore functions as a constantly synthesized glucose sensor regulating man operon expression. Deletion of ptsI (encodes the general PTS component EI) also inhibits man expression and the ΔptsI mutant was most strongly impeded in glucose utilization. The residual glucose uptake probably owes to three GlcU-like non-PTS transporters. The successful heterelogous complementation of the E. coli LJ140 strain, wich is unable to transport glucose, suggests that the L. monocytogenes GlcU proteins, GlcU1, GlcU2 and GlcU3 (identified by sequences homology to GlcU proteins in other firmicutes) are indeed capable of transporting glucose.A potential role of PTS and non-PTS components in PrfA regulation was studied in the L. monocytogenes AML73 strain (contains a Phly-gus fusion) and in the ΔmanL, ΔmanM, ΔmpoB, ΔmpoA, ΔptsI, glcU mutants derived from it. For that purpose, I carried out β-D-glucuronidase activity tests with bacteria grown either in liquid or on solid medium and qRT-PCR experiments (expression of actA and hly genes). Interestingly, deletion of ptsI, manL, manM and mpoB caused elevated PrfA activity (2- to -14 fold) and elevated expression of virulence gene expression (actA and hly) in the ΔmanL, ΔmanM and ΔmpoB mutants was observed. Nevertheless, glcU inactivation and mpoA deletion had no effect on PrfA activity. The elevated PrfA activity disappeared when the prfA gene was also deleted in the ΔmanL, ΔmanM and ΔmpoB mutants, confirming that the stimulatory effect of the various mutations on virulence gene expression is PrfA-dependent. All mutants exhibiting elevated virulence gene expression contain no or only little unphosphorylated EIIABMan, which we therefore suspect to play a major role in glucose-mediated PrfA inhibition. The effect of the PTS mutations was also tested in in vitro host cells infection assays (Caco-2, Jeg-3 cells) and in an in vivo mouse model. Deletion of ptsI led to elevated infection of the host cells, which probably owes to the elevated synthesis of the InlA protein.