Cadmium, ATPase-P , Levure (Du transport à la toxicité )

Mes travaux ont consisté en l'étude fonctionnelle de l'A TPase-CdL+, CadA de Listeria monocytogenes e't en compréhension des mécanismes de toxicité et de réponse vis-à-vis du cadmium chez Saccharomyces cerevisiae. 'originalité de ce travail repose sur l'utilisation d'un phénotype de sensibilité au cadmium associé à l'expression de CadA chez S. cerevisiae. Ce phénotype s'est révélé être un système de criblage pertinent pour l'identification des acides aminés essentiels au fonctionnement de CadA et un système d'étude de la toxicité du cadmium chez la levure. L'ATPase-P CadA transporte les ions Cd2+ d'un côté à l'autre d'une membrane en utilisant l'hydrolyse de l'ATPomme source d'énergie. La mutagenèse dirigée des acides aminés membranaires de CadA a permis de mettre en évidence que 2 ions Cd2+ seraient transportés par A TP hydrolysé et que les sites de transport du métal impliquent les cystéines et la proline du motif CPC (TM6), un aspartate (TM8), un glutamate (TM4) et une méthionine (TM3). l'expression de CadA rend la levure S. cerevisiae hypersensible au cadmium. Une souche sauvage croît en présence de lOOIlM de cadmium alors que la souche exprimant CadA est incapable de croître en présence de IIlM de cadmium. Ce phénotype a permis de montrer que l'entrée du cadmium se fait par un transporteur de manganèse et que la sensibilité au cadmium résulte d'une accumulation de cadmium dans le réticulum endoplasmique (RE), accumulation réalisée par CadA. Dans le RE, le cadmium altère le processus de repliement, ce à quoi la levure répond en déclenchant la réponse UPR. Cette réponse, aussi observée chez une levure normale fait du RE une cible majeure de la toxicité du cadmium.Two projects has been developed during my PhD. One consisting in the functional study of CadA, the Cd2+ -A TPase from Listeria monocytogenes, the other one was focused on the toxicity of cadmium and the associated response of the yeast Saccharomyces cerevisiae. This two studies used a a phenotype of sensitivity to cadmium induced by CadA expression in yeast. This phenotype was used as a screening tool to identify essential amino acids of Cd transport byCadA and to study cadmium toxicity and the corresponding yeast cellular response. CadA actively transports Cd using A TP hydrolysis as energy source. Directed mutagenesis of the membranous amino-acids revealed that Cd transport pathway implied four transmembrane segments (Tm) and more precisely the cysteine C354, C356 and pro line P355 of the CPC motif located in Tm6, aspartate D692 in Tm8, glutamate EI64 in Tm4 and methionine Ml49 in Tm5. From our studies, 2 Cd ions would be translocated for each hydrolysis A TP. Expression of CadA in the yeast Saccharomyces cerevisiae induces an hypersensitivity to Cd. A wild type cell can grow up to IOOIlM cadmium whereas CadA expressing yeast cannot grow with IIlM cadmium in the culture medium. This cadmium sensitivity was due to the localisation of CadA in the endoplasmic reticulum membrane. Transport of cadmium in this compartment poduces an accumulation of misfolded proteins that induces the Unfolded Protein Response (UPR). As UPR also occurs in a wild type yeast exposed to low Cd concentration, one can point out endoplasmic reticulum as a extremely sensitive cellular compartment. UPR also appears as an early response to Cd as it happens far before any visible signs oftoxicity.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

CadA, the Cd2+-ATPase from Listeria monocytogenes, can use Cd2+ as co-substrate.

International audienceCadA is a membrane protein of the P-type ATPase family which is the major determinant of the resistance to Cd2+ in Listeria monocytogenes. During its catalytic cycle, CadA undergoes auto-phosphorylation from ATP at Asp398, which allows Cd2+ translocation across the membrane. In the reverse mode, Asp398 is phosphorylated from Pi. From the data obtained so far, the CadA catalytic mechanism is similar to that proposed for the sarcoplasmic reticulum Ca2+-ATPase, the model of the P-type ATPase family. We show here that CadA is sensitive to two different ranges of Cd2+ concentration. The 0.1-10 microM range of added CdCl2 corresponds to Cd2+ binding at the transport site of unphosphorylated CadA which induces the reaction of the enzyme with ATP and impairs its reaction with Pi. The 0.1-1 mM range of added CdCl2 could correspond to Cd2+ binding to the transport site accessible from the extracellular medium. In addition, although it is widely accepted that the actual substrate of P-type ATPases is the MgATP complex, we show here that CadA can also perform its cycle in the absence of Mg2+, using CdATP in the place of MgATP at the catalytic site

The cadmium transport sites of CadA, the Cd2+-ATPase from Listeria monocytogenes.

International audienceCadA, the Cd(2+)-ATPase from Listeria monocytogenes, belongs to the Zn(2+)/Cd(2+)/Pb(2+)-ATPase bacterial subfamily of P(1B)-ATPases that ensure detoxification of the bacteria. Whereas it is the major determinant of Listeria resistance to Cd(2+), CadA expressed in Saccharomyces cerevisiae severely decreases yeast tolerance to Cd(2+) (Wu, C. C., Bal, N., Pérard, J., Lowe, J., Boscheron, C., Mintz, E., and Catty, P. (2004) Biochem. Biophys. Res. Commun. 324, 1034-1040). This phenotype, which reflects in vivo Cd(2+)-transport activity, was used to select from 33 point mutations, shared out among the eight transmembrane (TM) segments of CadA, those that affect the activity of the protein. Six mutations affecting CadA were found: M149A in TM3; E164A in TM4; C354A, P355A, and C356A in TM6; and D692A in TM8. Functional studies of the six mutants produced in Sf9 cells revealed that Cys(354) and Cys(356) in TM6 as well as Asp(692) in TM8 and Met(149) in TM3 could participate at the Cd(2+)-binding site(s). In the canonical Cys-Pro-Cys motif of P(1B)-ATPases, the two cysteines act at distinct steps in the transport mechanism, Cys(354) being directly involved in Cd(2+) binding, while Cys(356) seems to be required for Cd(2+) occlusion. This confirms an earlier observation that the two equivalent Cys of Ccc2, the yeast Cu(+)-ATPase, also act at different steps. In TM4, Glu(164), which is conserved among P(1B)-ATPases, may be required for Cd(2+) release. Finally, analysis of the role of Cd(2+) in the phosphorylation from ATP and from P(i) of the mutants suggests that two Cd(2+) ions are involved in the reaction cycle of CadA

Endoplasmic reticulum is a major target of cadmium toxicity in yeast

International audienceCadmium (Cd2+) is a very toxic metal that causes DNA damage, oxidative stress and apoptosis. Despite many studies, the cellular and molecular mechanisms underlying its high toxicity are not clearly understood. We show here that very low doses of Cd2+ cause ER stress in Saccharomyces cerevisiae as evidenced by the induction of the unfolded protein response (UPR) and the splicing of HAC1 mRNA. Furthermore, mutant strains (Δire1 and Δhac1) unable to induce the UPR are hypersensitive to Cd2+, but not to arsenite and mercury. The full functionality of the pathways involved in ER stress response is required for Cd2+ tolerance. The data also suggest that Cd2+-induced ER stress and Cd2+ toxicity are a direct consequence of Cd2+ accumulation in the ER. Cd2+ does not inhibit disulfide bond formation but perturbs calcium metabolism. In particular, Cd2+ activates the calcium channel Cch1/Mid1, which also contributes to Cd2+ entry into the cell. The results reinforce the interest of using yeast as a cellular model to study toxicity mechanisms in eukaryotic cells

Role of meprins to protect the gut mucosa from colonization by adherent-invasive E. coli

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Role of meprins to protect the gut mucosa from colonization by adherent-invasive E. coli

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Flowers of ruderal species are numerous but small, short and low‐rewarding

International audienceWeed species are ecological models that recently received considerable attention due to their particular strategies linked to their ruderal-competitive traits. They are known to have the potential to provide additional floral resources for insects in flower-poor agroecosystems. However, their floral traits are much more scarcely studied than those of plants found in other habitats, such as grasslands. The aim of this study was to describe the floral phenotype of weeds and to determine to what extent their floral traits match their ecological strategies as described based on leaf traits. We cultivated 19 forb weeds from perennial agroecosystems, previously identified in Mediterranean fields, in a greenhouse for seven months and collected data on 12 floral and five leaf traits. We tested whether these traits covaried and exhibited an ecological strategy at the phenotype scale. We found that in matters of flower production, weed species face a tradeoff: either numerous small, low-stature flowers with small quantities of pollen and nectar, or few, large, higher-held flowers with more pollen and nectar. The floral traits were found to reflect Grime's CSR strategies: the weed species producing fewer but costlier flowers belonged to C-strategy species, whereas those producing more but less costly flowers belonged to species dominated by an R strategy. These findings indicate that the potential of weeds as floral resources for insects is related to their ecological strategies, which are known to be affected by agricultural practices that filter species composition. This implies that, as for the provision of other ecosystem services, weed communities can be managed to select species with floral traits matching the requirements of flower-visiting insects like pollinators or parasitoid wasps

Meprin treatment affect mannose residue recognition by AIEC and AIEC-induced IL-8 secretion by T84 cells.

<p>A, ability of type 1 pili to bind D-mannose residues as determined by a yeast aggregation test. AIEC LF82 bacteria were treated with 100 µg/ml of meprin α or β at 37°C for 120 min. A fixed amount of inactivated yeast cells (<i>Saccharomyces cerevisiae</i>) suspension and decreasing concentrations of treated and untreated bacteria were mixed, and the loss of the ability to form homogenous aggregation was used as the read-out for impaired type 1 pili-yeast interaction. B, Amount of IL-8 secreted by uninfected or AIEC LF82- or type 1 pili negative mutant LF82-Δ<i>fimA</i>-infected T84 cells, at 24 h post-infection. AIEC LF82 and LF82-Δ<i>fimA</i> bacteria were treated with 100 µg/ml of meprins. Il-8 secretion was determined by ELISA. Data are expressed as fold increase in the amount of secreted IL-8 ± SEM by T84 cells infected with untreated or treated bacteria relative to non infected cells. Student's <i>t</i>-test, * <i>P</i><0.05 for comparison between IL-8 secretion induced by untreated versus meprin-treated AIEC LF82 or LF82-Δ<i>fimA</i> bacteria. C, LF82-Δ<i>fimA</i> bacteria were pretreated with exogenous meprin α or meprin β at 100 µg/ml and undifferentiated T84 cells were infected at a MOI of 10. The number of associated bacteria was determined. Results are expressed as the percentage of cell-associated bacteria pretreated with exogenous meprins relative to untreated bacteria, defined as 100%.D, effect of meprins on recombinant human IL-8. Recombinant human IL-8 (110 ng/ml) was treated with meprin α or β (100 µg/ml), electroblotted and detected with mouse anti-human IL-8.</p

Proteolytic activity of meprins α and β on AIEC LF82 outer membrane proteins and flagellin.

<p>Total protein extracts from untreated or meprin-treated (100 µg/mL) whole bacteria were immunoblotted for OmpA and OmpC/F (A) or Flagellin (B). The inner membrane protein Lep was used as internal control. Amounts of proteins were quantified by using <i>Image J</i> software. Results are expressed as protein amount relative to Lep. Data are mean ± SEM for at least three independent experiments. Student's <i>t</i>-test, * <i>P</i><0.05.</p

Intestinal meprin α and meprin β mRNA.

<p>A and B, Mep1A (A) and Mep1B (B) mRNA levels were determined by TaqMan quantitative real time PCR and are displayed as amounts relative to the intestinal epithelial marker villin-1. Healthy controls (hc) were compared with ulcerative colitis (UC) and Crohn's disease (CD) patients. CD patient biopsies were separated into groups with normal appearance or with macroscopic inflammation, as determined by an experienced endoscopist. Statistics were performed using GraphPad Prism 5.0 Software, and <i>P</i> values were calculated with the non-parametric Mann-Whitney test. C and D, Mep1A (C) and Mep1B (D) mRNA levels in C57Bl/6J mouse ileum and colon uninfected or infected with AIEC LF82 bacteria. The effect of AIEC LF82 infection on meprin expression was determined in mouse ileum and colon by quantitative real time PCR. Data are displayed as meprin amounts relative to the housekeeping TATA box binding protein (TBP) gene. Statistics were performed using GraphPad Prism 5.0 Software, and <i>P</i> values were calculated with the one-way ANOVA test. Dot plots show individual samples with relative mRNA (cDNA) amounts on a linear scale. Horizontal bars represent the median.</p