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Enterococcus faecalis, an organism generally not pathogenic for healthy humans, has the potential to cause disease in susceptible hosts. While it seems to be equipped to interact with and circumvent host immune defense, most of the molecular and cellular mechanisms underlying the enterococcal infectious process remain elusive. Here, we investigated the role of the Enterococcal Leucine Rich protein A (ElrA), an internalin-like protein of E. faecalis also known as a virulence factor. ElrA was previously shown to prevent adhesion to macrophages. We show that ElrA does not inhibit the basic phagocytic process, but is able to prevent sensing and migration of macrophages toward E. faecalis. Presence or absence of FHL2, a eukaryotic partner of ElrA, does not affect the ElrA-dependent mechanism preventing macrophage migration. However, we highlight a partial contribution of FHL2 in ElrA-mediated virulence in vivo. Our results indicate that ElrA plays at least a dual role of which anti-phagocytic activity may contribute to dissemination of extracellular E. faecalis during infection.

International audience; LeuRS (leucyl-tRNA synthetase) catalyses the esterification of tRNAsLeu with leucine. This family of enzymes is divided into prokaryotic and eukaryal/archaeal groups according to the presence and position of specific insertions and extensions. In the present study, we investigated the function of LSD1 (leucine-specific domain 1), which is naturally present in eukaryal/archaeal LeuRSs, but absent from prokaryotic LeuRSs. When mutated in their common domain, the eukaryal and archaeal LeuRSs exhibited defects in the first reaction step of amino acid activation with variations of leucine or ATP-binding strength, whereas the tRNA aminoacylation was moderately affected. When the eukaryal extension was mutated, severe tRNA charging defects were observed, suggesting that eukaryotes evolved this LSD1 extension in order to improve the aminoacylation reaction step. The results also showed that the LSD1s from organisms of both groups are dispensable for post-transfer editing. Together, the data provide us with a further understanding of the organization and structure of LeuRS domains.

The basis for the differential repressive effects of antiestrogens on transactivation by estrogen receptor-α (ERα) remains incompletely understood. Here, we show that the full antiestrogen ICI182,780 and, to a lesser extent, the selective ER modulator raloxifene (Ral), induce accumulation of exogenous ERα in a poorly soluble fraction in transiently transfected HepG2 or stably transfected MDA-MB231 cells and of endogenous receptor in MCF7 cells. ERα remained nuclear in HepG2 cells treated with either compound. Replacement of selected hydrophobic residues of ERα ligand-binding domain helix 12 (H12) enhanced receptor solubility in the presence of ICI182,780 or Ral. These mutations also increased transcriptional activity with Ral or ICI182,780 on reporter genes or on the endogenous estrogen target gene TFF1 in a manner requiring the integrity of the N-terminal AF-1 domain. The antiestrogen-specific effects of single mutations suggest that they affect receptor function by mechanisms other than a simple decrease in hydrophobicity of H12, possibly due to relief from local steric hindrance between these residues and the antiestrogen side chains. Fluorescence anisotropy experiments indicated an enhanced regional stabilization of mutant ligand-binding domains in the presence of antiestrogens. H12 mutations also prevent the increase in bioluminescence resonance energy transfer between ERα monomers induced by Ral or ICI182,780 and increase intranuclear receptor mobility in correlation with transcriptional activity in the presence of these antiestrogens. Our data indicate that ICI182,780 and Ral locally alter the ERα ligand binding structure via specific hydrophobic residues of H12 and decrease its transcriptional activity through tighter association with an insoluble nuclear structure.

International audience; NGFI-B is a ligand-independent orphan nuclear receptor of the NR4A subfamily that displays important functional differences with its homolog Nurr1. In particular, the NGFI-B ligand-binding domain (LBD) exhibits only modest activity in cell lines in which the Nurr1 LBD strongly activates transcription. To gain insight into the structural basis for the distinct activation potentials, we determined the crystal structure of the NGFI-B LBD at 2.4-angstroms resolution. Superimposition with the Nurr1 LBD revealed a significant shift of the position of helix 12, potentially caused by conservative amino acids exchanges in helix 3 or helix 12. Replacement of the helix 11-12 region of Nurr1 with that of NGFI-B dramatically reduces the transcriptional activity of the Nurr1 LBD. Similarly, mutation of Met414 in helix 3 to leucine or of Leu591 in helix 12 to isoleucine (the corresponding residues found in NGFI-B) significantly affects Nurr1 transactivation. In comparison, swapping the helix 11-12 region of Nurr1 into NGFI-B results in a modest increase of activity. These observations reveal a high sensitivity of LBD activity to changes that influence helix 12 positioning. Furthermore, mutation of hydrophobic surface residues in the helix 11-12 region (outside the canonical co-activator surface constituted by helices 3, 4, and 12) severely affects Nurr1 transactivation. Together, our data suggest that a novel co-regulator surface that includes helix 11 and a specifically positioned helix 12 determine the cell type-dependent activities of the NGFI-B and the Nurr1 LBD.

International audience; Listeria monocytogenes can use two different surface proteins, internalin (InlA) and InlB, to invade mammalian cells. The exact role of these invasiveness factors in vivo remains to be determined. In cultured cells, InlA is necessary to promote Listeria entry into human epithelial cells, such as Caco-2 cells, whereas InlB is necessary to promote Listeria internalization in several other cell types, including hepatocytes, fibroblasts, and epithelioid cells, such as Vero, HeLa, CHO, or Hep-2 cells. We have recently reported that the InlA receptor on Caco-2 cells is the cell adhesion molecule E-cadherin and demonstrated that nonpermissive fibroblasts become permissive for internalin-mediated entry when transfected with the gene coding for LCAM, the chicken homolog of the human E-cadherin gene. In this study, we demonstrate for the first time that the internalin protein alone is sufficient to promote internalization into cells expressing its receptor. Indeed, internalin confers invasiveness to both Enterococcus faecalis and internalin-coated latex beads. As shown by transmission electron microscopy, these beads were phagocytosed via a "zipper" mechanism similar to that observed during the internalin-E-cadherin-mediated entry of Listeria. Moreover, a functional analysis of internalin demonstrates that its amino-terminal region, encompassing the leucine-rich repeat (LRR) region and the inter-repeat (IR) region, is necessary and sufficient to promote bacterial entry into cells expressing its receptor. Several lines of evidence suggest that the LRR region would interact directly with E-cadherin, whereas the IR region would be required for a proper folding of the LRR region.

International audience; The design of a defined synthetic minimal medium for Campylobacter jejuni strain NCTC 11168 that includes only inorganic salts and necessary amino acids and vitamins is useful in physiological assays and responses to exogeneous agents. In silico genomic analysis of biosynthesis pathways was preliminarily performed prior to experimental assays to determine (i) amino acids and vitamins necessary for improving the growth of C. jejuni strains, and (ii) the most appropriate sources of carbon, nitrogen and sulfur. The different sources of carbon, nitrogen and sulfur were analyzed by comparing growth parameters. A new minimal medium that contains inorganic salts, the amino acids l-cysteine, l-leucine, l-methionine and l-aspartic acid (nitrogen source), the vitamin niacinamide and lactate as a carbon source, named MCLMAN (medium cysteine leucine methionine aspartic acid niacinamide), was checked on some C. jejuni strains and showed similar growth ratios and final biomass when compared to the most frequently used medium, MEM (modified Eagle's medium), primarily designed for eukaryote cell culture and more complex than MCLMAN. Our results show that C. jejuni presents auxotrophy for cysteine and methionine and can be inhibited by ammonium sulfate. A simple minimal medium containing few amino acids and vitamins will facilitate physiological studies of different functions in C. jejuni strains submitted to different stresses.

Summary The major capsid protein of the cauliflower mosaic virus (CaMV) is processed in vivo . The viral aspartic proteinase that catalyses this maturation has been characterized previously and is coded by the CaMV gene V. This virus has a second capsid protein, a minor component, encoded by gene III. This protein, P3, is also processed at its C-terminus in vivo . To determine whether P3 is matured by the CaMV proteinase P5, we expressed, in Saccharomyces cerevisiae , P3, P5 and a fusion protein P7-P4, containing potential sites of cleavage. P5 was found to be involved in maturation of P7-P4 but did not cleave P3. The latter result was confirmed by experiments carried out with an in vitro translation system (the reticulocyte lysate) and with preparations of replication complexes purified from infected plants. Moreover, N-(L-3- trans -carboxyoxiran-2-carbonyl)-L-leu cyl-amido(4-guanido)butane, a specific inhibitor of cysteine proteinases, inhibited the maturation of P3, suggesting that the two CaMV capsid proteins are not processed by the same proteolytic event.

A glycine-leucine-rich peptide was isolated from norepinephrine-stimulated skin secretions of the Sante Fe frog Leptodactylus laticeps (Leptodactylidae) whose primary structure (Gly-Leu-Val-Asn-Gly-Leu-Leu-Ser-Ser-Val-Leu-Gly-Gly-Gly-Gln-Gly-Gly-Gly -Gly-Leu-Leu-Gly-Gly-Ile-Leu) contains the (GXXXG)(3) motif found in the plasticins, previously identified only in phyllomedusid frogs (Hylidae). Circular dichroism studies showed that the secondary structure of the peptide, termed plasticin-L1, was markedly solvent-dependent displaying a random coil conformation in water, a beta-sheet structure in methanol, and an alpha-helical conformation in 50% trifluoroethanol-water. A synthetic replicate of the peptide did not inhibit the growth of Escherichia coli or Staphylococcus aureus or lyse human erythrocytes at concentrations up to 500 mu M. At relatively high concentrations (> 1 microM), the peptide produced a significant (P < 0.05), although modest (139% of basal rate at 3 mu M), increase in the rate of glucose-induced release of insulin from rat clonal BRIN-BD11 beta cells without increasing the rate of release of lactate dehydrogenase. A peptide, termed ocellatin-L2 was also identified in the skin secretion that was identical to the previously described ocellatin-L1 except for the substitution Asn(23) to Asp. Ocellatin-L2 was devoid of antimicrobial and hemolytic activity but also showed significant activity in stimulating insulin release from BRIN-BD11 cells (181% of basal rate at 3 microM). (C) 2009 Elsevier Inc. All rights reserved.

International audience; Myosin Storage Myopathies (MSM) have emerged as a new group of inherited myopathies with heterogenous clinical severity and age of onset. We have identified in a woman and her daughter, a pLeu1793Pro mutation in MYH7. This mutation has already been reported to be associated with MSM presenting as neonatal hypotony. Our index case complained of proximal muscle weakness at age 30. Her daughter presented at birth with a cardiomyopathy without any skeletal muscle involvement. This report underlines the clinical variability of MSM even with a given mutation or in a same family.

Aims: Short branched-chain fatty acids (BCFAs) are cheese flavour compounds, which result from the conversion of branched-chain amino acids (BCAAs). In Swiss cheese, the production of short BCFAs is mainly performed by Propionibacterium freudenreichii and is strain dependent. Our aim was to investigate the possible links between the biosynthesis of short BCFAs and membrane BCFAs in P. freudenreichii. Methods and Results: Short and membrane BCFAs were analysed by gas chromatography- mass spectrometry. Two strains differing in their capacities to release short BCFAs were selected. Tri-deuterated-labelled leucine was used in both strains as a precursor of short extracellular iso-BCFAs and of membrane iso-BCFAs. The proportions of anteiso : iso BCFAs synthesized varied as function of the BCAAs provided in the growth medium, from 72 : 28 to 100 : 0, with leucine and valine, and with isoleucine as sole BC precursors, respectively. The branching pattern of short BCFAs exactly matched that of membrane BCFAs, whatever the exogenous BCAAs provided. Conclusions: The biosynthesis of short BCFAs is closely related to that of membrane BCFAs in P. freudenreichii. Significance and Impact of the Study: The biosynthesis of short BCFAs in P. freudenreichii depends more on the strain than on the presence of exogenous BC precursors.

International audience; The marine cyanobacterium Prochlorococcus, the most abundant phototrophic organism on Earth, numerically dominates the phytoplankton in nitrogen (N)-depleted oceanic gyres. Alongside inorganic N sources such as nitrite and ammonium, natural populations of this genus also acquire organic N, specifically amino acids. Here, we investigated using isotopic tracer and flow cytometric cell sorting techniques whether amino acid uptake by Prochlorococcus is subject to a diel rhythmicity, and if so, whether this was linked to a specific cell cycle stage. We observed, in contrast to diurnally similar methionine uptake rates by Synechococcus cells, obvious diurnal rhythms in methionine uptake by Prochlorococcus cells in the tropical Atlantic. These rhythms were confirmed using reproducible cyclostat experiments with a light-synchronized axenic Prochlorococcus (PCC9511 strain) culture and (35)S-methionine and (3)H-leucine tracers. Cells acquired the tracers at lower rates around dawn and higher rates around dusk despite >10(4) times higher concentration of ammonium in the medium, presumably because amino acids can be directly incorporated into protein. Leucine uptake rates by cells in the S+G(2) cell cycle stage were consistently 2.2 times higher than those of cells at the G(1) stage. Furthermore, S+G(2) cells upregulated amino acid uptake 3.5 times from dawn to dusk to boost protein synthesis prior to cell division. Because Prochlorococcus populations can account from 13% at midday to 42% at dusk of total microbial uptake of methionine and probably of other amino acids in N-depleted oceanic waters, this genus exerts diurnally variable, strong competitive pressure on other bacterioplankton populations.

International audience; BACKGROUND: Mutations in the leucine-rich, glioma-inactivated 1 (LGI1) gene have been implicated in autosomal dominant lateral temporal epilepsy. OBJECTIVE: To describe the clinical and genetic findings in 2 families with autosomal dominant lateral temporal epilepsy and the functional consequences of 2 novel mutations in LGI1. DESIGN: Clinical, genetic, and functional investigations. SETTING: University hospital. Patients Two French families with autosomal dominant lateral temporal epilepsy. Main Outcome Measure Mutation analysis. RESULTS: Two novel disease-linked mutations, p.Leu232Pro and c.431 + 1G>A, were identified in LGI1. We demonstrated that the c.431 + 1G>A mutation causes the deletion of exons 3 and 4 of the LGI1 transcript and showed that the p.Leu232Pro mutation dramatically decreases secretion of the mutant protein by mammalian cells. CONCLUSION: Our data indicate that LGI1 is a secreted protein and suggest that LGI1-related epilepsy results from a loss of function.

The role of Leu 505 of Nox2 on the NADPH oxidase activation process was investigated. An X-CGD PLB-985 cell line expressing the Leu505Arg Nox2 mutant was obtained, exactly mim- icking the phenotype of a previously published X91- CGD case. In a reconstituted cell-free system (CFS), NADPH oxidase and iodonitrotetrazolium (INT) re- ductase activities were partially maintained concom- itantly with a partial cytosolic factors translocation to the plasma membrane. This suggests that assembly and electron transfer from NADPH occurred par- tially in the Leu505Arg Nox2 mutant. Moreover, in a simplified CFS using purified mutant cytochrome b558 and recombinant p67 phox , p47 phox , and Rac1proteins, we found that the Km for NADPH and for NADH was about three times higher than those of purified WT cytochrome b558, indicating that the Leu505Arg mutation induces a slight decrease of the affinity for NADPH and NADH. In addition, oxidase activity can be extended by increasing the amount of p67 phox in the simplified CFS assay. However, the maximal reconstituted oxidase activity using WT pu- rified cytochrome b558 could not be reached using mutant cytochrome b558. In a three-dimensional model of the C-terminal tail of Nox2, Leu 505 appears to have a strategic position just at the entry of the NADPH binding site and at the end of the -helical loop (residues 484-504), a potential cytosolic factor binding region. The Leu505Arg mutation seems to affect the oxidase complex activation process through alteration of cytosolic factors binding and more particularly the p67 phox interaction with cyto- chrome b558, thus affecting NADPH access to its binding site. J. Leukoc. Biol. 81: 238-249; 2007.

Centronuclear myopathy is a genetically heterogeneous congenital myopathy. Whilst mutations in the myotubularin (MTM1) gene are implicated in the X-linked variant, mutations in the dynamin 2 (DNM2) gene have been recently associated with dominant inheritance. We report a 16-year-old girl with clinical features of a congenital myopathy and external ophthalmoplegia. Multiple central nuclei affecting up to 50% of fibres and central accumulation of oxidative enzyme stains were the most prominent findings on muscle biopsy obtained at 1 year. However, some core-like areas appeared on repeat biopsy 8 years later; in addition, muscle MRI was compatible with the pattern we previously reported in patients with mutations in the skeletal muscle ryanodine receptor (RYR1) gene. Mutational analysis identified a de novo dominant RYR1 missense mutation (c.12335C>T; Ser4112Leu) affecting a highly conserved domain of the protein. Our findings expand the phenotypical spectrum associated with RYR1 mutations and indicate that RYR1 screening should be considered in centronuclear myopathy patients without MTM1 or DNM2 mutations; muscle MRI may aid selection of appropriate genetic testing.

International audience; Sulpha drugs act as competitive inhibitors of p-amino benzoic acid, an intermediate in the de novo folate pathway. Dihydropteroate synthase condenses sulpha drugs into sulpha-dihydropteroate (sulpha-DHP), which competes with dihydrofolate, the dihydrofolate reductase (DHFR) substrate. This designates DHFR as a possible target of sulpha-DHP. We suggest here that Plasmodium vivax DHFR is indeed the in vivo target of sulpha drugs. The wild-type DHFR expressed in Saccharomyces cerevisiae leads to cell growth inhibition, while sensitivity to the drug is exacerbated in the mutants. Contrary to what is observed with sulphanilamide, methotrexate is less effective on P. vivax-DHFR mutants than on wild-type mutant.

International audience; Yeast vacuoles are highly dynamic and flexible organelles. In a previous paper, we have shown that subtle, often unrecognised amino acid limitations lead to much lower final cell densities in cultures of different commonly used auxotrophic Saccharomyces cerevisiae strains (Cakar et al., Biotechnol. Lett. 21 (1999) 611). Here, we demonstrate for two of these strains, CEN.PK 113.6B and CBS7752, that such subtle leucine limitations also affect the number and morphology of vacuoles, and that these changes are correlated with the cell cycle in batch cultures in a similar way as is known from synchronized cultures. Morphological aspects were studied by electron microscopy, using advanced high pressure freezing/freeze-substitution techniques for sample preparation that so far have been barely successful in yeast. Cells of leucine-limited cultures had single, large vacuoles with a hexagonal tonoplast pattern and were partially arrested in G1 phase. To relieve leucine-limitation, additional leucine was supplied extracellularly via the medium or intracellularly via enhanced leucine biosynthesis due to plasmid-based expression of a leucine marker gene. Such cultures reached more than two-fold higher final optical densities in stationary phase. Cells in later growth phase were characterized by fragmented vacuoles lacking any tonoplast pattern and by a smaller proportion of cells in G1 phase. These drastic effects of subtle leucine limitation on cell physiology, vacuolar morphology and cell cycle distribution present a note of caution for morphological and cell cycle studies in yeast.

International audience; We investigated whether the toxicity-associated receptor-binding domain of the non-immunogenic Escherichia coli heat-stable enterotoxin (STh) as a fusion with a carrier protein and the inclusion of an appropriate spacer are critical factors for eliciting antibody responses against the native toxin. The immunological properties of three toxic and one non-toxic fusion proteins, consisting of STh N-terminally joined to the C-terminus of the major subunit ClpG of E. coli CS31A fimbriae, were compared. In contrast to the non-toxic hybrid STh with glycine and leucine simultaneously substituted for the receptor-interacting Pro(13) and Ala(14) amino acids, the toxic chimeras responded by producing high serum levels of anti-STh antibodies in immunized animals. On the other hand, only the toxic ClpG-STh construct with the natural peptide 47KSGPESM(53) of Pro-STh as spacer stimulated STh-neutralizing responses against both native toxin and enterotoxigenic live E. coli cells. Altogether, these findings suggest a close relationship between conformational similarity to the native structure of STh and the ability to elicit specific antibody responses against STh.

International audience; In Podospora anserina a linear plasmid with telomeric ends behaves as an artificial acentric minichromosome. Transformation is at least 100 times more efficient than with integrative vectors. Genomic DNA was inserted in this plasmid in vitro and the mixture used to transform a leu1-1 strain. Many fungal clones containing the leu1 gene as a genomic insert in the linear plasmid were identified. The leu1 gene was rescued as a circular plasmid in Escherichia coli demonstrating that a direct cloning procedure can be applied for the fungus P. anserina. The conservation of telomeric sequences among filamentous fungi suggests that a telomere-based linear plasmid could provide a general cloning vector for filamentous fungi.

International audience; The growth hormone receptor (GHR) is a member of the cytokine receptor family. Its function is to mediate cellular responses upon binding of growth hormone. Ligand binding induces dimerization and activation of the GHR. One mechanism by which the GHR is rapidly inactivated involves the ubiquitin conjugation system, a system implicated in the degradation of cytosolic and nuclear proteins. We have shown previously that the ubiquitin-conjugating system mediates internalization of the GHR. Here, we present evidence that in addition to the ubiquitin-dependent endocytosis signal, the cytosolic tail of the GHR contains a di-leucine motif. Upon truncation of the GHR at amino acid residue 349, this di-leucine motif is activated and mediates ubiquitin-independent internalization of the receptor. Di-leucine-mediated GHR internalization requires functional clathrin-coated pits and results in GHR transport to the lysosome. Although the full-length GHR internalizes independent of the di-leucine motif, this motif may function in internalization of GHR isoforms.

International audience; Interleukin 2 (IL-2) interacts with a receptor (IL-2R) composed of three subunits (IL-2R alpha, IL-2R beta and IL-2R gamma). IL-2R beta plays a critical role in signal transduction. An anti-human IL-2 mAb (H2-8) produced after immunization with peptide 1-30 of IL-2 was found to recognize the region occupied by Asp20, at the exposed interface between alpha-helices A and C. Muteins at position 17 and 20 are not recognized by mAb H2-8. mAb H2-8 specifically inhibits the IL-2 proliferation of TS1beta cells which are dependent on the expression of human IL-2R beta chain for IL-2 proliferation. Substitution at internal position Leu17 demonstrates that this position is essential for IL-2 binding and IL-2 bioactivity. New IL-2 mutants at position Asp20 have been analysed. Substitutions Asp --> Asn, Asp --> Lys, Asp --> Leu, show a correlation between diminished affinity for IL-2 receptor and reduced bioactivity measured on TS1beta cells. Mutein Asp Arg lose affinity for IL-2R and bioactivity simultaneously. Furthermore, during the course of the study we have found that mutein Asp20 --> Leu is an IL-2 antagonist. The biological effects of mAb H2-8 and the properties of new mutants at positions 17 and 20 demonstrate that this region of alpha helix-A is involved in IL-2-IL-2R beta interactions.

International audience; Most conditions detected by expanded newborn screening result from deficiency of one of the enzymes that degrade acyl-coenzyme A (CoA) esters in mitochondria. The role of acyl-CoAs in the pathophysiology of these disorders is poorly understood, in part because CoA esters are intracellular and samples are not generally available from human patients. We created a mouse model of one such condition, deficiency of 3-hydroxy-3-methylglutaryl-CoA lyase (HL), in liver (HLLKO mice). HL catalyses a reaction of ketone body synthesis and of leucine degradation. Chronic HL deficiency and acute crises each produced distinct abnormal liver acyl-CoA patterns, which would not be predictable from levels of urine organic acids and plasma acylcarnitines. In HLLKO hepatocytes, ketogenesis was undetectable. Carboxylation of [2-(14)C] pyruvate diminished following incubation of HLLKO hepatocytes with the leucine metabolite 2-ketoisocaproate (KIC). HLLKO mice also had suppression of the normal hyperglycemic response to a systemic pyruvate load, a measure of gluconeogenesis. Hyperammonemia and hypoglycemia, cardinal features of many inborn errors of acyl-CoA metabolism, occurred spontaneously in some HLLKO mice and were inducible by administering KIC. KIC loading also increased levels of several leucine-related acyl-CoAs and reduced acetyl-CoA levels. Ultrastructurally, hepatocyte mitochondria of KIC-treated HLLKO mice show marked swelling. KIC-induced hyperammonemia improved following administration of carglumate (N-carbamyl-L-glutamic acid), which substitutes for the product of an acetyl-CoA-dependent reaction essential for urea cycle function, demonstrating an acyl-CoA-related mechanism for this complication.

International audience; Internalin, a surface protein essential for entry of Listeria monocytogenes EGD into epithelial cells, was used as an antigen to raise nine monoclonal antibodies. These monoclonal antibodies recognized seven distinct epitopes which were located in three different regions of the protein. Three of them inhibited internalinmediated entry and recognized the amino-terminal leucine-rich repeat region of the protein, suggesting that this region is essential for entry.