Etude structurale et fonctionnelle des antigènes PfEMP1 et AMA1 de Plasmodium

Le paludisme est une maladie infectieuse qui reste associée à une mortalité élevée. L'agent infectieux responsable est le parasite Plasmodium. Notre intérêt s'est porté sur la protéine PfEMP1 codée par le gène var O. L'expression hétérologue des différents domaines de PfEMP1 var O, qui représente une difficulté majeure dans l'étude de ce type de protéine, a été étudiée dans les systèmes E. coli et baculovirus. Les domaines recombinants var O NTS-DBL1a1 et DBL2b-C2 ont été produits dans les deux systèmes sous forme soluble. L'étude de ces protéines par protéolyse ménagée et dichroïsme circulaire a montré que chaque protéine était repliée de manière équivalente dans les deux systèmes d'expression. Les anticorps monoclonaux obtenus chez la souris contre ces domaines recombinants reconnaissent la surface des globules rouges infectés exprimant le variant O. D'autre part, les anticorps polyclonaux murins dirigés contre le domaine NTS-DBL1a1 bactérien et baculovirus inhibent la formation des rosettes du variant O. Nous avons également poursuivi une étude structurale sur la protéine AMA1 de P. vivax, dont la structure cristalline a été récemment résolue au laboratoire, afin d'analyser l'antigénicité de cette protéine. Nous avons déterminé par cristallographie des rayons , la structure d'un complexe entre l'antigène PvAMA1 et le fragment Fab de l'anticorps monoclonal murin F8.12.19, qui montre une réactivité croisée sur d'autres espèces Plasmodium, ainsi qu'un second complexe entre le Fab F8.12.19 et l'antigène AMA1 de P. falciparum. La structure de ces deux complexes montre que l'anticorps s'adapte aux deux antigènes homologues malgré les différences en acides aminés.PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Phage T4 early promoters are resistant to inhibition by the anti-sigma factor AsiA.

Phage T4 early promoters are transcribed in vivo and in vitro by the Escherichia coli RNA polymerase holoenzyme Esigma(70). We studied in vitro the effects of the T4 anti-sigma(70) factor AsiA on the activity of several T4 early promoters. In single-round transcription, promoters motB, denV, mrh.2, motA wild type and UP element-deleted motA are strongly resistant to inhibition by AsiA. The alpha-C-terminal domain of Esigma(70) is crucial to this resistance. DNase I footprinting of Esigma(70) and Esigma(70)AsiA on motA and mrh.2 shows extended contacts between the holoenzyme with or without AsiA and upstream regions of these promoters. A TG --> TC mutation of the extended -10 motif in the motA UP element-deleted promoter strongly increases susceptibility to inhibition by AsiA, but has no effect on the motA wild-type promoter: either the UP element or the extended -10 site confers resistance to AsiA. Potassium permanganate reactivity shows that the two structure elements are not equivalent: with AsiA, the motA UP element-deleted promoter opens more slowly whereas the motA TC promoter opens like the wild type. Changes in UV laser photoreactivity at position +4 on variants of motA reveal an analogous distinction in the roles of the extended -10 and UP promoter elements

Residues in the HIV-1 capsid assembly inhibitor binding site are essential for maintaining the assembly-competent quaternary structure of the capsid protein.

International audienceMorphogenesis of infectious HIV-1 involves budding of immature virions followed by proteolytic disassembly of the Gag protein shell and subsequent assembly of processed capsid proteins (CA) into the mature HIV-1 core. The dimeric interface between C-terminal domains of CA (C-CA) has been shown to be important for both immature and mature assemblies. We previously reported a CA-binding peptide (CAI) that blocks both assembly steps in vitro. The three-dimensional structure of the C-CA/CAI complex revealed an allosteric effect of CAI that alters the C-CA dimer interface. Based on this structure, we now investigated the phenotypes of mutations in the binding pocket. CA variants carrying mutations Y169A, L211A, or L211S had a reduced affinity for CAI and were unable to form mature-like particles in vitro. These mutations also blocked morphological conversion to mature virions in tissue culture and abolished infectivity. X-ray crystallographic analyses of the variant C-CA domains revealed that these alterations induced the same allosteric change at the dimer interface observed in the C-CA/CAI complex. These results point to a role of key interactions between conserved amino acids in the CAI binding pocket of C-CA in maintaining the correct conformation necessary for mature core assembly

Biochemical and biophysical characterisation of DBL1α1-varO, the rosetting domain of PfEMP1 from the VarO line of Plasmodium falciparum

International audienceRosetting of erythrocytes infected with Plasmodium falciparum is frequently observed in children with severe malaria. This adhesion phenomenon has been linked to the DBL1␣ domain of P. falciparum erythrocyte membrane protein 1 (PfEMP1) in three laboratory clones: FCR3S1.2, IT4R29 and Palo Alto varO. Here, we compare the soluble recombinant NTS-DBL1␣ 1-varO domain (NTS: N-terminal segment) obtained from E. coli, Pichia pastoris and baculovirus/insect cell expression systems. In each case, the presence of NTS was necessary for obtaining a soluble product. Successful expression in E. coli required maltosebinding protein as an N-terminal fusion partner. Each expression system produced an identical, correctly folded protein, as judged by biochemical and biophysical characterisations, and by the capacity to elicit antibodies that react with the surface of VarO-infected erythrocytes and disrupt VarO rosettes. Binding studies using surface plasmon resonance (SPR) techniques showed that NTS-DBL1␣ 1 produced in E. coli binds to heparin with micromolar affinity. IC 50 constants for other sulphated oligosaccharides were determined using SPR by measuring their competitive binding to the soluble protein in the presence of immobilized heparin. The affinity to NTS-DBL1␣ 1 was related to the degree of sulphation of the oligosaccharide, although the position of the sulphate groups on the sugar rings was also important. VarO rosettes could be disrupted by sulphated oligosaccharides with an efficacy that correlated with their binding affinity to recombinant NTS-DBL1␣ 1. Thus high yields of soluble NTS-DBL1␣ 1 with native conformation have been produced, opening novel perspectives for both structure-function studies and vaccine development

Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor

We thank Emmanuel DEJEAN for continuous support, Kelly GARNIER for help with thermal-shift assay and the CALIXAR team for helpful discussions. We would like to thank Patrick SCHULTZ for initial electron microscopy work and discussions and Alice ROTHNIE for critical reading of the manuscript. We thank Dr. Francisco CIRUELA ALFEREZ (University of Barcelona) for kindly providing HEK-293 cells stably expressing the A2AR-Nanoluc fusion protein and Ivan GUDELJ for help with mass spectrometry measurements.International audienceStructural studies of integral membrane proteins have been limited by the intrinsic conformational flexibility and the need to stabilize the proteins in solution. Stabilization by mutagenesis was very successful for structural biology of G protein-coupled receptors (GPCRs). However, it requires heavy protein engineering and may introduce structural deviations. Here we describe the use of specific calixarenes-based detergents for native GPCR stabilization. Wild type, full length human adenosine A(2A) receptor was used to exemplify the approach. We could stabilize native, glycosylated, non-aggregated and homogenous A(2A)R that maintained its ligand binding capacity. The benefit of the preparation for fragment screening, using the Saturation-Transfer Difference nuclear magnetic resonance (STD-NMR) experiment is reported. The binding of the agonist adenosine and the antagonist caffeine were observed and competition experiments with CGS-21680 and ZM241385 were performed, demonstrating the feasibility of the STD-based fragment screening on the native A(2A) receptor. Interestingly, adenosine was shown to bind a second binding site in the presence of the agonist CGS-21680 which corroborates published results obtained with molecular dynamics simulation. Fragment-like compounds identified using STD-NMR showed antagonistic effects on A(2A)R in the cAMP cellular assay. Taken together, our study shows that stabilization of native GPCRs represents an attractive approach for STD-based fragment screening and drug design

Hydrogenated diglucose detergents for membrane-protein extraction and stabilization

International audienceWe report herein the design and synthesis of a novel series of alkyl glycosides detergents consisting of a nonionic polar headgroup that comprises two glucose moieties in a branched arrangement (DG), onto which octane-, decane- and dodecanethiols were grafted leading to ODG, DDG, and DDDG detergents. Micellization in aqueous solution was studied by isothermal titration calorimetry (ITC), 1H NMR spectroscopy, and surface tensiometry (SFT). Critical micellar concentration values were found to decrease by a factor of ~10 for each pair of methylene groups added to the alkyl chain, ranging from ~0.05 mM to 9 mM for DDDG and ODG, respectively. Dynamic light scattering (DLS) and analytical ultracentrifugation sedimentation velocity (AUC-SV) experiments were used to investigate the size and the composition of the micellar aggregates, showing that the aggregation number significantly increased from ~40 for ODG to ~80 for DDDG. All new compounds were able to solubilize MPs from bacterial membranes, insect cells, as well as Madin Darby Canine Kidney (MDCK) cells. In particular, native human adenosine receptor (A2AR) and bacterial transporter BmrA were solubilized efficiently. A striking thermostability improvement of +13 and +9°C when ODG and DDG were respectively applied to wild type and full length A2AR. Taken together, this novel detergent series shows promising detergent potency for solubilization and stabilization of MPs and, thus, makes a valuable addition to the chemical toolbox available for extracting and handling these important but challenging membrane protein targets

Immunogenicity of the Plasmodium falciparum PfEMP1-VarO Adhesin: Induction of Surface-Reactive and Rosette-Disrupting Antibodies to VarO Infected Erythrocytes

International audienceAdhesion of Plasmodium falciparum-infected red blood cells (iRBC) to human erythrocytes (i.e. rosetting) is associated with severe malaria. Rosetting results from interactions between a subset of variant PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) adhesins and specific erythrocyte receptors. Interfering with such interactions is considered a promising intervention against severe malaria. To evaluate the feasibility of a vaccine strategy targetting rosetting, we have used here the Palo Alto 89F5 VarO rosetting model. PfEMP1-VarO consists of five Duffy-Binding Like domains (DBL1-5) and one Cysteine-rich Interdomain Region (CIDR1). The binding domain has been mapped to DBL1 and the ABO blood group was identified as the erythrocyte receptor. Here, we study the immunogenicity of all six recombinant PfEMP1-VarO domains and the DBL1- CIDR1 Head domain in BALB/c and outbred OF1 mice. Five readouts of antibody responses are explored: ELISA titres on the recombinant antigen, VarO-iRBC immunoblot reactivity, VarO-iRBC surface-reactivity, capacity to disrupt VarO rosettes and the capacity to prevent VarO rosette formation. For three domains, we explore influence of the expression system on antigenicity and immunogenicity. We show that correctly folded PfEMP1 domains elicit high antibody titres and induce a homogeneous response in outbred and BALB/c mice after three injections. High levels of rosette-disrupting and rosette-preventing antibodies are induced by DBL1 and the Head domain. Reduced-alkylated or denatured proteins fail to induce surface-reacting and rosette-disrupting antibodies, indicating that surface epitopes are conformational. We also report limited cross-reactivity between some PfEMP1 VarO domains. These results highlight the high immunogenicity of the individual domains in outbred animals and provide a strong basis for a rational vaccination strategy targeting rosetting

An In Vivo and In Vitro Model of Plasmodium falciparum Rosetting and Autoagglutination Mediated by varO, a Group A var Gene Encoding a Frequent Serotype▿

In the Saimiri sciureus monkey, erythrocytes infected with the varO antigenic variant of the Plasmodium falciparum Palo Alto 89F5 clone bind uninfected red blood cells (rosetting), form autoagglutinates, and have a high multiplication rate, three phenotypic characteristics that are associated with severe malaria in human patients. We report here that varO parasites express a var gene having the characteristics of group A var genes, and we show that the varO Duffy binding-like 1α1 (DBL1α1) domain is implicated in the rosetting of both S. sciureus and human erythrocytes. The soluble varO N-terminal sequence (NTS)-DBL1α1 recombinant domain, produced in a baculovirus-insect cell system, induced high titers of antibodies that reacted with varO-infected red blood cells and disrupted varO rosettes. varO parasites were culture adapted in vitro using human erythrocytes. They formed rosettes and autoagglutinates, and they had the same surface serotype and expressed the same varO gene as the monkey-propagated parasites. To develop an in vitro model with highly homogeneous varO parasites, rosette purification was combined with positive selection by panning with a varO NTS-DBL1α1-specific mouse monoclonal antibody. The single-variant, clonal parasites were used to analyze seroprevalence for varO at the village level in a setting where malaria is holoendemic (Dielmo, Senegal). We found 93.6% (95% confidence interval, 89.7 to 96.4%) seroprevalence for varO surface-reacting antibodies and 86.7% (95% confidence interval, 82.8 to 91.6%) seroprevalence for the recombinant NTS-DBL1α1 domain, and virtually all permanent residents had seroconverted by the age of 5 years. These data imply that the varO model is a relevant in vivo and in vitro model for rosetting and autoagglutination that can be used for rational development of vaccine candidates and therapeutic strategies aimed at preventing malaria pathology

Influence of the expression system on the antibody response of animals immunised with PfEMP1-VarO DBL1 recombinant constructs.

<p><b>(A and B)</b> Titration curves of individual bleed 2 sera collected from five outbred (OF1-1 to -5) (<b>A</b>) or five inbred (BALB/c-1 to -5) mice immunised with bDBL1 (<b>B</b>) on the immunising antigen (bDBL1). OF1 mice responded poorly. <b>(C)</b> Titration curves of serum from a rabbit immunised with bDBL1 compared to the pool of bleed 4 sera from OF1 mice immunised with bDBL1; antigen used in the ELISA: bDBL1. <b>(D)</b> Titration curve of rabbit IgG purified from the serum shown in (<b>C)</b>; antigen used in the ELISA: bDBL1. <b>(E to H)</b> Cross-checking on the homologous and heterologous expression product: titration curves of individual bleed 4 sera from OF1 mice immunised with bDBL1 on bDBL1 <b>(E)</b> and eDBL1 <b>(G)</b>; titration curves of individual bleed 4 sera from OF1 mice immunised with eDBL1 on eDBL1 <b>(F)</b> and bDBL1 <b>(H)</b>.</p

Cross-reactivity of mouse sera raised to DBL1 constructs with bDBL2.

<p>ELISA assays of OF1 mouse antisera against recombinant antigens. (<b>A</b>) Reactivity of bleed 4 antisera against bDBL1, eDBL1, bDBL2 and bCIDR (dilution 1/10,000) with the cognate and heterologous antigens. Symbols for antigens are indicated on the right. (<b>B and C</b>) Titration curves of bleed 4 sera against bDBL1 <b>(B)</b> or eDBL1 <b>(C)</b> on eDBL1, bDBL1 and bDBL2 antigens. Symbols for antigens are indicated on the right.</p