129 results on '"Barale, Jean-Christophe"'
Search Results
2. A surrogate marker of piperaquine-resistant Plasmodium falciparum malaria: a phenotype–genotype association study
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Witkowski, Benoit, Duru, Valentine, Khim, Nimol, Ross, Leila S, Saintpierre, Benjamin, Beghain, Johann, Chy, Sophy, Kim, Saorin, Ke, Sopheakvatey, Kloeung, Nimol, Eam, Rotha, Khean, Chanra, Ken, Malen, Loch, Kaknika, Bouillon, Anthony, Domergue, Anais, Ma, Laurence, Bouchier, Christiane, Leang, Rithea, Huy, Rekol, Nuel, Grégory, Barale, Jean-Christophe, Legrand, Eric, Ringwald, Pascal, Fidock, David A, Mercereau-Puijalon, Odile, Ariey, Frédéric, and Ménard, Didier
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- 2017
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- View/download PDF
3. 3D structures of the Plasmodium vivax subtilisin-like drug target SUB1 reveal conformational changes to accommodate a substrate-derived α-ketoamide inhibitor
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Martinez, Mariano, Batista, Fernando, Maurel, Manon, Bouillon, Anthony, Ortega Varga, Laura, Wehenkel, Anne Marie, Le Chevalier-Sontag, Lucile, Blondel, Arnaud, Haouz, Ahmed, Hernandez, Jean-François, Alzari, Pedro, Barale, Jean-Christophe, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Université de Montpellier (UM), Bioinformatique structurale - Structural Bioinformatics, Cristallographie (Plateforme) - Crystallography (Platform), ANR, CNRS et Institut Pasteur, For this work, A. Bouillon and M. Martinez were supported by Agence Nationale pour la Recherche (ANR) grant ANR-11-RPIB-002 and FB and LO were supported by grant ANR-19-CE18-0010. LO was also supported by ANR-17-CE11-0030 and by the ‘URGENCE COVID-19’ fundraising campaign of Institut Pasteur. Part of this work and M. Maurel were supported by Carnot Chimie Balard Institute through ANR program 11 CARN 0001-01., ANR-11-RPIB-0002,MaPI,Développement d'un composé 'leader' à l'origine d'une nouvelle génération d'anti-paludiques: une approche multi-cibles(2011), ANR-19-CE18-0010,SPIM,Synthèse, biologie et structure pour guider l'optimisation d'Inhibiteurs de la peptidase SUB1 de Plasmodium, une cible potentielle pour le traitement du paludisme(2019), and ANR-17-CE11-0030,NICOFIVE,Découverte de modulateurs allostériques ciblant les récepteurs nicotiniques alpha5(2017)
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Plasmodium ,malaria ,[CHIM.CRIS]Chemical Sciences/Cristallography ,therapeutic target ,subtilisin ,[SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology ,[CHIM.THER]Chemical Sciences/Medicinal Chemistry ,crystalography - Abstract
International audience; The constant selection and propagation of multi-resistant Plasmodium sp. parasites require the identification of new antimalarial candidates involved in as-yet untargeted metabolic pathways. Subtilisin-like protease 1 (SUB1) belongs to a new generation of drug targets because it plays a crucial role during egress of the parasite from infected host cells at different stages of its life cycle. SUB1 is characterized by an unusual pro-region that tightly interacts with its cognate catalytic domain, thus precluding 3D structural analysis of enzyme–inhibitor complexes. In the present study, to overcome this limitation, stringent ionic conditions and controlled proteolysis of recombinant full-length P. vivax SUB1 were used to obtain crystals of an active and stable catalytic domain (PvS1 Cat ) without a pro-region. High-resolution 3D structures of PvS1 Cat , alone and in complex with an α-ketoamide substrate-derived inhibitor (MAM-117), showed that, as expected, the catalytic serine of SUB1 formed a covalent bond with the α-keto group of the inhibitor. A network of hydrogen bonds and hydrophobic interactions stabilized the complex, including at the P1′ and P2′ positions of the inhibitor, although P′ residues are usually less important in defining the substrate specificity of subtilisins. Moreover, when associated with a substrate-derived peptidomimetic inhibitor, the catalytic groove of SUB1 underwent significant structural changes, particularly in its S4 pocket. These findings pave the way for future strategies for the design of optimized SUB1-specific inhibitors that may define a novel class of antimalarial candidates.
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- 2023
4. Plasmodium falciparum Subtilisin-Like Protease 2, a Merozoite Candidate for the Merozoite Surface Protein 1-42 Maturase
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Barale, Jean-Christophe, Blisnick, Thierry, Fujioka, Hisashi, Alzari, Pedro M., Aikawa, Masamishi, Braun-Breton, Catherine, and Langsley, Gordon
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- 1999
5. Mammalian Subtilisin/Kexin Isozyme SKI-1: A Widely Expressed Proprotein Convertase with a Unique Cleavage Specificity and Cellular Localization
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Seidah, Nabil G., Mowla, Seyed J., Hamelin, Josee, Mamarbachi, Aida M., Benjannet, Suzanne, Toure, Barry B., Basak, Ajoy, Munzer, Jon Scott, Marcinkiewicz, Jadwiga, Zhong, Mei, Barale, Jean-Christophe, Lazure, Claude, Murphy, Richard A., Chretien, Michel, and Marcinkiewicz, Mieczyslaw
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- 1999
6. A small-molecule cell-based screen led to the identification of biphenylimidazoazines with highly potent and broad-spectrum anti-apicomplexan activity
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Moine, Espérance, Denevault-Sabourin, Caroline, Debierre-Grockiego, Françoise, Silpa, Laurence, Gorgette, Olivier, Barale, Jean-Christophe, Jacquiet, Philippe, Brossier, Fabien, Gueiffier, Alain, Dimier-Poisson, Isabelle, and Enguehard-Gueiffier, Cécile
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- 2015
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7. Prevalence and characterization of piperaquine, mefloquine and artemisinin derivatives triple-resistant Plasmodium falciparum in Cambodia.
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Mairet-Khedim, Mélissa, Roesch, Camille, Khim, Nimol, Srun, Sreynet, Bouillon, Anthony, Kim, Saorin, Ke, Sopheakvatey, Kauy, Chhayleang, Kloeung, Nimol, Eam, Rotha, Khean, Chanra, Kul, Chanvong, Chy, Sophy, Leang, Rithea, Ringwald, Pascal, Barale, Jean-Christophe, and Witkowski, Benoit
- Subjects
ARTEMISININ derivatives ,MEFLOQUINE ,PLASMODIUM falciparum ,ARTEMISININ ,FUNGICIDE resistance - Abstract
Background In early 2016, in Preah Vihear, Northern Cambodia, artesunate/mefloquine was used to cope with dihydroartemisinin/piperaquine-resistant Plasmodium falciparum parasites. Following this policy, P. falciparum strains harbouring molecular markers associated with artemisinin, piperaquine and mefloquine resistance have emerged. However, the lack of a viable alternative led Cambodia to adopt artesunate/mefloquine countrywide, raising concerns about a surge of triple-resistant P. falciparum strains. Objectives To assess the prevalence of triple-resistant parasites after artesunate/mefloquine implementation countrywide in Cambodia and to characterize their phenotype. Methods For this multicentric study, 846 samples were collected from 2016 to 2019. Genotyping of molecular markers associated with artemisinin, piperaquine and mefloquine resistance was coupled with phenotypic analyses. Results Only four triple-resistant P. falciparum isolates (0.47%) were identified during the study period. These parasites combined the pfk13 polymorphism with pfmdr1 amplification, pfpm2 amplification and/or pfcrt mutations. They showed significantly higher tolerance to artemisinin, piperaquine and mefloquine and also to the mefloquine and piperaquine combination. Conclusions The use of artesunate/mefloquine countrywide in Cambodia has not led to a massive increase of triple-resistant P. falciparum parasites. However, these parasites circulate in the population, and exhibit clear resistance to piperaquine, mefloquine and their combination in vitro. This study demonstrates that P. falciparum can adapt to more complex drug associations, which should be considered in future therapeutic designs. [ABSTRACT FROM AUTHOR]
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- 2023
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8. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria
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Aney, Frederic, Witkowski, Benoit, Amaratunga, Chanaki, Beghain, Johann, Langlois, Anne-Claire, Khim, Nimol, Kim, Saorin, Duru, Valentine, Bouchier, Christiane, Ma, Laurence, Lim, Pharath, Leang, Rithea, Duong, Socheat, Sreng, Sokunthea, Suon, Seila, Chuor, Char Meng, Bout, Denis Mey, Menard, Sandie, Rogers, William O., Genton, Blaise, Fandeur, Thierry, Miotto, Olivo, Ringwald, Pascal, Bras, Jacques Le, Berry, Antoine, Barale, Jean-Christophe, Fairhurst, Rick M., Benoit-Vical, Francoise, Mercereau-Puijalon, Odile, and Menard, Didier
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Drug resistance in microorganisms -- Research ,Molecular biology -- Research ,Plasmodium falciparum -- Physiological aspects -- Health aspects ,Microbiological research ,Environmental issues ,Science and technology ,Zoology and wildlife conservation - Abstract
Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain ('K13-propeller') with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread., The emergence of Plasmodium falciparum resistance to artemisinin derivatives (ART) in Cambodia threatens the world's malaria control and elimination efforts (1,2). The risk of ART-resistant parasites spreading from western Cambodia [...]
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- 2014
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9. Plasmodium chabaudi p68 Serine Protease Activity Required for Merozoite Entry into Mouse Erythrocytes
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Breton, Catherine Braun, Blisnick, Thierry, Jouin, Helene, Barale, Jean Christophe, Rabilloud, Thierry, Rabilloud, Thierry, and Langsley, Gordon
- Published
- 1992
10. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria
- Author
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Ariey, Frédéric, Witkowski, Benoit, Amaratunga, Chanaki, Beghain, Johann, Langlois, Anne-Claire, Khim, Nimol, Kim, Saorin, Duru, Valentine, Bouchier, Christiane, Ma, Laurence, Lim, Pharath, Leang, Rithea, Duong, Socheat, Sreng, Sokunthea, Suon, Seila, Chuor, Char Meng, Bout, Denis Mey, Ménard, Sandie, Rogers, William O., Genton, Blaise, Fandeur, Thierry, Miotto, Olivo, Ringwald, Pascal, Le Bras, Jacques, Berry, Antoine, Barale, Jean-Christophe, Fairhurst, Rick M., Benoit-Vical, Françoise, Mercereau-Puijalon, Odile, and Ménard, Didier
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- 2014
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11. Artemisinin-independent inhibitory activity of Artemisia sp. infusions against different Plasmodium stages including relapse-causing hypnozoites.
- Author
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Ashraf, Kutub, Tajeri, Shahin, Arnold, Christophe-Sébastien, Amanzougaghene, Nadia, Franetich, Jean-François, Vantaux, Amélie, Soulard, Valérie, Bordessoulles, Mallaury, Cazals, Guillaume, Bousema, Teun, van Gemert, Geert-Jan, Le Grand, Roger, Dereuddre-Bosquet, Nathalie, Barale, Jean-Christophe, Witkowski, Benoit, Snounou, Georges, Duval, Romain, Botté, Cyrille Y., and Mazier, Dominique
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- 2022
- Full Text
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12. Trypanosoma cruzi proline racemases are involved in parasite differentiation and infectivity
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Chamond, Nathalie, Goytia, Maira, Coatnoan, Nicolas, Barale, Jean-Christophe, Cosson, Alain, Degrave, Wim M., and Minoprio, Paola
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- 2005
13. Three multigene families in Plasmodium parasites: facts and questions
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Mercereau-Puijalon, Odile, Barale, Jean-Christophe, and Bischoff, Emmanuel
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- 2002
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14. CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use
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Mercereau-Puijalon Odile, Akanmori Bartholomew, Barale Jean-Christophe, Loizon Séverine, Jublot Delphine, Vigan-Womas Inès, Boeuf Philippe, and Behr Charlotte
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Immunologic diseases. Allergy ,RC581-607 - Abstract
Abstract Background Real-time PCR is becoming a common tool for detecting and quantifying expression profiling of selected genes. Cytokines mRNA quantification is widely used in immunological research to dissect the early steps of immune responses or pathophysiological pathways. It is also growing to be of clinical relevancy to immuno-monitoring and evaluation of the disease status of patients. The techniques currently used for "absolute quantification" of cytokine mRNA are based on a DNA standard curve and do not take into account the critical impact of RT efficiency. Results To overcome this pitfall, we designed a strategy using external RNA as standard in the RT-PCR. Use of synthetic RNA standards, by comparison with the corresponding DNA standard, showed significant variations in the yield of retro-transcription depending the target amplified and the experiment. We then developed primers to be used under one single experimental condition for the specific amplification of human IL-1β, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-γ, MIF, TGF-β1 and TNF-α mRNA. We showed that the beta-2 microglobulin (β2-MG) gene was suitable for data normalisation since the level of β2-MG transcripts in naïve PBMC varied less than 5 times between individuals and was not affected by LPS or PHA stimulation. The technique, we named CyProQuant-PCR (Cytokine Profiling Quantitative PCR) was validated using a kinetic measurement of cytokine transcripts under in vitro stimulation of human PBMC by lipopolysaccharide (LPS) or Staphylococcus aureus strain Cowan (SAC). Results obtained show that CyProQuant-PCR is powerful enough to precociously detect slight cytokine induction. Finally, having demonstrated the reproducibility of the method, it was applied to malaria patients and asymptomatic controls for the quantification of TGF-β1 transcripts and showed an increased capacity of cells from malaria patients to accumulate TGF-β1 mRNA in response to LPS. Conclusion The real-time RT-PCR technique based on a RNA standard curve, CyProQuant-PCR, outlined here, allows for a genuine absolute quantification and a simultaneous analysis of a large panel of human cytokine mRNA. It represents a potent and attractive tool for immunomonitoring, lending itself readily to automation and with a high throughput. This opens the possibility of an easy and reliable cytokine profiling for clinical applications.
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- 2005
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15. In vitro activity of ferroquine against artemisinin-based combination therapy (ACT)-resistant Plasmodium falciparum isolates from Cambodia.
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Mairet-Khedim, Mélissa, Nardella, Flore, Khim, Nimol, Kim, Saorin, Kloeung, Nimol, Ke, Sopheakvatey, Kauy, Chhayleang, Eam, Rotha, Khean, Chanra, Pellet, Alain, Leboulleux, Didier, Leang, Rithea, Ringwald, Pascal, Barale, Jean Christophe, Leroy, Didier, Menard, Didier, and Witkowski, Benoit
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PLASMODIUM falciparum ,GENE amplification ,MALARIA ,HAPLOTYPES - Abstract
Background: Cambodia is the epicentre of resistance emergence for virtually all antimalarial drugs. Selection and spread of parasites resistant to artemisinin-based combination therapy (ACT) is a major threat for malaria elimination, hence the need to renew the pool of effective treatments.Objectives: To determine whether ACT resistance haplotypes could have an effect on ferroquine in vitro antimalarial activity.Methods: In vitro susceptibility to ferroquine was measured for 80 isolates from Cambodia characterized for their molecular resistance profile to artemisinin, piperaquine and mefloquine.Results: Among the 80 isolates tested, the overall median (IQR) IC50 of ferroquine was 10.9 nM (8.7-18.3). The ferroquine median (IQR) IC50 was 8.9 nM (8.1-11.8) for Pfk13 WT parasites and was 12.9 nM (9.5-20.0) for Pfk13 C580Y parasites with no amplification of Pfpm2 and Pfmdr1 genes. The median (IQR) IC50 of ferroquine for Pfk13 C580Y parasites with amplification of the Pfpm2 gene was 17.2 nM (14.5-20.5) versus 9.1 nM (7.9-10.7) for Pfk13 C580Y parasites with amplification of the Pfmdr1 gene.Conclusions: Ferroquine exerts promising efficacy against ACT-resistant isolates. Whereas Pfpm2 amplification was associated with the highest parasite tolerance to ferroquine, the susceptibility range observed was in accordance with those measured in ACT resistance-free areas. This enables consideration of ferroquine as a relevant therapeutic option against ACT-resistant malaria. [ABSTRACT FROM AUTHOR]- Published
- 2019
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16. METHODS FOR DETECTING VIRULENT PLASMODIUM, FOR EVALUATING PLASMODIUM VIRULENCE, AND FOR SCREENING NEW DRUGS EMPLOYING THE 3'UTR OF PLASMODIUM SUB2 AND THE PLASMODIUM SUB2 SERINE PROTEASE: WO2006/120579
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Barale, Jean-Christophe, Uzureau, Pierrick, and Breton, Catherine Braun
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Microbiologie et protistologie [parasitologie hum. et anim.] ,Parasitologie ,Mutagenèse et technologie génétique - Abstract
info:eu-repo/semantics/published
- Published
- 2006
17. CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use
- Author
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Boeuf, Philippe, Vigan-Womas, Inès, Jublot, Delphine, Loizon, Séverine, Barale, Jean-Christophe, Akanmori, Bartholomew Dicky, Mercereau-Puijalon, Odile, Behr, Charlotte, Bases génétiques et moléculaires des interactions de la cellule eucaryote (BGMICE), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Structure et régulation de l'expression des anticorps et des récepteurs des lymphocytes T, Immunology Unit, Noguchi Memorial Institute for Medical Research [Accra, Ghana] (NMIMR), University of Ghana-University of Ghana, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
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Adult ,Lipopolysaccharides ,lcsh:Immunologic diseases. Allergy ,Enzyme-Linked Immunosorbent Assay ,Transforming Growth Factor beta1 ,Automation ,MESH: Transforming Growth Factor beta1 ,MESH: Gene Expression Profiling ,Transforming Growth Factor beta ,MESH: Automation ,MESH: Reverse Transcriptase Polymerase Chain Reaction ,MESH: Child ,Humans ,RNA, Messenger ,Malaria, Falciparum ,Child ,Macrophage Migration-Inhibitory Factors ,Cells, Cultured ,MESH: Transforming Growth Factor beta ,MESH: RNA, Messenger ,MESH: Cytokines ,MESH: Humans ,Reverse Transcriptase Polymerase Chain Reaction ,Tumor Necrosis Factor-alpha ,Methodology Article ,Gene Expression Profiling ,MESH: Malaria, Falciparum ,MESH: DNA ,MESH: Enzyme-Linked Immunosorbent Assay ,MESH: Adult ,DNA ,Reference Standards ,MESH: Gene Expression Regulation ,[SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology ,Gene Expression Regulation ,MESH: Tumor Necrosis Factor-alpha ,MESH: Macrophage Migration-Inhibitory Factors ,Cytokines ,MESH: Reference Standards ,MESH: Lipopolysaccharides ,lcsh:RC581-607 ,MESH: Cells, Cultured - Abstract
Background Real-time PCR is becoming a common tool for detecting and quantifying expression profiling of selected genes. Cytokines mRNA quantification is widely used in immunological research to dissect the early steps of immune responses or pathophysiological pathways. It is also growing to be of clinical relevancy to immuno-monitoring and evaluation of the disease status of patients. The techniques currently used for "absolute quantification" of cytokine mRNA are based on a DNA standard curve and do not take into account the critical impact of RT efficiency. Results To overcome this pitfall, we designed a strategy using external RNA as standard in the RT-PCR. Use of synthetic RNA standards, by comparison with the corresponding DNA standard, showed significant variations in the yield of retro-transcription depending the target amplified and the experiment. We then developed primers to be used under one single experimental condition for the specific amplification of human IL-1β, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-γ, MIF, TGF-β1 and TNF-α mRNA. We showed that the beta-2 microglobulin (β2-MG) gene was suitable for data normalisation since the level of β2-MG transcripts in naïve PBMC varied less than 5 times between individuals and was not affected by LPS or PHA stimulation. The technique, we named CyProQuant-PCR (Cytokine Profiling Quantitative PCR) was validated using a kinetic measurement of cytokine transcripts under in vitro stimulation of human PBMC by lipopolysaccharide (LPS) or Staphylococcus aureus strain Cowan (SAC). Results obtained show that CyProQuant-PCR is powerful enough to precociously detect slight cytokine induction. Finally, having demonstrated the reproducibility of the method, it was applied to malaria patients and asymptomatic controls for the quantification of TGF-β1 transcripts and showed an increased capacity of cells from malaria patients to accumulate TGF-β1 mRNA in response to LPS. Conclusion The real-time RT-PCR technique based on a RNA standard curve, CyProQuant-PCR, outlined here, allows for a genuine absolute quantification and a simultaneous analysis of a large panel of human cytokine mRNA. It represents a potent and attractive tool for immunomonitoring, lending itself readily to automation and with a high throughput. This opens the possibility of an easy and reliable cytokine profiling for clinical applications.
- Published
- 2005
18. Gene targeting demonstrates that the Plasmodium berghei subtilisin PbSUB2 is essential for red cell invasion and reveals spontaneous genetic recombination events
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Uzureau, Pierrick, Barale, Jean-Christophe, Janse, Chris J, Waters, Andrew P, Breton, Catherine Braun, Uzureau, Pierrick, Barale, Jean-Christophe, Janse, Chris J, Waters, Andrew P, and Breton, Catherine Braun
- Abstract
The Plasmodium merozoite proteases involved in the crucial process of erythrocyte invasion are promising targets for novel malaria control strategies. We report here the characterization of the subtilisin-like protease SUB2 from the rodent parasites Plasmodium berghei and Plasmodium yoelii, leading the way to in vivo functional studies of this enzyme. The kinetics of expression and subcellular localization imply a central role for SUB2 in erythrocyte invasion. Through the use of gene targeting strategies, we assessed the relevance of P. berghei SUB2 for the intraerythrocytic cycle. The selection of recombinant Pbsub2-TrimycDuoXpress-tagged parasites and the proper expression of the modified coding region demonstrate that the Pbsub2 locus is accessible to genetic modifications. However, Pbsub2 knock-out parasites were not recovered, confirming the importance of PbSUB2 for P. berghei merozoite stages, and supporting the fact that its Plasmodium falciparum SUB2 orthologue is an attractive drug target candidate. Finally, we identify revertant parasites that have lost the integrated selection cassette while conserving a Pbsub2-tagged gene. These spontaneous reversion events should overcome the scarcity of selectable markers available for this parasite, giving access to multiple gene tagging strategies, which, together with the validation of a TrimycDuoXpress tag, would represent valuable new tools for studying the biology of P. berghei., Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., FLWIN, info:eu-repo/semantics/published
- Published
- 2004
19. Pfsbp1, a Maurer's cleft Plasmodium falciparum protein, is associated with the erythrocyte skeleton.
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Blisnick, Thierry, Morales Betoulle, M E, Barale, Jean-Christophe, Uzureau, Pierrick, Berry, Lynne, Desroses, S, Fujioka, H, Mattei, D, Breton, Catherine Braun, Blisnick, Thierry, Morales Betoulle, M E, Barale, Jean-Christophe, Uzureau, Pierrick, Berry, Lynne, Desroses, S, Fujioka, H, Mattei, D, and Breton, Catherine Braun
- Abstract
Antibodies from hyperimmune monkey sera, selected by absorption to Plasmodium falciparum-infected erythrocytes, and elution at acidic pH, allowed us to characterize a novel parasite protein, Pfsbp1 (P. falciparum skeleton binding protein 1). Pfsbp1 is an integral membrane protein of parasite-induced membranous structures associated with the erythrocyte plasma membrane and referred to as Maurer's clefts. The carboxy-terminal domain of Pfsbp1, exposed within the cytoplasm of the host cell, interacts with a 35 kDa erythrocyte skeletal protein and might participate in the binding of the Maurer's clefts to the erythrocyte submembrane skeleton. Antibodies to the carboxy- and amino-terminal domains of Pfsbp1 labelled similar vesicular structures in the cytoplasm of Plasmodium chabaudi and Plasmodium berghei-infected murine erythrocytes, suggesting that the protein is conserved among malaria species, consistent with an important role of Maurer's cleft-like structures in the intraerythrocytic development of malaria parasites., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published
- Published
- 2000
20. Screening and Evaluation of Inhibitors of Plasmodium falciparum Merozoite Egress and Invasion Using Cytometry.
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Bouillon, Anthony, Gorgette, Olivier, Mercereau-Puijalon, Odile, and Barale, Jean-Christophe
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- 2013
- Full Text
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21. A malaria phosphatidylinositol-specific phospholipase C: a possible role in merozoite maturation and erythrocyte invasion
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Braun-Breton, Catherine, Langsley, Gordon, Barale, Jean-Christophe, Pereira Da Silva, Luiz, Parasitologie Expérimentale, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
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Software_OPERATINGSYSTEMS ,ComputerSystemsOrganization_COMPUTERSYSTEMIMPLEMENTATION ,malaria ,ComputerSystemsOrganization_SPECIAL-PURPOSEANDAPPLICATION-BASEDSYSTEMS ,[SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology ,phospholipase ,invasion ,ComputingMilieux_MISCELLANEOUS ,merozoite - Abstract
International audience; No resume
- Published
- 1990
22. Human peroxiredoxin 6 is essential for malaria parasites and provides a host-based drug target.
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Wagner, Matthias Paulus, Formaglio, Pauline, Gorgette, Olivier, Dziekan, Jerzy Michal, Huon, Christèle, Berneburg, Isabell, Rahlfs, Stefan, Barale, Jean-Christophe, Feinstein, Sheldon I., Fisher, Aron B., Ménard, Didier, Bozdech, Zbynek, Amino, Rogerio, Touqui, Lhousseine, and Chitnis, Chetan E.
- Abstract
The uptake and digestion of host hemoglobin by malaria parasites during blood-stage growth leads to significant oxidative damage of membrane lipids. Repair of lipid peroxidation damage is crucial for parasite survival. Here, we demonstrate that Plasmodium falciparum imports a host antioxidant enzyme, peroxiredoxin 6 (PRDX6), during hemoglobin uptake from the red blood cell cytosol. PRDX6 is a lipid-peroxidation repair enzyme with phospholipase A 2 (PLA 2) activity. Inhibition of PRDX6 with a PLA 2 inhibitor, Darapladib, increases lipid-peroxidation damage in the parasite and disrupts transport of hemoglobin-containing vesicles to the food vacuole, causing parasite death. Furthermore, inhibition of PRDX6 synergistically reduces the survival of artemisinin-resistant parasites following co-treatment of parasite cultures with artemisinin and Darapladib. Thus, PRDX6 is a host-derived drug target for development of antimalarial drugs that could help overcome artemisinin resistance. [Display omitted] • Human peroxiredoxin 6 (PRDX6) is internalized by P. falciparum blood stages • Inhibition of host PRDX6 causes lethal lipid-peroxidation damage in the parasite • Co-treatment with artemisinin and PRDX6 inhibitors overcomes artemisinin resistance • Targeting of a host enzyme like PRDX6 may prevent development of drug resistance Wagner et al. find that Plasmodium falciparum blood stages internalize human PRDX6, which repairs lipid-peroxidation damage. PRDX6 inhibitors prevent lipid repair and kill the parasite. Co-treatment of artemisinin-resistant strains with PRDX6 inhibitors and artemisinin restores susceptibility to artemisinin. PRDX6 thus provides a promising host-based target for anti-malaria drug development. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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- View/download PDF
23. Computational Design of Protein-Based Inhibitors of Plasmodium vivax Subtilisin-Like 1 Protease.
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Bastianelli, Giacomo, Bouillon, Anthony, Nguyen, Christophe, Le-Nguyen, Dung, Nilges, Michael, and Barale, Jean-Christophe
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THERAPEUTIC use of proteins ,MALARIA treatment ,PLASMODIUM vivax ,SUBTILISINS ,ERYTHROCYTES - Abstract
Background: Malaria remains a major global health concern. The development of novel therapeutic strategies is critical to overcome the selection of multiresistant parasites. The subtilisin-like protease (SUB1) involved in the egress of daughter Plasmodium parasites from infected erythrocytes and in their subsequent invasion into fresh erythrocytes has emerged as an interesting new drug target. Findings: Using a computational approach based on homology modeling, protein–protein docking and mutation scoring, we designed protein–based inhibitors of Plasmodium vivax SUB1 (PvSUB1) and experimentally evaluated their inhibitory activity. The small peptidic trypsin inhibitor EETI-II was used as scaffold. We mutated residues at specific positions (P4 and P1) and calculated the change in free-energy of binding with PvSUB1. In agreement with our predictions, we identified a mutant of EETI-II (EETI-II-P4LP1W) with a Ki in the medium micromolar range. Conclusions: Despite the challenges related to the lack of an experimental structure of PvSUB1, the computational protocol we developed in this study led to the design of protein-based inhibitors of PvSUB1. The approach we describe in this paper, together with other examples, demonstrates the capabilities of computational procedures to accelerate and guide the design of novel proteins with interesting therapeutic applications. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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24. Cytometric measurement of in vitro inhibition of Plasmodium falciparum field isolates by drugs: a new approach for re-invasion inhibition study.
- Author
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Varela, Marie Louise, Razakandrainibe, Romy, Aldebert, Delphine, Barale, Jean Christophe, and Jambou, Ronan
- Abstract
Background: A flow cytometric method is proposed to study in vitro drug sensitivity of Plasmodium falciparum. Standard [
3 H]-hypoxanthine incorporation assay gives only information on inhibition of maturation by drugs. This method is usable on field isolates and provides data on both inhibition of maturation and re-invasion. Methods: The method is based on the staining of parasites with hydroethidine (HE) and thiazole orange (TO) which allow differential identification of early, trophozoite and late stage of the parasite by flow cytometry. Late stages of the parasites are obtained by incubation in culture for 24 hours. Reinvasion is followed by culturing parasitized red blood cells for 24 h more. Results: Compared to the standard [3 H]-hypoxanthine incorporation assay, it gave similar results as expressed by 50% inhibitory concentrations for chloroquine of laboratory strains and “field” isolates. The effect of quinine on the schizont-ring transition was also explored using this method. First data on the inhibition of re-invasion induced by quinine are presented for both P. falciparum-cultured strains and field isolates. Discussion: This method is simple to use event for field isolate study. It is suitable to analyse effect of drugs on steps of the parasite life cycle different for the maturation one. Using this method quinine was found to have a inhibitory effect on re-invasion of red cells by Plasmodium. [ABSTRACT FROM AUTHOR]- Published
- 2014
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25. Whole Pichia pastoris Yeast Expressing Measles Virus Nucleoprotein as a Production and Delivery System to Multimerize Plasmodium Antigens.
- Author
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Jacob, Daria, Ruffie, Claude, Dubois, Myriam, Combredet, Chantal, Amino, Rogerio, Formaglio, Pauline, Gorgette, Olivier, Pehau-Arnaudet, Gérard, Guery, Charline, Puijalon, Odile, Barale, Jean-Christophe, Ménard, Robert, Tangy, Frédéric, and Sala, Monica
- Subjects
PICHIA pastoris ,MEASLES virus ,NUCLEOPROTEINS ,PLASMODIUM ,LABORATORY mice ,PARASITIC disease treatment ,MEASLES vaccines - Abstract
Yeasts are largely used as bioreactors for vaccine production. Usually, antigens are produced in yeast then purified and mixed with adjuvants before immunization. However, the purification costs and the safety concerns recently raised by the use of new adjuvants argue for alternative strategies. To this end, the use of whole yeast as both production and delivery system appears attractive. Here, we evaluated Pichia pastoris yeast as an alternative vaccine production and delivery system for the circumsporozoite protein (CS) of Plasmodium, the etiologic agent of malaria. The CS protein from Plasmodium berghei (Pb) was selected given the availability of the stringent C57Bl/6 mouse model of infection by Pb sporozoites, allowing the evaluation of vaccine efficacy in vivo. PbCS was multimerized by fusion to the measles virus (MV) nucleoprotein (N) known to auto-assemble in yeast in large-size ribonucleoprotein rods (RNPs). Expressed in P. pastoris, the N-PbCS protein generated highly multimeric and heterogenic RNPs bearing PbCS on their surface. Electron microscopy and immunofluorescence analyses revealed the shape of these RNPs and their localization in peripheral cytoplasmic inclusions. Subcutaneous immunization of C57Bl/6 mice with heat-inactivated whole P. pastoris expressing N-PbCS RNPs provided significant reduction of parasitemia after intradermal challenge with a high dose of parasites. Thus, in the absence of accessory adjuvants, a very low amount of PbCS expressed in whole yeast significantly decreased clinical damages associated with Pb infection in a highly stringent challenge model, providing a proof of concept of the intrinsic adjuvancy of this vaccine strategy. In addition to PbCS multimerization, the N protein contributed by itself to parasitemia delay and long-term mice survival. In the future, mixtures of whole recombinant yeasts expressing relevant Plasmodium antigens would provide a multivalent formulation applicable for antigen combination screening and possibly for large-scale production, distribution and delivery of a malaria vaccine in developing countries. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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26. Computational Reverse-Engineering of a Spider-Venom Derived Peptide Active Against Plasmodium falciparum SUB1.
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Bastianelli, Giacomo, Bouillon, Anthony, Nguyen, Christophe, Crublet, Elodie, Pêtres, Stéphane, Gorgette, Olivier, Le-Nguyen, Dung, Barale, Jean-Christophe, and Nilges, Michael
- Subjects
SPIDER venom ,ANTI-infective agents ,PEPTIDES ,AMINO acid sequence ,PLASMODIUM falciparum ,MALARIA ,PARASITES ,ENZYME activation - Abstract
Background: Psalmopeotoxin I (PcFK1), a protein of 33 aminoacids derived from the venom of the spider Psalmopoeus Cambridgei, is able to inhibit the growth of Plasmodium falciparum malaria parasites with an IC
50 in the low micromolar range. PcFK1 was proposed to act as an ion channel inhibitor, although experimental validation of this mechanism is lacking. The surface loops of PcFK1 have some sequence similarity with the parasite protein sequences cleaved by PfSUB1, a subtilisin-like protease essential for egress of Plasmodium falciparum merozoites and invasion into erythrocytes. As PfSUB1 has emerged as an interesting drug target, we explored the hypothesis that PcFK1 targeted PfSUB1 enzymatic activity. Findings: Molecular modeling and docking calculations showed that one loop could interact with the binding site of PfSUB1. The calculated free energy of binding averaged 25.01 kcal/mol, corresponding to a predicted low-medium micromolar constant of inhibition. PcFK1 inhibited the enzymatic activity of the recombinant PfSUB1 enzyme and the in vitro P.falciparum culture in a range compatible with our bioinformatics analysis. Using contact analysis and free energy decomposition we propose that residues A14 and Q15 are important in the interaction with PfSUB1. Conclusions: Our computational reverse engineering supported the hypothesis that PcFK1 targeted PfSUB1, and this was confirmed by experimental evidence showing that PcFK1 inhibits PfSUB1 enzymatic activity. This outlines the usefulness of advanced bioinformatics tools to predict the function of a protein structure. The structural features of PcFK1 represent an interesting protein scaffold for future protein engineering. [ABSTRACT FROM AUTHOR]- Published
- 2011
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27. On the Knorr Synthesis of 6-Bromo-4-methylquinolin-2(1H)-one.
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Wlodarczyk, Nicolas, Simenel, Catherine, Delepierre, Muriel, Barale, Jean-Christophe, and Janin, Yves L.
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MICROBIOLOGICAL synthesis ,COMMUNICABLE diseases ,QUINOLINE ,ANILIDES ,RING formation (Chemistry) ,ETHYL acetoacetate ,MAGNESIUM sulfate - Abstract
In the course of our work on infectious diseases, we were led to prepare 6-bromo-2-chloro-4-methylquinoline as a starting material. Since surprisingly little has been reported in the literature, the two synthetic steps to this compound were investigated. The synthesis involves a condensation between b-keto esters and 4-bromoaniline and the cyclization of the resulting anilides into 6-bromoquinolin- 2(1H)-one, otherwise known as the Knorr reaction. The
1 H NMR monitoring of the first step allowed us to optimize the conditions leading specifically to the anilide without the occurrence of the alternative crotonate. To illustrate the scope of our finding, few additional anilides featuring electron-attracting groups were prepared. The study of their cyclization revealed some unsuspected steric effect governing this second step. Aside from rectifying a few claims in this chemistry, this study led to a three-step preparation of 6-bromo-2-chloro-4-methylquinoline in 48% overall yield from 4- bromoaniline. [ABSTRACT FROM AUTHOR]- Published
- 2011
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28. Reduced Artemisinin Susceptibility of Plasmodium falciparumRing Stages in Western Cambodia
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Witkowski, Benoit, Khim, Nimol, Chim, Pheaktra, Kim, Saorin, Ke, Sopheakvatey, Kloeung, Nimol, Chy, Sophy, Duong, Socheat, Leang, Rithea, Ringwald, Pascal, Dondorp, Arjen M., Tripura, Rupam, Benoit-Vical, Françoise, Berry, Antoine, Gorgette, Olivier, Ariey, Frédéric, Barale, Jean-Christophe, Mercereau-Puijalon, Odile, and Menard, Didier
- Abstract
ABSTRACTThe declining efficacy of artemisinin derivatives against Plasmodium falciparumin western Cambodia is a major concern. The knowledge gap in the understanding of the mechanisms involved hampers designing monitoring tools. Here, we culture-adapted 20 isolates from Pailin and Ratanakiri (areas of artemisinin resistance and susceptibility in western and eastern Cambodia, respectively) and studied their in vitroresponse to dihydroartemisinin. No significant difference between the two sets of isolates was observed in the classical isotopic test. However, a 6-h pulse exposure to 700 nM dihydroartemisinin (ring-stage survival assay -RSA]) revealed a clear-cut geographic dichotomy. The survival rate of exposed ring-stage parasites (ring stages) was 17-fold higher in isolates from Pailin (median, 13.5%) than in those from Ratanakiri (median, 0.8%), while exposed mature stages were equally and highly susceptible (0.6% and 0.7%, respectively). Ring stages survived drug exposure by cell cycle arrest and resumed growth upon drug withdrawal. The reduced susceptibility to artemisinin in Pailin appears to be associated with an altered in vitrophenotype of ring stages from Pailin in the RSA.
- Published
- 2012
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29. The Mouse Homeoprotein mLIM-3 Is Expressed Early in Cells Derived from the Neuroepithelium and Persists in Adult Pituitary.
- Author
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SEIDAH, NABIL G., BARALE, JEAN-CHRISTOPHE, MARCINKIEWICZ, MIECZSLAW, MATTEI, MARIE-GENEVIÈVE, DAY, ROBERT, and CHRÉTIEN, MICHEL
- Published
- 1994
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30. Centromeric Plasmids and Artificial Chromosomes: New Kids on the Plasmodium Transfection Block.
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Barale, Jean-Christophe and Ménard, Robert
- Subjects
PLASMIDS ,ARTIFICIAL chromosomes ,GENE transfection ,PLASMODIUM ,HOSTS (Biology) ,BACTERIAL antigens ,BIOLOGICAL variation ,MICROBIAL genetics - Abstract
In this issue of Cell Host & Microbe, Iwanaga and colleagues () report on the construction of plasmids and artificial chromosomes that are stably maintained throughout the Plasmodium life cycle. These new tools will have multiple applications, from episome-based genetic strategies to studies on telomere biology and antigenic variation. [Copyright &y& Elsevier]
- Published
- 2010
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31. The LIM homeobox protein mLIM3/Lhx3 induces expression of the prolactin gene by a Pit‐1/GHF‐1‐independent pathway in corticotroph AtT20 cells
- Author
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Girardin, Stephen E, Benjannet, Suzanne, Barale, Jean-Christophe, Chrétien, Michel, and Seidah, Nabil G
- Abstract
mLIM3, a member of the LIM homeobox family, was recently demonstrated to be critical for proliferation and differentiation of the pituitary cell lineage. Using a pool of degenerate oligonucleotides we determined the DNA sequence ANNAGGAAA(T/C)GA(C/G)AA as the set preferentially recognized by mLIM3. A nearly identical sequence is found in the prolactin (PRL) promoter, within a 15‐mer stretch from nucleotides (nts) −218 to −204 which is highly conserved between human, rat, and bovine. In order to test the hypothesis of a transcriptional effect of mLIM3 on the prolactin promoter, stable transfectants of mLIM3 cDNA in AtT20 tumor cells revealed that PRL mRNA expression was induced in 3 separate stable clones. Gel retardation experiments performed using nuclear extracts isolated from one of the AtT20/mLIM3 stable transfectants revealed affinity towards the 15‐mer element of the PRLpromoter. From these results, we propose that the PRLpromoter element (nts −218 to −204) could be functional in vivo. Finally, we demonstrate that in AtT20 cells prolactin mRNA expression is not induced by the Pit‐1/GHF‐1 pathway and that growth hormone mRNA is not detected concomitantly with prolactin. We conclude that mLIM3 may play a key role in inducing PRLgene expression in lactotrophs by binding to a conserved motif close to a Pit‐1/GHF‐1 site within the proximal PRLpromoter.
- Published
- 1998
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32. A novel Plasmodium-specific prodomain fold regulates the malaria drug target SUB1 subtilase.
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Giganti, David, Bouillon, Anthony, Tawk, Lina, Robert, Fabienne, Martinez, Mariano, Crublet, Elodie, Weber, Patrick, Girard-Blanc, Christine, Petres, Stéphane, Haouz, Ahmed, Hernandez, Jean-François, Mercereau-Puijalon, Odile, Alzari, Pedro M., and Barale, Jean-Christophe
- Published
- 2014
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33. LANCL1, an erythrocyte protein recruited to the Maurer's clefts during Plasmodium falciparum development
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Blisnick, Thierry, Vincensini, Laetitia, Barale, Jean Christophe, Namane, Abdelkader, and Braun Breton, Catherine
- Subjects
- *
PLASMODIUM falciparum , *PROTOZOAN diseases , *MEMBRANE proteins , *ERYTHROCYTES - Abstract
Abstract: As the malarial parasite Plasmodium falciparum develops inside the erythrocyte, parasite-derived membrane structures, referred to as Maurer''s clefts, play an important role in parasite development by delivering parasite proteins to the host cell surface, and participating in the assembly of the cytoadherence complex, essential for the pathogenesis of cerebral malaria. PfSBP1 is an integral membrane protein of the clefts, interacting with an erythrocyte cytosolic protein, identified here as the human Lantibiotic synthetase component C-like protein LANCL1. LANCL1 is specifically recruited to the surface of Maurer''s clefts in P. falciparum mature blood stages. We propose that the interaction between PfSBP1 and LANCL1 is central for late steps of the parasite development to prevent premature rupture of the red blood cell membrane. [Copyright &y& Elsevier]
- Published
- 2005
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34. A Key Role for Plasmodium Subtilisin-like SUB1 Protease in Egress of Malaria Parasites from Host Hepatocytes.
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Tawk, Lina, Lacroix, Céline, Gueirard, Pascale, Kent, Robyn, Gorgette, Olivier, Thiberge, Sabine, Mercereau-Puijalon, Odile, Ménard, Robert, and Barale, Jean-Christophe
- Subjects
- *
PLASMODIUM , *PLASMODIIDAE , *PROTEOLYTIC enzymes , *HYDROLASES , *MALARIA - Abstract
In their mammalian host, Plasmodium parasites have two obligatory intracellular development phases, first in hepatocytes and subsequently in erythrocytes. Both involve an orchestrated process of invasion into and egress from host cells. The Plasmodium SUB1 protease plays a dual role at the blood stage by enabling egress of the progeny merozoites from the infected erythrocyte and priming merozoites for subsequent erythrocyte invasion. Here, using conditional mutagenesis in P. berghei, we show that SUB1 plays an essential role at the hepatic stage. Stage-specific sub1 invalidation during prehepatocytic development showed that SUB1-deficient parasites failed to rupture the parasitophorous vacuole membrane and to egress from hepatocytes. Furthermore, mechanically released parasites were not adequately primed and failed to establish a blood stage infection in vivo. The critical involvement of SUB1 in both pre-erythrocytic and erythrocytic developmental phases qualifies SUB1 as an attractive multistage target for prophylactic and therapeutic anti-Plasmodium intervention strategies. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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35. In vitro activity of ferroquine against artemisinin-based combination therapy (ACT)-resistant Plasmodium falciparum isolates from Cambodia
- Author
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Jean Christophe Barale, Chanra Khean, Nimol Khim, Sopheakvatey Ke, Saorin Kim, Benoit Witkowski, Nimol Kloeung, Didier Leboulleux, Pascal Ringwald, Didier Leroy, Didier Menard, Chhayleang Kauy, Rithea Leang, Rotha Eam, Flore Nardella, Alain Pellet, Melissa Mairet-Khedim, Malaria Molecular Epidemiology, Institut Pasteur du Cambodge, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Biologie des Interactions Hôte-Parasite - Biology of Host-Parasite Interactions, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Evotec, National Center of Parasitology (CNM), National Malaria Center [Phnom Penh], National Center for Malariology, Ministry of Health [Phnom Penh], World Health Organisation (WHO), Organisation Mondiale de la Santé / World Health Organization Office (OMS / WHO), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Malaria : parasites et hôtes - Malaria : parasites and hosts, Institut Pasteur [Paris], Medicines for Malaria Venture (MMV), Université de Genève (UNIGE), This study was supported by Medicines for Malaria Venture (project RD/13/0002). M. M.-K. and B. W. were supported by 5% initiative [molecular and in vitro surveillance of ACT partner drug efficacy in the Greater Mekong Subregion (MIVS-ACT), grant #15SANIN211]. F. N. was supported by Foundation Pierre Ledoux., We would like to thank the patients who participated in TESs and the Cambodian Health Centre medical staff for their contribution, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), Université de Genève = University of Geneva (UNIGE), Barale, Jean-Christophe, and Malaria Molecular Epidemiology (MMEU)
- Subjects
0301 basic medicine ,Microbiology (medical) ,Combination therapy ,Metallocenes ,Plasmodium falciparum ,030231 tropical medicine ,Drug Resistance ,Antimalarials ,Inhibitory Concentration 50 ,03 medical and health sciences ,0302 clinical medicine ,Parasitic Sensitivity Tests ,Piperaquine ,medicine ,Humans ,Parasite hosting ,Pharmacology (medical) ,[SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology ,Ferrous Compounds ,Malaria, Falciparum ,Artemisinin ,Pharmacology ,biology ,Mefloquine ,Haplotype ,medicine.disease ,biology.organism_classification ,Virology ,Artemisinins ,3. Good health ,030104 developmental biology ,Infectious Diseases ,Aminoquinolines ,Drug Therapy, Combination ,Cambodia ,[SDV.MP.PAR] Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology ,Malaria ,medicine.drug - Abstract
Background Cambodia is the epicentre of resistance emergence for virtually all antimalarial drugs. Selection and spread of parasites resistant to artemisinin-based combination therapy (ACT) is a major threat for malaria elimination, hence the need to renew the pool of effective treatments. Objectives To determine whether ACT resistance haplotypes could have an effect on ferroquine in vitro antimalarial activity. Methods In vitro susceptibility to ferroquine was measured for 80 isolates from Cambodia characterized for their molecular resistance profile to artemisinin, piperaquine and mefloquine. Results Among the 80 isolates tested, the overall median (IQR) IC50 of ferroquine was 10.9 nM (8.7–18.3). The ferroquine median (IQR) IC50 was 8.9 nM (8.1–11.8) for Pfk13 WT parasites and was 12.9 nM (9.5–20.0) for Pfk13 C580Y parasites with no amplification of Pfpm2 and Pfmdr1 genes. The median (IQR) IC50 of ferroquine for Pfk13 C580Y parasites with amplification of the Pfpm2 gene was 17.2 nM (14.5–20.5) versus 9.1 nM (7.9–10.7) for Pfk13 C580Y parasites with amplification of the Pfmdr1 gene. Conclusions Ferroquine exerts promising efficacy against ACT-resistant isolates. Whereas Pfpm2 amplification was associated with the highest parasite tolerance to ferroquine, the susceptibility range observed was in accordance with those measured in ACT resistance-free areas. This enables consideration of ferroquine as a relevant therapeutic option against ACT-resistant malaria.
- Published
- 2019
36. In Silico Screening on the Three-dimensional Model of the Plasmodium vivax SUB1 Protease Leads to the Validation of a Novel Anti-parasite Compound.
- Author
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Bouillon, Anthony, Giganti, David, Benedet, Christophe, Gorgette, Olivier, Pêtres, Stéphane, Crublet, Elodie, Girard-Blanc, Christine, Witkowski, Benoit, Ménard, Didier, Nilges, Michael, Mercereau-Puijalon, Odile, Stoven, Véronique, and Barale, Jean-Christophe
- Subjects
- *
DRUG resistance in microorganisms , *PLASMODIUM falciparum , *MALARIA , *SERINE proteinases , *ERYTHROCYTES , *MEROZOITES - Abstract
Widespread drug resistance calls for the urgent development of new antimalarials that target novel steps in the life cycle of Plasmodium falciparum and Plasmodium vivax. The essential subtilisin-like serine protease SUB1 of Plasmodium merozoites plays a dual role in egress from and invasion into host erythrocytes. It belongs to a new generation of attractive drug targets against which specific potent inhibitors are actively searched. Wecharacterize here the P. vivax SUB1 enzyme and show that it displays a typical auto-processing pattern and apical localization in P. vivax merozoites. To search for small PvSUB1 inhibitors, we took advantage of the similarity of SUB1 with bacterial subtilisins and generated P. vivax SUB1 three-dimensional models. The structure-based virtual screening of a large commercial chemical compounds library identified 306 virtual best hits, of which 37 were experimentally confirmed inhibitors and 5 had Ki values of <50μM for PvSUB1. Interestingly, they belong to different chemical families. The most promising competitive inhibitor of PvSUB1(compound 2) was equally active on PfSUB1 and displayed anti-P. falciparum and Plasmodium berghei activity in vitro and in vivo, respectively. Compound 2 inhibited the endogenous PfSUB1 as illustrated by the inhibited maturation of its natural substrate PfSERA5 and inhibited parasite egress and subsequent erythrocyte invasion. These data indicate that the strategy of in silico screening of three-dimensional models to select for virtual inhibitors combined with stringent biological validation successfully identified several inhibitors of the PvSUB1 enzyme. The most promising hit proved to be a potent cross-inhibitor of PlasmodiumSUB1, laying the groundwork for the development of a globally active small compound antimalarial. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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- View/download PDF
37. Prodomain-driven enzyme dimerization: a pH-dependent autoinhibition mechanism that controls Plasmodium Sub1 activity before merozoite egress.
- Author
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Martinez M, Bouillon A, Brûlé S, Raynal B, Haouz A, Alzari PM, and Barale J-C
- Subjects
- Animals, Humans, Subtilisin metabolism, Merozoites physiology, Dimerization, Protozoan Proteins metabolism, Plasmodium falciparum metabolism, Erythrocytes parasitology, Hydrogen-Ion Concentration, Plasmodium, Malaria, Falciparum parasitology
- Abstract
Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBCs) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes. Over the last two decades, it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi-step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto-maturation in the endoplasmic reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergoes further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the PV. Here, we report the crystal structure of full-length P. falciparum Sub1 (PfS1
FL ) and demonstrate, through structural, biochemical, and biophysical studies, that the atypical Plasmodium- specific Sub1 PD directly promotes the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, explaining how PmX processing and full activation of Sub1 can occur in different cellular compartments, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition that plays a key role in P. falciparum merozoites egress from infected host cells.IMPORTANCEMalaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin-like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is required in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here, we show that the unusual, parasite-specific Sub1 prodomain is directly responsible for its acidic-dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites., Competing Interests: The authors declare no conflict of interest.- Published
- 2024
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38. 3D structures of the Plasmodium vivax subtilisin-like drug target SUB1 reveal conformational changes to accommodate a substrate-derived α-ketoamide inhibitor.
- Author
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Martinez M, Batista FA, Maurel M, Bouillon A, Ortega Varga L, Wehenkel AM, Le Chevalier-Sontag L, Blondel A, Haouz A, Hernandez JF, Alzari PM, and Barale JC
- Subjects
- Plasmodium vivax, Enzyme Inhibitors pharmacology, Protozoan Proteins chemistry, Subtilisin, Antimalarials pharmacology, Antimalarials chemistry
- Abstract
The constant selection and propagation of multi-resistant Plasmodium sp. parasites require the identification of new antimalarial candidates involved in as-yet untargeted metabolic pathways. Subtilisin-like protease 1 (SUB1) belongs to a new generation of drug targets because it plays a crucial role during egress of the parasite from infected host cells at different stages of its life cycle. SUB1 is characterized by an unusual pro-region that tightly interacts with its cognate catalytic domain, thus precluding 3D structural analysis of enzyme-inhibitor complexes. In the present study, to overcome this limitation, stringent ionic conditions and controlled proteolysis of recombinant full-length P. vivax SUB1 were used to obtain crystals of an active and stable catalytic domain (PvS1
Cat ) without a pro-region. High-resolution 3D structures of PvS1Cat , alone and in complex with an α-ketoamide substrate-derived inhibitor (MAM-117), showed that, as expected, the catalytic serine of SUB1 formed a covalent bond with the α-keto group of the inhibitor. A network of hydrogen bonds and hydrophobic interactions stabilized the complex, including at the P1' and P2' positions of the inhibitor, although P' residues are usually less important in defining the substrate specificity of subtilisins. Moreover, when associated with a substrate-derived peptidomimetic inhibitor, the catalytic groove of SUB1 underwent significant structural changes, particularly in its S4 pocket. These findings pave the way for future strategies for the design of optimized SUB1-specific inhibitors that may define a novel class of antimalarial candidates.- Published
- 2023
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39. Artemisinin-independent inhibitory activity of Artemisia sp. infusions against different Plasmodium stages including relapse-causing hypnozoites.
- Author
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Ashraf K, Tajeri S, Arnold CS, Amanzougaghene N, Franetich JF, Vantaux A, Soulard V, Bordessoulles M, Cazals G, Bousema T, van Gemert GJ, Le Grand R, Dereuddre-Bosquet N, Barale JC, Witkowski B, Snounou G, Duval R, Botté CY, and Mazier D
- Subjects
- Antimalarials chemistry, Artemisinins chemistry, Erythrocytes drug effects, Erythrocytes parasitology, Hepatocytes drug effects, Hepatocytes parasitology, Humans, Life Cycle Stages drug effects, Malaria drug therapy, Malaria parasitology, Parasitic Sensitivity Tests, Plant Extracts chemistry, Plasmodium growth & development, Antimalarials pharmacology, Artemisia chemistry, Artemisinins pharmacology, Plant Extracts pharmacology, Plasmodium drug effects
- Abstract
Artemisinin-based combination therapies (ACT) are the frontline treatments against malaria worldwide. Recently the use of traditional infusions from Artemisia annua (from which artemisinin is obtained) or Artemisia afra (lacking artemisinin) has been controversially advocated. Such unregulated plant-based remedies are strongly discouraged as they might constitute sub-optimal therapies and promote drug resistance. Here, we conducted the first comparative study of the anti-malarial effects of both plant infusions in vitro against the asexual erythrocytic stages of Plasmodium falciparum and the pre-erythrocytic (i.e., liver) stages of various Plasmodium species. Low concentrations of either infusion accounted for significant inhibitory activities across every parasite species and stage studied. We show that these antiplasmodial effects were essentially artemisinin-independent and were additionally monitored by observations of the parasite apicoplast and mitochondrion. In particular, the infusions significantly incapacitated sporozoites, and for Plasmodium vivax and P. cynomolgi , disrupted the hypnozoites. This provides the first indication that compounds other than 8-aminoquinolines could be effective antimalarials against relapsing parasites. These observations advocate for further screening to uncover urgently needed novel antimalarial lead compounds., (© 2021 Ashraf et al.)
- Published
- 2021
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40. A Worldwide Map of Plasmodium falciparum K13-Propeller Polymorphisms.
- Author
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Ménard D, Khim N, Beghain J, Adegnika AA, Shafiul-Alam M, Amodu O, Rahim-Awab G, Barnadas C, Berry A, Boum Y, Bustos MD, Cao J, Chen JH, Collet L, Cui L, Thakur GD, Dieye A, Djallé D, Dorkenoo MA, Eboumbou-Moukoko CE, Espino FE, Fandeur T, Ferreira-da-Cruz MF, Fola AA, Fuehrer HP, Hassan AM, Herrera S, Hongvanthong B, Houzé S, Ibrahim ML, Jahirul-Karim M, Jiang L, Kano S, Ali-Khan W, Khanthavong M, Kremsner PG, Lacerda M, Leang R, Leelawong M, Li M, Lin K, Mazarati JB, Ménard S, Morlais I, Muhindo-Mavoko H, Musset L, Na-Bangchang K, Nambozi M, Niaré K, Noedl H, Ouédraogo JB, Pillai DR, Pradines B, Quang-Phuc B, Ramharter M, Randrianarivelojosia M, Sattabongkot J, Sheikh-Omar A, Silué KD, Sirima SB, Sutherland C, Syafruddin D, Tahar R, Tang LH, Touré OA, Tshibangu-wa-Tshibangu P, Vigan-Womas I, Warsame M, Wini L, Zakeri S, Kim S, Eam R, Berne L, Khean C, Chy S, Ken M, Loch K, Canier L, Duru V, Legrand E, Barale JC, Stokes B, Straimer J, Witkowski B, Fidock DA, Rogier C, Ringwald P, Ariey F, and Mercereau-Puijalon O
- Subjects
- Algorithms, Artemisinins therapeutic use, Asia, Southeastern, China, Endemic Diseases, Genotype, Humans, Lactones therapeutic use, Malaria, Falciparum drug therapy, Malaria, Falciparum parasitology, Plasmodium falciparum drug effects, Sequence Analysis, DNA, Artemisinins pharmacology, Drug Resistance genetics, Lactones pharmacology, Mutation, Plasmodium falciparum genetics, Polymorphism, Genetic, Protozoan Proteins genetics
- Abstract
Background: Recent gains in reducing the global burden of malaria are threatened by the emergence of Plasmodium falciparum resistance to artemisinins. The discovery that mutations in portions of a P. falciparum gene encoding kelch (K13)-propeller domains are the major determinant of resistance has provided opportunities for monitoring such resistance on a global scale., Methods: We analyzed the K13-propeller sequence polymorphism in 14,037 samples collected in 59 countries in which malaria is endemic. Most of the samples (84.5%) were obtained from patients who were treated at sentinel sites used for nationwide surveillance of antimalarial resistance. We evaluated the emergence and dissemination of mutations by haplotyping neighboring loci., Results: We identified 108 nonsynonymous K13 mutations, which showed marked geographic disparity in their frequency and distribution. In Asia, 36.5% of the K13 mutations were distributed within two areas--one in Cambodia, Vietnam, and Laos and the other in western Thailand, Myanmar, and China--with no overlap. In Africa, we observed a broad array of rare nonsynonymous mutations that were not associated with delayed parasite clearance. The gene-edited Dd2 transgenic line with the A578S mutation, which expresses the most frequently observed African allele, was found to be susceptible to artemisinin in vitro on a ring-stage survival assay., Conclusions: No evidence of artemisinin resistance was found outside Southeast Asia and China, where resistance-associated K13 mutations were confined. The common African A578S allele was not associated with clinical or in vitro resistance to artemisinin, and many African mutations appear to be neutral. (Funded by Institut Pasteur Paris and others.).
- Published
- 2016
- Full Text
- View/download PDF
41. Whole Pichia pastoris yeast expressing measles virus nucleoprotein as a production and delivery system to multimerize Plasmodium antigens.
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Jacob D, Ruffie C, Dubois M, Combredet C, Amino R, Formaglio P, Gorgette O, Pehau-Arnaudet G, Guery C, Puijalon O, Barale JC, Ménard R, Tangy F, and Sala M
- Subjects
- Animals, Drug Discovery, Fluorescent Antibody Technique, Malaria Vaccines administration & dosage, Measles virus metabolism, Mice, Mice, Inbred C57BL, Microscopy, Electron, Nucleoproteins metabolism, Protozoan Proteins isolation & purification, Ribonucleoproteins biosynthesis, Bioreactors, Drug Delivery Systems methods, Malaria Vaccines biosynthesis, Pichia metabolism, Plasmodium berghei chemistry, Protozoan Proteins metabolism
- Abstract
Yeasts are largely used as bioreactors for vaccine production. Usually, antigens are produced in yeast then purified and mixed with adjuvants before immunization. However, the purification costs and the safety concerns recently raised by the use of new adjuvants argue for alternative strategies. To this end, the use of whole yeast as both production and delivery system appears attractive. Here, we evaluated Pichia pastoris yeast as an alternative vaccine production and delivery system for the circumsporozoite protein (CS) of Plasmodium, the etiologic agent of malaria. The CS protein from Plasmodium berghei (Pb) was selected given the availability of the stringent C57Bl/6 mouse model of infection by Pb sporozoites, allowing the evaluation of vaccine efficacy in vivo. PbCS was multimerized by fusion to the measles virus (MV) nucleoprotein (N) known to auto-assemble in yeast in large-size ribonucleoprotein rods (RNPs). Expressed in P. pastoris, the N-PbCS protein generated highly multimeric and heterogenic RNPs bearing PbCS on their surface. Electron microscopy and immunofluorescence analyses revealed the shape of these RNPs and their localization in peripheral cytoplasmic inclusions. Subcutaneous immunization of C57Bl/6 mice with heat-inactivated whole P. pastoris expressing N-PbCS RNPs provided significant reduction of parasitemia after intradermal challenge with a high dose of parasites. Thus, in the absence of accessory adjuvants, a very low amount of PbCS expressed in whole yeast significantly decreased clinical damages associated with Pb infection in a highly stringent challenge model, providing a proof of concept of the intrinsic adjuvancy of this vaccine strategy. In addition to PbCS multimerization, the N protein contributed by itself to parasitemia delay and long-term mice survival. In the future, mixtures of whole recombinant yeasts expressing relevant Plasmodium antigens would provide a multivalent formulation applicable for antigen combination screening and possibly for large-scale production, distribution and delivery of a malaria vaccine in developing countries.
- Published
- 2014
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42. Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in western Cambodia.
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Witkowski B, Khim N, Chim P, Kim S, Ke S, Kloeung N, Chy S, Duong S, Leang R, Ringwald P, Dondorp AM, Tripura R, Benoit-Vical F, Berry A, Gorgette O, Ariey F, Barale JC, Mercereau-Puijalon O, and Menard D
- Subjects
- Antimalarials therapeutic use, Artemisinins therapeutic use, Cambodia, Cell Cycle Checkpoints drug effects, Malaria, Falciparum parasitology, Parasitic Sensitivity Tests, Plasmodium falciparum isolation & purification, Antimalarials pharmacology, Artemisinins pharmacology, Drug Resistance, Malaria, Falciparum drug therapy, Plasmodium falciparum drug effects
- Abstract
The declining efficacy of artemisinin derivatives against Plasmodium falciparum in western Cambodia is a major concern. The knowledge gap in the understanding of the mechanisms involved hampers designing monitoring tools. Here, we culture-adapted 20 isolates from Pailin and Ratanakiri (areas of artemisinin resistance and susceptibility in western and eastern Cambodia, respectively) and studied their in vitro response to dihydroartemisinin. No significant difference between the two sets of isolates was observed in the classical isotopic test. However, a 6-h pulse exposure to 700 nM dihydroartemisinin (ring-stage survival assay -RSA]) revealed a clear-cut geographic dichotomy. The survival rate of exposed ring-stage parasites (ring stages) was 17-fold higher in isolates from Pailin (median, 13.5%) than in those from Ratanakiri (median, 0.8%), while exposed mature stages were equally and highly susceptible (0.6% and 0.7%, respectively). Ring stages survived drug exposure by cell cycle arrest and resumed growth upon drug withdrawal. The reduced susceptibility to artemisinin in Pailin appears to be associated with an altered in vitro phenotype of ring stages from Pailin in the RSA.
- Published
- 2013
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43. Screening and evaluation of inhibitors of Plasmodium falciparum merozoite egress and invasion using cytometry.
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Bouillon A, Gorgette O, Mercereau-Puijalon O, and Barale JC
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- Cell Culture Techniques, Erythrocytes drug effects, Humans, Merozoites metabolism, Parasitic Sensitivity Tests methods, Schizonts drug effects, Schizonts metabolism, Antimalarials pharmacology, Erythrocytes parasitology, Flow Cytometry, Merozoites drug effects, Plasmodium falciparum drug effects, Plasmodium falciparum growth & development
- Abstract
Drug discovery programs heavily rely on assays adequately monitoring the activity of the drug on the -parasite stage targeted. So far, assays used to screen molecules active against Plasmodium falciparum parasites have mostly been based on measuring growth inhibition of asexual blood stages. We have developed a robust protocol allowing for monitoring parasite egress at the late schizont stage and subsequent erythrocyte invasion. This cytometry-based methodology uses nucleic acid labelling by the dye YOYO-1 and synchronized in vitro culture of P.falciparum exposed to inhibitors during the late phase of the intraerythrocytic cycle and the reinvasion process. This cytometry-based method is quick, accurate and allows for distinguishing egress from reinvasion on thousands of events. The throughput is also increased, as the assay can be scaled up for medium throughput screening for compounds that inhibit either the egress of merozoites or their entry into host erythrocytes.
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- 2013
- Full Text
- View/download PDF
44. FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei.
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Lacroix C, Giovannini D, Combe A, Bargieri DY, Späth S, Panchal D, Tawk L, Thiberge S, Carvalho TG, Barale JC, Bhanot P, and Ménard R
- Subjects
- Animals, Anopheles parasitology, Gene Knockout Techniques, Mice, Rats, Rats, Wistar, Recombination, Genetic, Sporozoites physiology, Genetic Engineering methods, Mutagenesis, Site-Directed methods, Plasmodium berghei genetics
- Abstract
We describe here a highly efficient procedure for conditional mutagenesis in Plasmodium. The procedure uses the site-specific recombination FLP-FRT system of yeast and targets the pre-erythrocytic stages of the rodent Plasmodium parasite P. berghei, including the sporozoite stage and the subsequent liver stage. The technique consists of replacing the gene under study by an FRTed copy (i.e., flanked by FRT sites) in the erythrocytic stages of a parasite clone that expresses the flip (FLP) recombinase stage-specifically--called the 'deleter' clone. We present the available deleter clones, which express FLP at different times of the parasite life cycle, as well as the schemes and tools for constructing new deleter parasites. We also outline and discuss the various strategies for exchanging a wild-type gene with an FRTed copy and for generating conditional gene knockout or knockdown parasite clones. Finally, we detail the protocol for obtaining sporozoites that lack a protein of interest and for monitoring sporozoite-specific DNA excision and depletion of the target protein. The protocol should allow the functional analysis of any essential protein in the sporozoite, liver stage or hepatic merozoite stages of rodent Plasmodium parasites.
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- 2011
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45. Drug inhibition of HDAC3 and epigenetic control of differentiation in Apicomplexa parasites.
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Bougdour A, Maubon D, Baldacci P, Ortet P, Bastien O, Bouillon A, Barale JC, Pelloux H, Ménard R, and Hakimi MA
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- Animals, Apicomplexa cytology, Apicomplexa enzymology, Cell Differentiation, Conserved Sequence, Gene Expression Regulation, Histone Deacetylase Inhibitors, Histone Deacetylases drug effects, Peptides, Cyclic pharmacology, Plasmodium enzymology, Plasmodium genetics, Toxoplasma enzymology, Toxoplasma genetics, Apicomplexa genetics, Histone Deacetylases genetics
- Abstract
Plasmodium and Toxoplasma are parasites of major medical importance that belong to the Apicomplexa phylum of protozoa. These parasites transform into various stages during their life cycle and express a specific set of proteins at each stage. Although little is yet known of how gene expression is controlled in Apicomplexa, histone modifications, particularly acetylation, are emerging as key regulators of parasite differentiation and stage conversion. We investigated the anti-Apicomplexa effect of FR235222, a histone deacetylase inhibitor (HDACi). We show that FR235222 is active against a variety of Apicomplexa genera, including Plasmodium and Toxoplasma, and is more potent than other HDACi's such as trichostatin A and the clinically relevant compound pyrimethamine. We identify T. gondii HDAC3 (TgHDAC3) as the target of FR235222 in Toxoplasma tachyzoites and demonstrate the crucial role of the conserved and Apicomplexa HDAC-specific residue TgHDAC3 T99 in the inhibitory activity of the drug. We also show that FR235222 induces differentiation of the tachyzoite (replicative) into the bradyzoite (nonreplicative) stage. Additionally, via its anti-TgHDAC3 activity, FR235222 influences the expression of approximately 370 genes, a third of which are stage-specifically expressed. These results identify FR235222 as a potent HDACi of Apicomplexa, and establish HDAC3 as a central regulator of gene expression and stage conversion in Toxoplasma and, likely, other Apicomplexa.
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- 2009
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46. Gene targeting demonstrates that the Plasmodium berghei subtilisin PbSUB2 is essential for red cell invasion and reveals spontaneous genetic recombination events.
- Author
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Uzureau P, Barale JC, Janse CJ, Waters AP, and Breton CB
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- Amino Acid Sequence, Animals, DNA, Protozoan chemistry, DNA, Protozoan isolation & purification, Gene Deletion, Gene Targeting methods, Genes, Protozoan, Mice, Molecular Sequence Data, Plasmodium berghei genetics, Recombinant Proteins metabolism, Sequence Alignment, Sequence Analysis, DNA, Subtilisins chemistry, Transfection, Erythrocytes parasitology, Plasmodium berghei enzymology, Plasmodium berghei pathogenicity, Recombination, Genetic, Subtilisins genetics, Subtilisins physiology
- Abstract
The Plasmodium merozoite proteases involved in the crucial process of erythrocyte invasion are promising targets for novel malaria control strategies. We report here the characterization of the subtilisin-like protease SUB2 from the rodent parasites Plasmodium berghei and Plasmodium yoelii, leading the way to in vivo functional studies of this enzyme. The kinetics of expression and subcellular localization imply a central role for SUB2 in erythrocyte invasion. Through the use of gene targeting strategies, we assessed the relevance of P. berghei SUB2 for the intraerythrocytic cycle. The selection of recombinant Pbsub2-TrimycDuoXpress-tagged parasites and the proper expression of the modified coding region demonstrate that the Pbsub2 locus is accessible to genetic modifications. However, Pbsub2 knock-out parasites were not recovered, confirming the importance of PbSUB2 for P. berghei merozoite stages, and supporting the fact that its Plasmodium falciparum SUB2 orthologue is an attractive drug target candidate. Finally, we identify revertant parasites that have lost the integrated selection cassette while conserving a Pbsub2-tagged gene. These spontaneous reversion events should overcome the scarcity of selectable markers available for this parasite, giving access to multiple gene tagging strategies, which, together with the validation of a TrimycDuoXpress tag, would represent valuable new tools for studying the biology of P. berghei.
- Published
- 2004
- Full Text
- View/download PDF
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