Probing druggability and biological function of essential proteins in Leishmania combining facilitated null mutant and plasmid shuffle analyses

Autor: Dacher, Mariko, Morales, Miguel A, Pescher, Pascale, Leclercq, Olivier, Rachidi, Najma, Prina, Eric, Cayla, Mathieu, Descoteaux, Albert, Späth, Gerald F
Přispěvatelé: Parasitologie moléculaire et Signalisation, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Armand Frappier (INRS-IAF), Institut National de la Recherche Scientifique [Québec] (INRS)-Réseau International des Instituts Pasteur (RIIP), This work was supported by the 7th Framework Programme of the European Community through a grant to the LEISHDRUG (223414) consortium, by the Agence Nationale de Recherche through a grant to the ANR-11-RPIB-0016 TRANSLEISH consortium, and the French Government's Investissements d'Avenir programme: Laboratoire d'Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (Grant No. ANR-10-LABX-62-IBEID). MD was the recipient of a Bourse de stage from the International Division of the Pasteur Institut and of a Bourse Fin de these scientifique from the Fondation de Recherche Médicale Equipe FRM programme (FDT20110922563), ANR-11-RPIB-0016,TRANSLEISH,Découverte de nouvelles protéines kinases chez Leishmania donovani à partir des inhibiteurs tête de série issus d'un criblage phénotypique(2011), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 223414,EC:FP7:HEALTH,FP7-HEALTH-2007-B,LEISHDRUG(2008), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS) - Institut Pasteur [Paris], Institut National de la Recherche Scientifique [Québec] (INRS) - Réseau International des Instituts Pasteur - Institut Armand Frappier, This work was supported bythe 7th Framework Programme of the European Communitythrough a grant to the LEISHDRUG (223414) consortium, bythe Agence Nationale de Recherche through a grant to theANR-11-RPIB-0016 TRANSLEISH consortium, and theFrench Government’s Investissements d’Avenir programme:Laboratoire d’Excellence ‘Integrative Biology of EmergingInfectious Diseases’ (Grant No. ANR-10-LABX-62-IBEID). MDwas the recipient of a Bourse de stage from the InternationalDivision of the Pasteur Institut and of a Bourse Fin de thesescientifique from the Fondation de Recherche MédicaleEquipe FRM programme (FDT20110922563)., ANR-11-RPIB-0016, TRANSLEISH, Découverte de nouvelles protéines kinases chez Leishmania donovani à partir des inhibiteurs tête de série issus d'un criblage phénotypique(2011), ANR-10-LABX-62-IBEID, IBEID, Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases'(2010), European Project : 223414, EC:FP7:HEALTH, FP7-HEALTH-2007-B, LEISHDRUG(2008)
Jazyk: angličtina
Rok vydání: 2014
Předmět:
Zdroj: Molecular Microbiology
Molecular Microbiology, Wiley, 2014, 93, pp.146-166. ⟨10.1111/mmi.12648⟩
Molecular Microbiology, 2014, 93, pp.146-166. ⟨10.1111/mmi.12648⟩
Molecular Microbiology, Wiley, 2014, 93 (1), pp.146-66. <10.1111/mmi.12648>
ISSN: 0950-382X
1365-2958
DOI: 10.1111/mmi.12648⟩
Popis: International audience; Leishmania parasites cause important human morbidity and mortality. Essential Leishmania genes escape genetic assessment by loss-of-function analyses due to lethal null mutant phenotypes, even though these genes and their products are biologically most significant and represent validated drug targets. Here we overcome this limitation using a facilitated null mutant approach applied for the functional genetic analysis of the MAP kinase LmaMPK4. This system relies on the episomal expression of the target gene from vector pXNG that expresses the Herpes simplex virus thymi-dine kinase gene thus rendering transgenic parasites susceptible for negative selection using the antiviral drug ganciclovir. Using this system we establish the genetic proof of LmaMPK4 as essential kinase in pro-mastigotes. LmaMPK4 structure/function analysis by plasmid shuffle allowed us to identify regulatory kinase sequence elements relevant for chemothera-peutic intervention. A partial null mutant, expressing an MPK4 derivative with altered ATP-binding properties , showed defects in metacyclogenesis, establishing a first link of MPK4 function to parasite differentiation. The approaches presented here are broadly applicable to any essential gene in Leishmania thus overcoming major bottlenecks for their functional genetic analysis and their exploitation for structure-informed drug development.
Databáze: OpenAIRE
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