EC Accepts, published online ahead of print on 2 October 2009

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Eukaryotic Cell doi:10.1128/EC.00196-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Leishmania major LmaMPK7 protein kinase activity inhibits intracellular growth of the pathogenic amastigote stage

Miguel A. Morales, Pascale Pescher, and Gerald F. Späth^*

Institut Pasteur, CNRS URA 2581, Laboratory of Parasite Virulence, and the Institut National de la Santé et de la Recherche Médicale (INSERM) AVENIR Program, 75015 Paris, France

^* To whom correspondence should be addressed. Email: gerald.spaeth{at}pasteur.fr.

During the infectious cycle, protozoan parasites of the genus Leishmania undergo several adaptive differentiation steps that are induced by environmental factors and crucial for parasite infectivity. The genetic analysis of signalling proteins underlying Leishmania stage differentiation is often rendered difficult due to lethal null mutant phenotypes. Here, we used a transgenic strategy to gain insight into the functions of the mitogen-activated protein kinases LmaMPK7 and LmaMPK10 on parasite virulence. We established L. major and L. donovani lines expressing episomal GFP-LmaMPK7 and GFP-LmaMPK10 fusion proteins. The transgenic lines were normal in promastigote morphology, growth and the ability to differentiate into metacyclic and amastigote stages. While parasites expressing GFP-LmaMPK10 showed normal infectivity in mouse footpad analysis and macrophage infection assays, GFP-LmaMPK7 transgenic parasites displayed a strong delay in lesion formation and reduced intracellular parasite growth. Significantly, the effects of GFP-LmaMPK7 on virulence and proliferation were exclusively due to protein kinase activity, as over-expression of two kinase-dead mutants had no effect on parasite infectivity. GFP-LmaMPK7 transgenic L. donovani revealed a reversible, stage-specific growth defect in axenic amastigotes that was independent from cell death, but linked to non-synchronous growth arrest and significant reduction of de novo protein biosynthesis. Our data suggest that LmaMPK7 protein kinase activity may be implicated in parasite growth control and thus relevant for the development of non-proliferating stages during the infectious cycle.