Over-expression of Leishmania major MAP kinases reveals stage-specific induction of phosphotransferase activity

Morales, Miguel A.; Renaud, Olivier; Faigle, Wolfgang; Shorte, Spencer; Späth, Gérald F.

doi:10.1016/j.ijpara.2007.03.006

Cited by 48 publications

(62 citation statements)

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“…We previously observed a robust increase in GFPLmaMPK7 protein kinase activity in axenic amastigotes compared to log-phase promastigotes (22). Hence, GFPK7 virulence attenuation may be a direct result of increased phosphotransferase activity.…”

Section: Resultsmentioning

confidence: 92%

“…L. major and L. donovani transgenic parasites overexpressing GFP-tagged LmaMPK7 and LmaMPK10 (termed GFPK7 and GFPK10, respectively) allowed us in previous studies to reveal an increased activity of these kinases in response to heat and pH shock and following amastigote differentiation (22). Here we investigated the effects of LmaMPK overexpression on parasite virulence by using experimental mouse infection (35,37).…”

Section: Resultsmentioning

confidence: 99%

“…L. donovani strain 1S2D (MHOM/SD/62/ 1S-CL2D) clone LdB was cultured as described previously (10,15,17,30). L. major and L. donovani pXG mock-transfected control and GFPK transgenic parasites expressing GFP-tagged LmaMPKs and inactive dead mutant kinase were generated as previously described (22). To control for nonspecific effects associated with the GFP tag, transgenic L. donovani lines expressing pLEXSYLmaMPK7 or vector alone were established.…”

Section: Methodsmentioning

confidence: 99%

“…One possible strategy to overcome this limitation and gain insight into the functions of these essential protein kinases is provided by the biochemical characterization of epitopetagged derivatives expressed in transgenic parasites. We previously utilized recombinant Leishmania MAPKs to study protein kinase activities (22). The expression of three green fluorescent protein (GFP)-tagged Leishmania major MAPKs, LmaMPK4, LmaMPK7, and LmaMPK10, allowed us to reveal environmentally and developmentally regulated phosphorylation and induction of phosphotransferase activity of these protein kinases after exposure to pH 5.5 and 34°C in transgenic L. major and in transgenic Leishmania donovani axenic amastigotes.…”

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confidence: 99%

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Leishmania majorMPK7 Protein Kinase Activity Inhibits Intracellular Growth of the Pathogenic Amastigote Stage

2010

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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. Genetic analyses of signaling proteins underlying Leishmania stage differentiation are often rendered difficult due to lethal null mutant phenotypes. Here we used a transgenic strategy to gain insight into the functions of the mitogenactivated Leishmania major protein kinases LmaMPK7 and LmaMPK10 in parasite virulence. We established L. major and Leishmania donovani lines expressing episomal green fluorescent protein (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 by 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 due exclusively to protein kinase activity, as the overexpression of two kinase-dead mutants had no effect on parasite infectivity. GFP-LmaMPK7 transgenic L. donovani cells revealed a reversible, stage-specific growth defect in axenic amastigotes that was independent of cell death but linked to nonsynchronous growth arrest and a 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 nonproliferating stages during the infectious cycle.

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Leishmania majorMPK7 Protein Kinase Activity Inhibits Intracellular Growth of the Pathogenic Amastigote Stage

2010

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“…The existence of MAPK homologues has been documented in certain parasitic protozoa. For instance ERK1 and ERK2 homologues of Giardia lamblia have been shown to play a critical role in trophozoite differentiation into cysts (Ellis et al, 2003), Pfmap2, a MAPK homologue in Plasmodium falciparum is essential for the completion of the asexual phase of the parasite lifecycle (Dorin-Semblat et al, 2006) and Leishmania major MAPK homologues exhibit an increased phosphotransferase activity in response to pH and temperature shift (Morales et al, 2007). On the other hand, E. histolytica has been shown to possess a single homologue of a typical MAPK gene (EhMAPK).…”

Section: Biology Of Entamoeba Histolyticamentioning

confidence: 99%