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Parasitology Section Summary During the life cycle Leishmania differentiate from flagellated forms, the promastigotes, to amastigotes carrying a rudimentary flagellum. Besides biochemical changes, this process involves a change in overall cell morphology including flagellar shortening. We could show that LmxMKK, a mitogen-activated protein kinase kinase homologue from Leishmania mexicana, is involved in the regulation of flagellar assembly and cell size. A deletion mutant for LmxMKK revealed motile flagella dramatically reduced in length and lacking the paraflagellar rod, a cytoskeletal structure adjacent to the axoneme in kinetoplastid flagella. Moreover, a fraction of the cells showed perturbance of the axonemal structure. Complementation of the deletion mutant with the wild-type gene restored typical promastigote morphology. LmxMKK is the first protein kinase known to be involved in organellar assembly. Introduction Leishmaniasis, an infection caused by protozoan parasites of the genus Leishmania, has been estimated with 12 million cases worldwide and 1.5 – 2 million new cases annually (World Health Organization 1998). Treatment for Leishmaniasis is unsatisfactory. Available drugs cause severe side effects, require hospitalisation, are too expensive or even lost their efficacy due to re- Signal transduction in Leishmania Zusammenfassung Im Verlauf des Lebenszyklus differenzieren Leishmanien von begeißelten Formen, den Promastigoten, zu Amastigoten, die nur rudimentäre Geißeln tragen. Neben biochemischen Veränderungen ist diese Umwandlung begleitet durch eine Änderung der gesamten Morphologie der Zelle einschließlich der Verkürzung der Geißel. Wir konnten zeigen, dass LmxMKK, ein Mitogen-aktivierte Protein (MAP) Kinase Kinase Homolog von Leishmania mexicana, an der Regulation des Aufbaus der Geißel und der Größe der Zelle beteiligt ist. Eine Deletionsmutante <strong>für</strong> LmxMKK zeigte bewegliche aber deutlich verkürzte Geißeln, denen der Paraxialstab, eine Struktur des Zytoskeletts entlang des Axonemas der Geißeln von Kinetoplastiden, fehlt. Darüber hinaus, zeigte ein Teil der Zellen einen gestörten Aufbau des Axonemas. Komplementation der Deletionsmutante mit dem Wildtypgen stellte die typische Morphologie der Promastigoten wieder her. LmxMKK ist die erste Proteinkinase <strong>für</strong> die eine Beteiligung am Aufbau eines Organells gezeigt wurde. 40 sistance development. Protein kinases are key-regulatory molecules in all eukaryotic cells including parasites like Leishmania. Specific inhibition of pathogen signal transduction involving protein kinases leading to growth arrest, inability to differentiate, and/or loss of adaptation to the mammalian host is therefore an ideal means to treat this infectious disease. Especially, protein kinases of the mitogen-activated protein (MAP) kinase signal transduction pathways are of significant importance for the proliferation, differentiation and adaptation to changing environments in eukaryotic cells. Hence, we concentrated on MAP kinases and their activating kinases, the MAP kinase kinases. The genes for nine MAP kinase and four MAP kinase kinase homologues from Leishmania mexicana were identified by a polymerase chain reaction based approach using degenerate oligonucleotide primers deduced from conserved regions of these proteins. A selection of these protein kinases were used for deletion analysis. Project Description and Results We generated a homozygous deletion mutant for LmxMKK, a homologue of MAP kinase kinases from Leishmania mexicana by homologous recombination. The promastigote parasites revealed a peculiar phenotype. They displayed a reduced cell size and a flagellum reduced to a maximum of 1/5 of the length of the flagellum of the wild-type (Fig. 1). Figure 1: Scanning electron micrograph of Leishmania mexicana wild-type (A) and LmxMKK null mutant (B) promastigotes at the same magnification. Bar, 10 µm. Determination of flagellar lengths revealed an average length of 1.76 µm and a size range from 0 to 5 µm. In the wild-type the average flagellar length is about 12 µm. The short flagella in the mutant were unable to move the cells through the medium. Instead all cells were found at the bottom of the culture flasks wiggling with their short flagella. Reintroduction of LmxMKK into the deletion mutant restored the wild-type phenotype corroborating a single gene deletion effect. Closer inspection of the mutant cells revealed additional defects (Fig. 2).
The paraflagellar rod, a crosshatched cytoskeletal structure running along the axoneme in kinetoplastid flagella is missing in 78.5 % of the mutant promastigotes. The rest displayed a rudimentary paraflagellar rod. Moreover, 11 % of all flagella lacked the central microtubule doublet of the canonical 9-plus-2-microtubule structure seen in wild-type flagella. Finally, the mutant promastigotes were found to accumulate membrane fragments in the flagellar pocket (Fig. 3). 41 Interestingly, the mutant promastigotes showed delayed lesion development in experimental infections of Balb/c mice. In wild-type Leishmania mexicana infections lesion development commences at about 5 weeks post infection whereas in the mutant first swelling of infected feet was observed at 30 weeks post infection. However, amastigotes found in these lesions appeared morphologically normal und transformed back into promastigotes with short flagella after cultivation under appropriate conditions. As flagella are widespread in eukaryotic cells from protists to mammals and a number of human disorders are caused by immotile or misassembled flagella, the study of signal transduction pathways regulating flagellar morphology is of substantial relevance. Here the uniflagellated parasite Leishmania could function as a suitable model organism. Selected Publications • Wiese M, Wang Q, Görcke I. (2003). Identification of mitogen-activated protein kinase homologues from Leishmania mexicana. Int. J. Parasitol. 33(14): 1577-1587 • Wiese M, Kuhn D, Grünfelder CG. (2003). Protein kinase involved in flagellar-length control. Eukaryot. Cell. Aug 2(4): 769-777 Investigators • Martin Wiese • Daniela Kuhn • Stephani Tenbreul Parasitology Section Figure 2: Ultrastructure of L. mexicana wild-type and LmxMKK null mutant (DLmxMKK) flagella. (A-D, G) Sections of wild-type and ∆LmxMKK flagella after high-pressure freezing (HPF), freeze-substitution, and Epon embedding; (A, C) wild-type longitudinal and transverse sections; (B, D, G) ∆LmxMKK longitudinal and transverse sections. (E, F, H) Chemically fixed transverse flagellar sections of the wild-type (E), and ∆LmxMKK (F, H). a, axoneme; p, paraflagellar rod; tj, tight junction. Bars, 0.25 µm. Figure 3: Ultrastructure of L. mexicana wild-type and LmxMKK null mutant (∆LmxMKK) cells. ∆LmxMKK promastigotes HPFcryofixed (A) or chemically fixed (B) containing large amounts of membrane fragments in their flagellar pockets. fp, flagellar pocket; k, kinetoplast; mf, membrane fragments; tz, transition zone. Bar, 0.5 µm.
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Education and Teaching Lars Volkman
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Diary 2004/2005 12.-14. November 20
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