Background In order to proceed through their life cycle, Leishmania parasites switch between sandflies and mammals. The flagellated promastigote cells transmitted by the insect vector are phagocytized by macrophages within the mammalian host and convert into the amastigote stage, which possesses a rudimentary flagellum only. During an earlier proteomic study of the stage differentiation of the parasite we identified a component of the outer dynein arm docking complex, a structure of the flagellar axoneme. The 70 kDa subunit of the outer dynein arm docking complex consists of three subunits altogether and is essential for the assembly of the outer dynein arm onto the doublet microtubule of the flagella. According to the nomenclature of the well-studied Chlamydomonas reinhardtii complex we named the Leishmania protein LdDC2. Methodology/Principal Findings This study features a characterization of the protein over the life cycle of the parasite. It is synthesized exclusively in the promastigote stage and localizes to the flagellum. Gene replacement mutants of lddc2 show reduced growth rates and diminished flagellar length. Additionally, the normally spindle-shaped promastigote parasites reveal a more spherical cell shape giving them an amastigote-like appearance. The mutants lose their motility and wiggle in place. Ultrastructural analyses reveal that the outer dynein arm is missing. Furthermore, expression of the amastigote-specific A2 gene family was detected in the deletion mutants in the absence of a stage conversion stimulus. In vitro infectivity is slightly increased in the mutant cell line compared to wild-type Leishmania donovani parasites. Conclusions/Significance Our results indicate that the correct assembly of the flagellum has a great influence on the investigated characteristics of Leishmania parasites. The lack of a single flagellar protein causes an aberrant morphology, impaired growth and altered infectiousness of the parasite.

Antigenic variation to fool the immune system is one of the molecular tricks Plasmodium uses to maintain infection in its human host. The exclusive expression of the surface-exposed PfEMP1 molecules, encoded by var genes, is the best example for this. Central questions regarding the dynamics of antigenic variation, namely the rate of switching and the regulation of var gene expression in Plasmodium falciparum, are yet unanswered. To elucidate the in vivo situation, we studied var gene switching by analysing the var transcripts from parasites isolated from 20 non-immune malaria patients as well as during subsequent in vitro generations. Parasites were found to be highly co-ordinated as the whole population isolated from individual patients usually expressed only one dominant - preferentially group A -var gene. While some isolates have very low switching rates, others switched their var gene expression in every generation. However, during extended cultivation the co-ordinated expression and switching is lost resulting in random expression of all var gene groups. Switching as observed on the RNA level was also supported on the protein level using PfEMP1-specific antibodies. The results suggest that var genes switch in an ordered, hierarchical manner at much higher rates than previously described.

Leishmania promastigote cells transmitted by their insect vector get phagocytosed by macrophages and convert into the amastigote form. In a recently performed proteomic study, a thymidine kinase (TK) was found to be preferentially expressed in amastigotes. Western blot analysis showing a marked increase in TK protein synthesis during stage differentiation from promastigotes to amastigotes confirmed this result. After comparison of the amino acid sequence of Leishmania donovani and Leishmania major thymidine kinases with thymidine kinases of other organisms the Leishmania protein has to be classified as a type II TK. Therefore, in accordance with the nomenclature of other thymidine kinases we named the Leishmania enzymes LdTK1 and LmTK1, respectively. The LdTK1 is localised within the cytoplasm of promastigotes. In amastigotes, increased expression and a clustered distribution of the protein can be observed. Lmtk1 single allele gene replacement mutants have significantly elongated flagellum. In contrast, lmtk1 double allele gene replacement mutants show a remarkably reduced flagellar length, diminished overall size and a deformed body shape. In addition, they have a 12-fold reduced growth rate. For both mutant strains, macrophage infectivity is clearly reduced compared to a L. major wildtype infection.

ABSTRACTLeishmaniapromastigote cells transmitted by the insect vector get phagocytosed by macrophages and convert into the amastigote form. During development and transformation, the parasites are exposed to various concentrations of reactive oxygen species, which can induce programmed cell death (PCD). We show that a mitochondrial peroxiredoxin (LdmPrx) protectsLeishmania donovanifrom PCD. Whereas this peroxiredoxin is restricted to the kinetoplast area in promastigotes, it covers the entire mitochondrion in amastigotes, accompanied by dramatically increased expression. A similar change in the expression pattern was observed during the growth ofLeishmaniafrom the early to the late logarithmic phase. Recombinant LdmPrx shows typical peroxiredoxin-like enzyme activity. It is able to detoxify organic and inorganic peroxides and prevents DNA from hydroxyl radical-induced damage. Most notably,Leishmaniaparasites overexpressing this peroxiredoxin are protected from hydrogen peroxide-induced PCD. This protection is also seen in promastigotes grown to the late logarithmic phase, also characterized by high expression of this peroxiredoxin. Apparently, the physiological role of this peroxiredoxin is stabilization of the mitochondrial membrane potential and, as a consequence, inhibition of PCD through removal of peroxides.

In order to proceed through their life cycle, protozoan parasites of the genus Leishmania cycle between sandflies and mammals. This change of environment correlates with the differentiation from the promastigote stage (insect form) to the amastigote stage (intracellular mammalian form). The molecular basis underlying this major transformation is poorly understood so far; however, heat shock protein 90 (HSP90) appears to play a pivotal role. To further elucidate this process we identified proteins expressed preferentially in either of the two life cycle stages. By using two-dimensional (2-D) gel electrophoresis we observed defined changes in the protein pattern. A total of approximately 2000 protein spots were visualized. Of these, 31 proteins were present only in promastigotes. The abundance of 65 proteins increased during heat-induced in vitro amastigote differentiation, while a decreased abundance is observed for four proteins late in amastigote differentiation. Further analyses using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and peptide mass fingerprinting 67 protein spots were identified representing 41 different proteins known from databases and eight hypothetical proteins. Further studies showed that most of the stage-specific proteins fall into five groups of functionally related proteins. These functional categories are: (i) stress response (e.g. heat, oxidative stress); (ii) cytoskeleton and cell membrane; (iii) energy metabolism and phosphorylation; (iv) cell cycle and proliferation; and (v) amino acid metabolism. Very similar changes in the 2-D protein pattern were obtained when in vitro amastigote differentiation was induced either by pharmacological inhibition of HSP90 or by a combination of heat stress and acidic pH supporting the critical role for HSP90 in life cycle control.

Determinaram-se as concentrações de glicose, ureia, GGT (gama glutamil transferase), CK (creatino quinase), AST (aspartato aminotransferase) e LDH (lactato desidrogenase) em equinos alimentados com dietas normal e suplementadas com gorduras. As amostras de sangue foram obtidas após os exercícios físicos. O delineamento experimental utilizado foi um ensaio rotativo em quadrado latino 4x4. Os quatro tratamentos foram: Tl - dieta normal; T2 - dieta normal, com 10% de óleo de milho (gordura insaturada); T3 - dieta normal, com 10% de gordura de coco (gordura saturada); T4 - dieta normal, com 5% de óleo de milho e 5% de gordura de coco. Os resultados encontrados para glicose, ureia e GGT não apresentaram diferenças significativas entre as dietas. Os resultados para a AST e LDH foram maiores nos equinos alimentados com dietas sem gordura, e os valores da CK, em UI/L. foram de 118,01 (Tl); 84,24 (T2); 60,37 (T3) e 76,28 (T4), sendo significativamente menores (P<0,05) nos animais suplementados com gordura saturada, sugerindo menor lesão às fibras musculares após os exercícios.

Determinaram-se as concentrações de glicose, ureia, GGT (gama glutamil transferase), CK (creatino quinase), AST (aspartato aminotransferase) e LDH (lactato desidrogenase) em equinos alimentados com dietas normal e suplementadas com gorduras. As amostras de sangue foram obtidas após os exercícios físicos. O delineamento experimental utilizado foi um ensaio rotativo em quadrado latino 4x4. Os quatro tratamentos foram: Tl - dieta normal; T2 - dieta normal, com 10% de óleo de milho (gordura insaturada); T3 - dieta normal, com 10% de gordura de coco (gordura saturada); T4 - dieta normal, com 5% de óleo de milho e 5% de gordura de coco. Os resultados encontrados para glicose, ureia e GGT não apresentaram diferenças significativas entre as dietas. Os resultados para a AST e LDH foram maiores nos equinos alimentados com dietas sem gordura, e os valores da CK, em UI/L. foram de 118,01 (Tl); 84,24 (T2); 60,37 (T3) e 76,28 (T4), sendo significativamente menores (P<0,05) nos animais suplementados com gordura saturada, sugerindo menor lesão às fibras musculares após os exercícios.This experiment determined lhe concentrations of blood glucose, urea, GGT (gama glutamyltransferase), creatine kinase (CK), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in horses after the exercises, with normal diet and additional dietary fat. It was used the 4x4 Latin square design. The four diets were Tl-normal diet; T2-normal diet with 10% com oil; T3-nonnal diet with 10% coconut oil; T4-nonnal diet with 5% com oil and 5% coconut oil. There were no signifi cam changes in glucose, urea and GGT leveis due to diet. The serum concentrations of AST and LDH were higher in horses fed a diet withoutfat, and the resultsfound to CK (Ul/L) were 118.01 (Tl); 84.24 (T2); 60.37 (T3) and 76.28 (T4). Serum leveis ofCK were significantiy smaller (P<0.05) in horses suppiemented with saturated fat, suggesting a smaller damage to the muscle fibers after the exercises.