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.

A novel form of acto-myosin regulation has been proposed in which polymerization of new actin filaments regulates motility of parasites of the apicomplexan class of protozoa. In vivo and in vitro parasite F-actin is very short and unstable, but the structural basis and details of filament dynamics remain unknown. Here, we show that long actin filaments can be obtained by polymerizing unlabeled rabbit skeletal actin (RS-actin) onto both ends of the short rhodamine-phalloidin-stabilized Plasmodium falciparum actin I (Pf-actin) filaments. Following annealing, hybrid filaments of micron length and "zebra-striped" appearance are observed by fluorescence microscopy that are stable enough to move over myosin class II motors in a gliding filament assay. Using negative stain electron microscopy we find that pure Pf-actin stabilized by jasplakinolide (JAS) also forms long filaments, indistinguishable in length from RS-actin filaments, and long enough to be characterized structurally. To compare structures in near physiological conditions in aqueous solution we imaged Pf-actin and RS-actin filaments by atomic force microscopy (AFM). We found the monomer stacking to be distinctly different for Pf-actin compared with RS-actin, such that the pitch of the double helix of Pf-actin filaments was 10% larger. Our results can be explained by a rotational angle between subunits that is larger in the parasite compared with RS-actin. Modeling of the AFM data using high-resolution actin filament models supports our interpretation of the data. The structural differences reported here may be a consequence of weaker inter-and intra-strand contacts, and may be critical for differences in filament dynamics and for regulation of parasite motility. Plasmodium falciparum (Pf)5 is a protozoan causing human malaria and belongs to the apicomplexan group of intracellular parasites. Apicomplexan motility and host cell invasion requires the parasite's own acto-myosin motor system (1-3). The organization of the acto-myosin machinery under the cell surface of Apicomplexa has been described by a linear model (4) (for review, see Refs. 3, 5-7). In this model, actin filaments are linked via receptor-ligand interactions to a substrate or the host cell surface, and the action of myosin motors moves the parasite forward. Interestingly, the treatment of the closely related apicomplexan parasite Toxoplasma gondii (Tg) with the actin filament-stabilizing toxin jasplakinolide (JAS) increased both actin polymerization and the speed of gliding (8 -10). Based on these observations, a novel form of acto-myosin regulation has been proposed in which the availability of actin filaments regulates apicomplexan motility (8).Many recent studies have since focused on actin filament dynamics in these parasites. In contrast to other cell types it appears that in Apicomplexa, actin exists primarily in a monomeric form (8,10,11). This is consistent with the fact that the apicomplexan actin, in vitro and in vivo, only forms very short and unstable filaments (8,9,12,13). Unlike ...

SummaryAntigenic 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 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) protects Leishmania donovani from 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 of Leishmania from 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, Leishmania parasites 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.Leishmania parasites affect more than 12 million people worldwide, with an estimated 2 million new cases each year (WHO World Health Report, 2004, http://www.who.int/whr /en). Depending on the species involved, symptoms range from the self-healing cutaneous form (Leishmania major) to the fatal visceral form (L. donovani). The parasite is transmitted as the infective promastigote form from the gut of its insect vector, female phlebotomine flies of the genera Phlebotomus and Lutzomyia, to mammalian hosts. Promastigotes get phagocytosed by macrophages and convert into the amastigote form, which is able to survive and replicate within phagolysosomes. During phagocytosis of Leishmania promastigotes, the macrophages produce different reactive oxygen species (ROS) to kill the parasites. ROS readily react with proteins, DNA, and lipids and have been implicated in a wide variety of cell functions, such as signal transduction, redox homeostasis, apoptosis, aging, tumor progression, and pathogen infection (9,19,42,58). Numerous reports have shown that Leishmania parasites are susceptible to ROS-and RNS (reactive nitrogen species)-mediated toxicity (41, 57). In order to survive and establish an infection, they have to cope with these pro-oxidants. In Trypanosomatidae, it was shown that peroxiredoxins are the major antioxidant enzymes that can use different ROS and RNS like H 2 O 2 , hydroperoxides, and ONOO as substrates (56). Peroxiredoxins are found in a great variety of organisms, where they fulfill distinct functions, such as detoxification, signaling, or differentiation (25). In different members of the family Trypanosomatidae, cytosolic, as well as mitochondrial, peroxiredoxins were found (6,10,11,21). Peroxire...

BackgroundIn 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 FindingsThis 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/SignificanceOur 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.