Signalling in Malaria Parasites the Malsig Consortium

Depending on their developmental stage in the life cycle, malaria parasites develop within or outside host cells, and in extremely diverse contexts such as the vertebrate liver and blood circulation, or the insect midgut and hemocoel. Cellular and molecular mechanisms enabling the parasite to sense and respond to the intra- and the extra-cellular environments are therefore key elements for the proliferation and transmission of Plasmodium, and therefore are, from a public health perspective, strategic targets in the fight against this deadly disease. The MALSIG consortium, which was initiated in February 2009, was designed with the primary objective to integrate research ongoing in Europe and India on i) the properties of Plasmodium signalling molecules, and ii) developmental processes occurring at various points of the parasite life cycle. On one hand, functional studies of individual genes and their products in Plasmodium falciparum (and in the technically more manageable rodent model Plasmodium berghei) are providing information on parasite protein kinases and phosphatases, and of the molecules governing cyclic nucleotide metabolism and calcium signalling. On the other hand, cellular and molecular studies are elucidating key steps of parasite development such as merozoite invasion and egress in blood and liver parasite stages, control of DNA replication in asexual and sexual development, membrane dynamics and trafficking, production of gametocytes in the vertebrate host and further parasite development in the mosquito. This article, which synthetically reviews such signalling molecules and cellular processes, aims to provide a glimpse of the global frame in which the activities of the MALSIG consortium will develop over the next three years.

A glimpse into the clinical proteome of human malaria parasites Plasmodium falciparum and Plasmodium vivax

Malaria causes a worldwide annual mortality of about a million people.Rapidly evolving drug-resistant species of the parasite have created a pressing need for the identification of new drug targets and vaccine candidates. By developing fractionation protocols to enrich parasites from low-parasitemia patient samples, we have carried out the first ever proteomics analysis of clinical isolates of early stages of Plasmodium falciparum (Pf) and P. vivax. Patient-derived malarial parasites were directly processed and analyzed using shotgun proteomics approach using high-sensitivity MS for protein identification. Our study revealed about 100 parasite-coded gene products that included many known drug targets such as Pf hypoxanthine guanine phosphoribosyl transferase, Pf L-lactate dehydrogenase, and Plasmepsins. In addition,our study reports the expression of several parasite proteins in clinical ring stages that have never been reported in the ring stages of the laboratory-cultivated parasite strain. This proof-of-principle study represents a noteworthy step forward in our understanding of pathways elaborated by the parasite within the malaria patient and will pave the way towards identification of new drug and vaccine targets that can aid malaria therapy.

Heat shock protein 90 from neglected protozoan parasites

Significant advances have been made in our understanding of heat shock protein 90 (Hsp90) in terms of its structure, biochemical characteristics, post-translational modifications, interactomes, regulation and functions. In addition to yeast as a model several new systems have now been examined including flies, worms, plants as well as mammalian cells. This review discusses themes emerging out of studies reported on Hsp90 from infectious disease causing protozoa. A common theme of sensing and responding to host cell microenvironment emerges out of analysis of Hsp90 in Malaria, Trypanosmiasis as well as Leishmaniasis. In addition to their functional roles, the potential of Hsp90 from these infectious disease causing organisms to serve as drug targets and the current status of this drug development endeavor are discussed. Finally, a unique and the only known example of a split Hsp90 gene from another disease causing protozoan Giardia lamblia and its evolutionary significance are discussed. Clearly studies on Hsp90 from protozoan parasites promise to reveal important new paradigms in Hsp90 biology while exploring its potential as an anti-infective drug target. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90). (C) 2011 Elsevier B.V. All rights reserved.

Construction of a Plasmodium falciparum Rab-interactome identifies CK1 and PKA as Rab-effector kinases in malaria parasites

Background information. The pathology causing stages of the human malaria parasite Plasmodium falciparum reside within red blood cells that are devoid of any regulated transport system. The parasite, therefore, is entirely responsible for mediating vesicular transport within itself and in the infected erythrocyte cytoplasm, and it does so in part via its family of 11 Rab GTPases. Putative functions have been ascribed to Plasmodium Rabs due to their homology with Rabs of yeast, particularly with Saccharomyces that has an equivalent number of rab/ypt genes and where analyses of Ypt function is well characterized. Results. Rabs are important regulators of vesicular traffic due to their capacity to recruit specific effectors. In order to identify P. falciparum Rab (PfRab) effectors, we first built a Ypt-interactome by exploiting genetic and physical binding data available at the Saccharomyces genome database (SGD). We then constructed a PfRab-interactome using putative parasite Rab-effectors identified by homology to Ypt-effectors. We demonstrate its potential by wet-bench testing three predictions; that casein kinase-1 (PfCK1) is a specific Rab5B interacting protein and that the catalytic subunit of cAMP-dependent protein kinase A (PfPKA-C) is a PfRab5A and PfRab7 effector. Conclusions. The establishment of a shared set of physical Ypt/PfRab-effector proteins sheds light on a core set Plasmodium Rab-interactants shared with yeast. The PfRab-interactome should benefit vesicular trafficking studies in malaria parasites. The recruitment of PfCK1 to PfRab5B+ and PfPKA-C to PfRab5A+ and PfRab7+ vesicles, respectively, suggests that PfRab-recruited kinases potentially play a role in early and late endosome function in malaria parasites.

Parasites exert conflicting selection pressures to affect reproductive asynchrony of their host plant in an obligate pollination mutualism

1. Plant reproductive phenology is generally viewed as an individual's strategy to maximize gamete exchange and propagule dispersal and is often considered largely dependent on patterns of floral initiation. Reproductive phenology, however, can be affected by proximate responses to pollinators, parasites and herbivores which could influence floral longevity or fruit development time. 2. We examined the influence of insect interactants on within-plant reproductive phenology in the fig-fig wasp nursery pollination mutualism in Ficus racemosa (Moraceae). Most figs support a wasp community comprised of a mutualistic pollinator, with several host-plant-specific non-pollinating herbivorous gallers and parasitoids. These wasps reproduce within enclosed inflorescences called syconia, which develop into fruit after pollination. While different wasp species oviposit into syconia at varying times during its ontogeny, all wasp progeny are constrained to exit syconia simultaneously just prior to fruit ripening. Developing larvae of early-ovipositing wasps may hasten syconium ontogeny through formation of earlier and larger nutrient sinks, whereas larvae of late-arriving parasites may lengthen syconium ontogeny to complete their development successfully. Seeds are also important nutrient sinks. The number of seeds and the type and number of developing wasps may therefore be expected to influence syconium development times, thereby affecting the reproductive synchrony of syconia on a plant. 3. Observations on naturally pollinated and parasitized syconia indicated that their seed and wasp content affected syconium development time. Experimental manipulations of syconia to produce only seeds or various combinations of wasps confirmed this finding. Early-ovipositing galler progeny reduced syconium development times, while gallers ovipositing concurrently with pollinators had no effect on syconium development. Late-ovipositing parasitoid progeny, the presence of only seeds within the syconium, or delayed pollination increased syconium development time. The differential development of syconia, which was influenced by mutualistic or parasitic progeny, accordingly contributed to within-tree reproductive asynchrony. 4. Synthesis. Individual reproductive units in fig trees called syconia, which also function as brood sites for pollinating and parasitic fig wasps, have plastic development durations dependent on pollination timing and species of wasps developing within them. Syconium development times are a likely compromise between conflicting demands from developing seeds and different wasp species.

High Temperatures Result in Smaller Nurseries which Lower Reproduction of Pollinators and Parasites in a Brood Site Pollination Mutualism

In a nursery pollination mutualism, we asked whether environmental factors affected reproduction of mutualistic pollinators, non-mutualistic parasites and seed production via seasonal changes in plant traits such as inflorescence size and within-tree reproductive phenology. We examined seasonal variation in reproduction in Ficus racemosa community members that utilise enclosed inflorescences called syconia as nurseries. Temperature, relative humidity and rainfall defined four seasons: winter; hot days, cold nights; summer and wet seasons. Syconium volumes were highest in winter and lowest in summer, and affected syconium contents positively across all seasons. Greater transpiration from the nurseries was possibly responsible for smaller syconia in summer. The 3-5 degrees C increase in mean temperatures between the cooler seasons and summer reduced fig wasp reproduction and increased seed production nearly two-fold. Yet, seed and pollinator progeny production were never negatively related in any season confirming the mutualistic fig-pollinator association across seasons. Non-pollinator parasites affected seed production negatively in some seasons, but had a surprisingly positive relationship with pollinators in most seasons. While within-tree reproductive phenology did not vary across seasons, its effect on syconium inhabitants varied with season. In all seasons, within-tree reproductive asynchrony affected parasite reproduction negatively, whereas it had a positive effect on pollinator reproduction in winter and a negative effect in summer. Seasonally variable syconium volumes probably caused the differential effect of within-tree reproductive phenology on pollinator reproduction. Within-tree reproductive asynchrony itself was positively affected by intra-tree variation in syconium contents and volume, creating a unique feedback loop which varied across seasons. Therefore, nursery size affected fig wasp reproduction, seed production and within-tree reproductive phenology via the feedback cycle in this system. Climatic factors affecting plant reproductive traits cause biotic relationships between plants, mutualists and parasites to vary seasonally and must be accorded greater attention, especially in the context of climate change.

Differences in host species relationships and biogeographic influences produce contrasting patterns of prevalence, community composition and genetic structure in two genera of avian malaria parasites in southern Melanesia

1. Host-parasite interactions have the potential to influence broadscale ecological and evolutionary processes, levels of endemism, divergence patterns and distributions in host populations. Understanding the mechanisms involved requires identification of the factors that shape parasite distribution and prevalence. 2. A lack of comparative information on community-level host-parasite associations limits our understanding of the role of parasites in host population divergence processes. Avian malaria (haemosporidian) parasites in bird communities offer a tractable model system to examine the potential for pathogens to influence evolutionary processes in natural host populations. 3. Using cytochrome b variation, we characterized phylogenetic diversity and prevalence of two genera of avian haemosporidian parasites, Plasmodium and Haemoproteus, and analysed biogeographic patterns of lineages across islands and avian hosts, in southern Melanesian bird communities to identify factors that explain patterns of infection. 4. Plasmodium spp. displayed isolation-by-distance effects, a significant amount of genetic variation distributed among islands but insignificant amounts among host species and families, and strong local island effects with respect to prevalence. Haemoproteus spp. did not display isolation-by-distance patterns, showed marked structuring of genetic variation among avian host species and families, and significant host species prevalence patterns. 5. These differences suggest that Plasmodium spp. infection patterns were shaped by geography and the abiotic environment, whereas Haemoproteus spp. infection patterns were shaped predominantly by host associations. Heterogeneity in the complement and prevalence of parasite lineages infecting local bird communities likely exposes host species to a mosaic of spatially divergent disease selection pressures across their naturally fragmented distributions in southern Melanesia. Host associations for Haemoproteus spp. indicate a capacity for the formation of locally co-adapted host-parasite relationships, a feature that may limit intraspecific gene flow or range expansions of closely related host species.

Heme biosynthesis by the malarial parasite - Import of delta-aminolevulinate dehydrase from the host red cell

The mouse and human malarial parasites, Plasmodium berghei and Plasmodium falciparum, respectively, synthesize heme de novo following the standard pathway observed in animals despite the availability of large amounts of heme, derived from red cell hemoglobin, which is stored as hemozoin pigment, The enzymes, delta-aminolevulinate dehydrase (ALAD), coproporphyrinogen oxidase, and ferrochelatase are present at strikingly high levels in the P, berghei infected mouse red cell in vivo, The isolated parasite has low levels of ALAD and the data clearly indicate it to be of red cell origin. The purified enzyme preparations from the uninfected red cell and the parasite are identical in kinetic properties, subunit molecular weight, cross-reaction with antibodies to the human enzyme, and N-terminal amino acid sequence. Immunogold electron microscopy of the infected culture indicates that the enzyme is present inside the parasite and, therefore, is not a contaminant, The parasite derives functional ALAD from the host and the enzyme binds specifically to isolated parasite membrane in vitro, suggestive of the involvement of a receptor in its translocation into the parasite, While, ALAD, coproporphyrinogen oxidase, and ferrochelatase from the parasite and the uninfected red cell supernatant have identical subunit molecular weights on SDS-polyacrylamide gel electrophoresis and show immunological cross-reaction with antibodies to the human enzymes, as revealed by Western analysis, the first enzyme of the pathway, namely, delta-aminolevulinate synthase (ALAS) in the parasite, unlike that of the red cell host, does not cross-react with antibodies to the human enzyme, However, ALAS enzyme activity in the parasite is higher than that of the infected red cell supernatant. We therefore conclude that the parasite, while making its own ALAS, imports ALAD and perhaps most of the other enzymes of the pathway from the host to synthesize heme de novo, and this would enable it to segregate this heme from the heme derived from red cell hemoglobin degradation, ALAS of the parasite and the receptor(s) involved in the translocation of the host enzymes into the parasite would be unique drug targets.

Identification and Characterization of a Novel Deoxyhypusine Synthase in Leishmania donovani

Deoxyhypusine synthase, an NAD(+)-dependent enzyme, catalyzes the first step in the post-translational synthesis of an unusual amino acid, hypusine (N-epsilon-(4-amino-2-hydroxybutyl)lysine), in the eukaryotic initiation factor 5A precursor protein. Two putative deoxyhypusine synthase (DHS) sequences have been identified in the Leishmania donovani genome, which are present on chromosomes 20: DHSL20 (DHS-like gene from chromosome 20) and DHS34 (DHS from chromosome 34). Although both sequences exhibit an overall conservation of key residues, DHSL20 protein lacks a critical lysine residue, and the recombinant protein showed no DHS activity in vitro. However, DHS34 contains the critical lysine residue, and the recombinant DHS34 effectively catalyzed deoxyhypusine synthesis. Furthermore, in vivo labeling confirmed that hypusination of eukaryotic initiation factor 5A occurs in intact Leishmania parasites. Interestingly, the DHS34 is much longer, with 601 amino acids, compared with the human DHS enzyme (369 amino acids) and contains several unique insertions. To study the physiological role of DHS34 in Leishmania, gene deletion mutations were attempted via targeted gene replacement. However, chromosomal null mutants of DHS34 could only be obtained in the presence of a DHS34-containing episome. The present data provide evidence that DHS34 is essential for L. donovani and that structural differences in the human and leishmanial DHS enzyme may be exploited for designing selective inhibitors against the parasite.

Metabolism of Plasmodium berghei. I. Krebs cycle

The free parasites of Plasmodium berghei, obtained from infected cells of rats using an antiserum method, were investigated to study the operation of Krebs cycle. P. berghei was found to respire only with succinate; pyruvate, and other substrates of the Krebs cycle were not oxidized. The presence of a succinate dehydrogenase and a functioning cytochrome oxidase system was demonstrated. Cell-free extracts of free parasites showed the presence of enzymes for the utilization of C4 dicarboxylic acids; other enzymes of the Krebs cycle could not be detected. P. berghei differs from other species of Plasmodium in this respect.

Metabolism of Plasmodium bergheistar, open: III. Carbon dioxide fixation and role of pyruvate and dicarboxylic acids

Formation of C4 dicarboxylic acids in Plasmodium berghei by carbon dioxide fixation reaction has been demonstrated by the use of labeled NaH14CO3. The reactions require glucose, which may be required not only as an energy source but also to contribute to the formation of pyruvate in the process of carbon dioxide fixation. Intracellular concentration of pyruvate may play an important role in the metabolism of P. berghei; an increased intracellular level of pyruvate seems to be a prerequisite before some of these reactions could be detected. The distribution of the label indicates extensive randomization of amino acids and suggests an extensive cycling of the amino acid and organic acid pools of the parasites. This investigation formed part of the thesis submitted in 1965 for the doctoral degree at the Indian Institute of Science, Bangalore 12, India, and was supported in part by the Council of Scientific and Industrial Research, India.

Metabolism of Plasmodium bergheistar, open: II. 32Pi incorporation into high-energy phosphates

Free parasites of Plasmodium berghei were found to incorporate labeled inorganic phosphate into high-energy phosphates by substrate linked and oxidative hosphorylation. But the parasites also appear to utilize the reserve ATP of the host cells when they are within the host cells which may indicate the dependence of the parasite on the host cells for provision of energy. This investigation formed part of the thesis submitted in 1965 for the doctoral degree at the Indian Institute of Science, Bangalore 12, India, and was supported in part by the Council of Scientific and Industrial Research, India.

Proteomics of Trypanosoma evansi Infection in Rodents

Background: Trypanosoma evansi infections, commonly called 'surra', cause significant economic losses to livestock industry. While this infection is mainly restricted to large animals such as camels, donkeys and equines, recent reports indicate their ability to infect humans. There are no World Animal Health Organization (WAHO) prescribed diagnostic tests or vaccines available against this disease and the available drugs show significant toxicity. There is an urgent need to develop improved methods of diagnosis and control measures for this disease. Unlike its related human parasites T. brucei and T. cruzi whose genomes have been fully sequenced T. evansi genome sequence remains unavailable and very little efforts are being made to develop improved methods of prevention, diagnosis and treatment. With a view to identify potential diagnostic markers and drug targets we have studied the clinical proteome of T. evansi infection using mass spectrometry (MS).Methodology/Principal Findings: Using shot-gun proteomic approach involving nano-lc Quadrupole Time Of Flight (QTOF) mass spectrometry we have identified over 160 proteins expressed by T. evansi in mice infected with camel isolate. Homology driven searches for protein identification from MS/MS data led to most of the matches arising from related Trypanosoma species. Proteins identified belonged to various functional categories including metabolic enzymes; DNA metabolism; transcription; translation as well as cell-cell communication and signal transduction. TCA cycle enzymes were strikingly missing, possibly suggesting their low abundances. The clinical proteome revealed the presence of known and potential drug targets such as oligopeptidases, kinases, cysteine proteases and more.Conclusions/Significance: Previous proteomic studies on Trypanosomal infections, including human parasites T. brucei and T. cruzi, have been carried out from lab grown cultures. For T. evansi infection this is indeed the first ever proteomic study reported thus far. In addition to providing a glimpse into the biology of this neglected disease, our study is the first step towards identification of diagnostic biomarkers, novel drug targets as well as potential vaccine candidates to fight against T. evansi infections.

Chaperone expression profiles correlate with distinct physiological states of Plasmodium falciparum in malaria patients

Background: Molecular chaperones have been shown to be important in the growth of the malaria parasite Plasmodium falciparum and inhibition of chaperone function by pharmacological agents has been shown to abrogate parasite growth. A recent study has demonstrated that clinical isolates of the parasite have distinct physiological states, one of which resembles environmental stress response showing up-regulation of specific molecular chaperones. Methods: Chaperone networks operational in the distinct physiological clusters in clinical malaria parasites were constructed using cytoscape by utilizing their clinical expression profiles. Results: Molecular chaperones show distinct profiles in the previously defined physiologically distinct states. Further, expression profiles of the chaperones from different cellular compartments correlate with specific patient clusters. While cluster 1 parasites, representing a starvation response, show up-regulation of organellar chaperones, cluster 2 parasites, which resemble active growth based on glycolysis, show up-regulation of cytoplasmic chaperones. Interestingly, cytoplasmic Hsp90 and its co-chaperones, previously implicated as drug targets in malaria, cluster in the same group. Detailed analysis of chaperone expression in the patient cluster 2 reveals up-regulation of the entire Hsp90-dependent pro-survival circuitries. In addition, cluster 2 also shows up-regulation of Plasmodium export element (PEXEL)-containing Hsp40s thought to have regulatory and host remodeling roles in the infected erythrocyte. Conclusion: In all, this study demonstrates an intimate involvement of parasite-encoded chaperones, PfHsp90 in particular, in defining pathogenesis of malaria.