Characterization of two malaria parasite organelle translation elongation factor G proteins: the likely targets of the anti-malarial fusidic acid

Malaria parasites harbour two organelles with bacteria-like metabolic processes that are the targets of many anti-bacterial drugs. One such drug is fusidic acid, which inhibits the translation component elongation factor G. The response of P. falciparum to fusidic acid was characterised using extended SYBR-Green based drug trials. This revealed that fusidic acid kills in vitro cultured P. falciparum parasites by immediately blocking parasite development. Two bacterial-type protein translation elongation factor G genes are identified as likely targets of fusidic acid. Sequence analysis suggests that these proteins function in the mitochondria and apicoplast and both should be sensitive to fusidic acid. Microscopic examination of protein-reporter fusions confirm the prediction that one elongation factor G is a component of parasite mitochondria whereas the second is a component of the relict plastid or apicoplast. The presence of two putative targets for a single inhibitory compound emphasizes the potential of elongation factor G as a drug target in malaria.     

Genomes and genome projects of protozoan parasites

Protozoan parasites are causing some of the most devastating diseases world-wide. It has now been recognised that a major effort is needed to be able to control or eliminate these diseases. Genome projects for the most important protozoan parasites have been initiated in the hope that the read-out of these projects will help to understand the biology of the parasites and identify new targets for urgently needed drugs. Here, I will review the current status of protozoan parasite genome projects, present findings obtained as a result of the availability of genomic data and discuss the potential impact of genome information on disease control.     

PLP-dependent enzymes as potential drug targets for protozoan diseases

The chemical properties of the B(6) vitamers are uniquely suited for wide use as cofactors in essential reactions, such as decarboxylations and transaminations. This review addresses current efforts to explore vitamin B(6) dependent enzymatic reactions as drug targets. Several current targets are described that are found amongst these enzymes. The focus is set on diseases caused by protozoan parasites. Comparison across a range of these organisms allows insight into the distribution of potential targets, many of which may be of interest in the development of broad range anti-protozoan drugs. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.     

Recent advances in the search for new anti-coccidial drugs

Coccidia provide a rich hunting ground for drug-designers, as there are significant biochemical differences between the parasites and their hosts. Recent years have brought the discovery of the plastid and its possible metabolic machinery, characterisation of acidocalcisomes, reports on the apparent absence from some coccidia of a typical mitochondrion, and the discovery of the mannitol cycle and shikimate pathway in the parasites. Moreover, modern technologies such as genomics and proteomics are bringing new insights into the biochemistry of coccidia and highlighting possible drug targets in abundance. A major issue for would-be drug discoverers is to decide upon the targets to prioritise. This review provides an update on recent findings on how coccidia differ biochemically from vertebrates. It includes discoveries within coccidian parasites themselves but also uses findings in Plasmodium to provide an overview of biochemical features that may be characteristics of many apicomplexan parasites and so potential targets for broad-spectrum drugs.     

Global protein expression analysis in apicomplexan parasites: current status

Members of the phylum Apicomplexa are important protozoan parasites that cause some of the most serious, and in some cases, deadly diseases in humans and animals. They include species from the genus Plasmodium, Toxoplasma, Eimeria, Neospora, Cryptosporidium, Babesia and Theileria. The medical, veterinary and economic impact of these pathogens on a global scale is enormous. Although chemo- and immuno-prophylactic strategies are available to control some of these parasites, they are inadequate. Currently, there is an urgent need to design new vaccines or chemotherapeutics for apicomplexan diseases. High-throughput global protein expression analyses using gel or non-gel based protein separation technologies coupled with mass spectrometry and bioinformatics provide a means to identify new drug and vaccine targets in these pathogens. Protein identification based proteomic projects in apicomplexan parasites is currently underway, with the most significant progress made in the malaria parasite, Plasmodium falciparum. More recently, preliminary two-dimensional gel electrophoresis maps of Toxoplasma gondii and Neospora caninum tachyzoites and Eimeria tenella sporozoites, have been produced, as well as for micronemes in E. tenella. In this review, the status of proteomics in the analysis of global protein expression in apicomplexan parasites will be compared and the challenges associated with these investigations discussed.     

The genetic toolbox for Leishmania parasites

Leishmania parasites cause a variety of devastating diseases in tropical areas around the world. Due to the lack of vaccines and limited availability of drugs, new therapeutic targets are urgently needed. A variety of genetic tools have been developed to investigate the complex biology of this parasite and its interactions with the host. One of the main techniques is the generation of knock-out parasites via targeted gene replacement, a process that takes advantage of the parasites ability to undergo homologous recombination. Studying the effect of gene deletions in vitro and in infectivity models in vivo allows understanding the function of a target gene and its potential as a therapeutic target. Other genetic manipulations available include episomal and chromosomal complementation and the generation of overproducer strains. However, there are also limitations, such as the lack of RNA interference machinery in most Leishmania species and limited options for inducible expression systems. The genomes of several Leishmania species have now been sequenced and will provide powerful resources in combination with the genetic tools that are available. The increasing knowledge of parasite biology and host parasite interactions derived from these studies will raise the number of potential therapeutic targets, which are sorely needed to combat leishmaniasis.     

Polyamine homoeostasis as a drug target in pathogenic protozoa: peculiarities and possibilities

New drugs are urgently needed for the treatment of tropical and subtropical parasitic diseases, such as African sleeping sickness, Chagas' disease, leishmaniasis and malaria. Enzymes in polyamine biosynthesis and thiol metabolism, as well as polyamine transporters, are potential drug targets within these organisms. In the present review, the current knowledge of unique properties of polyamine metabolism in these parasites is outlined. These properties include prozyme regulation of AdoMetDC (S-adenosylmethionine decarboxylase) activity in trypanosomatids, co-expression of ODC (ornithine decarboxylase) and AdoMetDC activities in a single protein in plasmodia, and formation of trypanothione, a unique compound linking polyamine and thiol metabolism in trypanosomatids. Particularly interesting features within polyamine metabolism in these parasites are highlighted for their potential in selective therapeutic strategies.     

Genetic regulation of protein synthesis in trypanosomes

It is becoming increasingly clear that parasitic protozoa remain a scourge to humans in the 21st century. The trypanosomes are a diverse group of insect-transmitted parasites that wiggle their way through multiple life cycle stages as they destroy human lives. Exquisitely detailed studies of these organisms reveal basic differences in gene expression that separate these single celled eukaryotes from multicellular eukaryotic organisms and have suggested numerous potential drug targets.     

Genomics of apicomplexan parasites

The increasing prevalence of infections involving intracellular apicomplexan parasites such as Plasmodium, Toxoplasma, and Cryptosporidium (the causative agents of malaria, toxoplasmosis, and cryptosporidiosis, respectively) represent a significant global healthcare burden. Despite their significance, few treatments are available; a situation that is likely to deteriorate with the emergence of new resistant strains of parasites. To lay the foundation for programs of drug discovery and vaccine development, genome sequences for many of these organisms have been generated, together with large-scale expression and proteomic datasets. Comparative analyses of these datasets are beginning to identify the molecular innovations supporting both conserved processes mediating fundamental roles in parasite survival and persistence, as well as lineage-specific adaptations associated with divergent life-cycle strategies. The challenge is how best to exploit these data to derive insights into parasite virulence and identify those genes representing the most amenable targets. In this review, we outline genomic datasets currently available for apicomplexans and discuss biological insights that have emerged as a consequence of their analysis. Of particular interest are systems-based resources, focusing on areas of metabolism and host invasion that are opening up opportunities for discovering new therapeutic targets.     

Falcipain cysteine proteases of malaria parasites: An update

BackgroundThe malaria parasite Plasmodium falciparum expresses four related papain-family cysteine proteases known as falcipains. These proteases play critical roles in the parasite life cycle, and as such are potential targets for new modes of antimalarial chemotherapy, as discussed in this review.Scope of reviewThis review summarizes available knowledge describing falcipain cysteine proteases of malaria parasites.Major conclusionsBased on available data the falcipains can be broken into two sub-families, the falcipain-1 and the falcipain-2/3 sub-families. Falcipain-1 has been difficult to study; it appears to play its most important roles in nonerythrocytic parasites, but not the erythrocytic stage responsible for human disease. Falcipain-2 and falcipain-3 have similar biochemical features, and are expressed sequentially during the erythrocytic cycle. Inhibition of either of these enzymes blocks hemoglobin hydrolysis and completion of the parasite developmental cycle. Knockout of falcipain-2 blocks hemoglobin hydrolysis, but parasites recover, presumably due to subsequent expression of falcipain-3. Knockout of falcipain-3 has not been possible, suggesting that the protease is essential for erythrocytic parasites. Determination of structures of falcipains and extensive chemistry efforts have facilitated identification of numerous small molecule falcipain inhibitors as potential new antimalarial agents. Other malaria parasites express close homologs of falcipain-1 and falcipain-2/3 proteases, suggesting that agents that target the falcipains will also be active against other human malaria parasites.General Significance. Falcipain-2 and falcipain-3 play vital roles during the erythrocytic stage of infection with P. falciparum and thus are promising targets for new agents to treat malaria.     

P25 and P28 proteins of the malaria ookinete surface have multiple and partially redundant functions

The ookinete surface proteins (P25 and P28) are proven antimalarial transmission-blocking vaccine targets, yet their biological functions are unknown. By using single (Sko) and double gene knock-out (Dko) Plasmodium berghei parasites, we show that P25 and P28 share multiple functions during ookinete/oocyst development. In the midgut of mosquitoes, the formation of ookinetes lacking both proteins (Dko parasites) is significantly inhibited due to decreased protection against lethal factors, including protease attack. In addition, Dko ookinetes have a much reduced capacity to traverse the midgut epithelium and to transform into the oocyst stage. P25 and P28 are partially redundant in these functions, since the efficiency of ookinete/oocyst development is only mildly compromised in parasites lacking either P25 or P28 (Sko parasites) compared with that of Dko parasites. The fact that Sko parasites are efficiently transmitted by the mosquito is a compelling reason for including both target antigens in transmission-blocking vaccines.     

Calcineurin is required for Leishmania major stress response pathways and for virulence in the mammalian host

Leishmania parasites must adapt to elevated temperatures and other environmental stresses during infection of their mammalian hosts. How these environmental cues are sensed is poorly understood. In this study we show that calcium uptake is required for parasite thermotolerance at 34-37°C. To identify potential downstream targets of calcium influx, a Leishmania major mutant lacking the essential regulatory subunit (CnB) of the Ca(2+) /calmodulin-dependent serine/threonine-specific phosphatase, calcineurin, was generated. The Δcnb mutant grew as well as wild-type parasites at 27°C and differentiated normally to infective metacyclic promastigotes. However, Δcnb parasites lost viability when exposed to increased temperature (34°C) and were hypersensitive to endoplasmic reticulum and membrane stress, induced by tunicamycin and inhibitors of sterol and sphingolipid biosynthesis respectively. Δcnb promastigotes were internalized by macrophages, but their differentiation to the heat adapted amastigote stage was delayed and the resulting parasites failed to proliferate. Strikingly, the Δcnb parasites were completely cleared by susceptible BALB/c mice. Complementation of Δcnb parasites with CnB restored thermotolerance and infectivity in both macrophages and animal models. Our results suggest that Ca(2+) influx and calcineurin signalling are required for both early and long-term adaptive parasite responses to environmental stresses encountered in the mammalian host.     

Schistosome glysoconjugates

Schistosomes are trematodes known as blood flukes that cause schistosomiasis in people and animals. The male and female worms reside mainly in intestinal veins where they lay eggs that result in a wide-ranging pathology in infected individuals. A growing body of evidence indicates that carbohydrates on glycoproteins, glycolipids and glycosaminoglycans synthesized by the parasite are targets of humoral immunity and may play a role in modulating host immune responses. Carbohydrate antigens may provide protective immunity against infection. In addition, recent evidence indicates that glycoconjugates and carbohydrate-binding proteins from the parasites and their hosts participate in egg adhesion and granuloma formation involved in disease pathology. This review will highlight our current knowledge of the glycoconjugates synthesized by the parasites and their immunological and biological properties. There is increasing anticipation in the field that information about the glycobiology of these parasites may lead to carbohydrate-based vaccines and diagnostics for the disease and perhaps new therapies for treating infected individuals.     

Mitochondria in malaria and related parasites: ancient,diverse and streamlined

Parasitic organisms have emerged from nearly every corner of the eukaryotic kingdom and hence display tremendous diversity of form and function. This diversity extends to their mitochondria and mitochondrion-derived organelles. While the principles of the chemiosmotic theory apply to all these pathogens, the differences from their hosts provide opportunities for therapeutic development. In this review we discuss examples of mitochondrial systems from a deep-branching phylum, Apicomplexa. Many important human pathogens, such as malaria parasites, belong to this phylum. Unique features of their mitochondria are validated targets for drugs that are selectively toxic to the parasites.     

The diversity and evolution of thioredoxin reductase: new perspectives

The thioredoxin system is a major line of cellular defence against oxygen damage. Two distinct thioredoxin reductases found in eukaryotes have different catalytic mechanisms and a mutually exclusive distribution reflecting a complex evolutionary history. Most eukaryotes, including several important parasites, contain a low molecular weight thioredoxin reductase, apparently of bacterial origin. By contrast, animals and apicomplexan protozoa, including Plasmodium, appear to have lost this enzyme. Instead, they contain a high molecular weight thioredoxin reductase, which shares common ancestry with glutathione reductase. This article reviews these fundamental differences between the thioredoxin reductases of some parasites and their hosts, discusses their phylogenetic relationships and considers the potential of the enzymes as therapeutic targets.     

Toxoplasma gondii: microneme protein MIC2

The phylum Apicomplexa contains parasites responsible for a variety of diseases including malaria, cryptosporidiosis, and toxoplasmosis. One of the common features of these parasites is that they contain a set of apical organelles whose sequential secretion is required for the invasion of host cells. Microneme proteins are the main adhesins involved in the attachment to the host cell surface by apicomplexans. The microneme protein MIC2, produced by Toxoplasma gondii, is conserved in apicomplexans and serves as a model to understand the first steps of invasion by the phylum. New data about the structure-function relationship of MIC2 reinforce the critical role of this protein in the successful invasion of cells by Toxoplasma and reveal potential therapeutic targets that may be used to control toxoplasmosis.     

Oxidative stress and antioxidant defenses: a target for the treatment of diseases caused by parasitic protozoa

Parasitic protozoa cause several diseases, affecting hundreds of millions, particularly in underdeveloped countries. Although these organisms are eukaryotic cells, some of them present major differences with their mammalian host in selected metabolic pathways. These differences may be exploited as targets for developing better pharmacological agents for the treatment of specific parasitic diseases. This review describes some of the differences in terms of antioxidant defenses between these organisms and their mammalian host, which may provide useful targets for the treatment of these diseases. Some of the potential targets are: (i). iron metabolism in Plasmodium, (ii). the presence of a Fe-containing form of superoxide dismutase in trypanosomatids and malaria-causing parasites, (iii). the unique trypanothione-dependent antioxidant metabolism in trypanosomatids, (iv). the ascorbate peroxidase found in Trypanosoma cruzi and perhaps present in other trypanosomatids.     

Volutin granules of Eimeria parasites are acidic compartments and have physiological and structural characteristics similar to acidocalcisomes

The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles, and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites.