Protein glycosylation in the malaria parasite.

The nature and extent of glycosylation in Plasmodium falciparum has long been controversial. It has been widely believed that O-glycosylation is the major carbohydrate modification in the intraerythrocytic stage of P. falciparum and that the parasite has no N-glycosylation capacity. Contrary to this, recent studies have demonstrated that P. falciparum has a low N-glycosylation capability, and O-glycosylation is either absent or present at an extremely low level, whereas glycosylphosphatidylinositol (GPI) anchor modification is common and is the major carbohydrate modification in parasite proteins. The GPI anchor moieties are essential for parasite survival. The parasite GPI anchors can activate signaling pathways in host cells, and thereby induce the expression of inflammatory cytokines, adhesion molecules and induced nitric oxide synthase (iNOS). This might cause erythrocyte sequestration, hypoglycemia, triglyceride lipogenesis and immune dysregulation. Thus, the parasite GPI anchor structure and biosynthetic pathways are attractive targets for antimalarial and/or antiparasite drug development, as discussed here by Channe Gowda and Eugene Davidson.     

Mining the Plasmodium genome database to define organellar function: what does the apicoplast do?

Apicomplexan species constitute a diverse group of parasitic protozoa, which are responsible for a wide range of diseases in many organisms. Despite differences in the diseases they cause, these parasites share an underlying biology, from the genetic controls used to differentiate through the complex parasite life cycle, to the basic biochemical pathways employed for intracellular survival, to the distinctive cell biology necessary for host cell attachment and invasion. Different parasites lend themselves to the study of different aspects of parasite biology: Eimeria for biochemical studies, Toxoplasma for molecular genetic and cell biological investigation, etc. The Plasmodium falciparum Genome Project contributes the first large-scale genomic sequence for an apicomplexan parasite. The Plasmodium Genome Database (http://PlasmoDB.org) has been designed to permit individual investigators to ask their own questions, even prior to formal release of the reference P. falciparum genome sequence. As a case in point, PlasmoDB has been exploited to identify metabolic pathways associated with the apicomplexan plastid, or 'apicoplast' - an essential organelle derived by secondary endosymbiosis of an alga, and retention of the algal plastid.     

Adenylyl and guanylyl cyclases from the malaria parasite Plasmodium falciparum

Completion of several malaria parasite genome sequences and advances in Plasmodium gene manipulation technology, will lead to significant advances in our knowledge of the biology of these organisms. Biochemical analysis of the cyclic nucleotide signalling pathways of P. falciparum has provided important information on malaria parasite development. The Plasmodium purine nucleotide cyclase enzymes have extremely unusual structures and the regulatory mechanisms controlling parasite enzyme activity are distinct from those operating on the analogous host molecules. Study of these enzymes could therefore lead to novel strategies for anti-malarial intervention in addition to providing unique insights into the intriguing biology of the parasite.     

High resolution 3D perspective of Plasmodium biology: advancing into a new era

Apicomplexan parasites exhibit a great variety of complex life cycles that require adaptation to different niches of parasitism. They invade different host cells and highjack their biological functions. Plasmodium falciparum, responsible for the deadliest form of human malaria, causes disease while completely remodeling the erythrocytes of its human host through mechanisms that are only partly understood. Recent developments in ultrastructural technologies offer new opportunities to investigate fundamental aspects in the biology of the parasite in a three-dimensional (3D) perspective. Here we bring together recent work on host cell invasion, hemoglobin uptake, protein export and nuclear dynamics. A comprehensive 3D view of the ultrastructural biology of the parasite may shed new light on cellular mechanisms that underlie the pathogenicity of P. falciparum.     

Detection of homologous oncogene sequences in the genome of Plasmodium falciparum

Homologous sequences of the acute RNA tumor virus oncogenes have been found to be highly conserved within vertebrates, insects and yeasts. In the present work, seven different oncogene DNA sequences have been used as probes to search for homologous sequences in the DNA of the protozoan Plasmodium falciparum. Both the v-fms v-Ha ras probes hybridized P. falciparum DNA. The oncogene study will allow an understanding of the biology of the parasite and particularly the host-parasite relationships which allow P. falciparum to develop, keeping the established harmony between the parasite and his host.     

Frequent recombination events generate diversity within the multi-copy variant antigen gene families of Plasmodium falciparum

The human malaria parasite Plasmodium falciparum utilises a mechanism of antigenic variation to avoid the antibody response of its human host and thereby generates a long-term, persistent infection. This process predominantly results from systematic changes in expression of the primary erythrocyte surface antigen, a parasite-produced protein called PfEMP1 that is encoded by a repertoire of over 60 var genes in the P. falciparum genome. var genes exhibit extensive sequence diversity, both within a single parasite's genome as well as between different parasite isolates, and thus provide a large repertoire of antigenic determinants to be alternately displayed over the course of an infection. Whilst significant work has recently been published documenting the extreme level of diversity displayed by var genes found in natural parasite populations, little work has been done regarding the mechanisms that lead to sequence diversification and heterogeneity within var genes. In the course of producing transgenic lines from the original NF54 parasite isolate, we cloned and characterised a parasite line, termed E5, which is closely related to but distinct from 3D7, the parasite used for the P. falciparum genome nucleotide sequencing project. Analysis of the E5 var gene repertoire, as well as that of the surrounding rif and stevor multi-copy gene families, identified examples of frequent recombination events within these gene families, including an example of a duplicative transposition which indicates that recombination events play a significant role in the generation of diversity within the antigen encoding genes of P. falciparum.     

Functional genomics of Plasmodium falciparum through transposon-mediated mutagenesis

Plasmodium falciparum is the causative agent for the most lethal form of human malaria, killing millions annually. Genetic analyses of P. falciparum have been relatively limited due to the lack of robust techniques to manipulate this parasite. Development of transfection technologies and whole genome analyses have helped in understanding the complex biology of this parasite. Even with this wealth of information functional genomics approaches are still very limited in P. falciparum due to the cumbersome and inefficient methods of genetic manipulation. This review focuses on a recently developed, highly efficient method for transposon-based mutagenesis and transgene expression in P. falciparum that will allow functional genomics studies to be performed proficiently on this deadly malaria parasite. By using a piggyBac-based transposition system, multiple random integrations have been obtained into the genome of the parasite. This technique could hence be employed to set up several biological screens in this lethal protozoan parasite that may lead to identification of novel drug targets and vaccine candidates.     

Illuminating Plasmodium falciparum-infected red blood cells

The malaria parasite undergoes a remarkable series of morphological transformations, which underpin its life in both human and mosquito hosts. The advent of molecular transfection technology coupled with the ability to introduce fluorescent reporter proteins that faithfully track and expose the activities of parasite proteins has revolutionized our view of parasite cell biology. The greatest insights have been realized in the erythrocyte stages of Plasmodium falciparum. P. falciparum invades and remodels the human erythrocyte: it feeds on haemoglobin, grows and divides, and subverts the physiology of its hapless host. Fluorescent proteins have been employed to track and dissect each of these processes and have revealed details and exposed new paradigms.     

Transfection of the human malaria parasite Plasmodium falciparum.

In the past few years, methods have been developed which allow the introduction of exogenous DNA into the human malaria parasite Plasmodium falciparum. This important technical advance known as parasite transfection, provides powerful new tools to study the function of Plasmodium proteins and their roles in biology and disease. Already it has allowed the analysis of promoter function and has been successfully applied to establish the role of particular molecules and/or mutations in the biology of this parasite. This review summarises the current state of the technology and how it has been applied to dissect the function of the P. falciparum genome.     

Apparent lack of N-glycosylation in the asexual intraerythrocytic stage of Plasmodium falciparum.

This study investigates protein glycosylation in the asexual intraerythrocytic stage of the malaria parasite, Plasmodium falciparum, and the presence in the infected erythrocyte of the respective precursors. In in vitro cultures, P. falciparum can be metabolically labeled with radioactive sugars, and its multiplication can be affected by glycosylation inhibitors, suggesting the capability of the parasite to perform protein-glycosylation reactions. Gel-filtration analysis of sugar-labeled malarial proteins before and after specific cleavage of N-glycans or O-glycans, respectively, revealed the majority of the protein-bound sugar label to be incorporated into O-glycans, but only little (7-12% of the glucosamine label) or no N-glycans were found. Analysis of the nucleotide sugar and sugar-phosphate fraction showed that radioactive galactose, glucosamine, fucose and ethanolamine were converted to their activated derivatives required for incorporation into protein. Mannose was mainly recovered as a bisphosphate, whereas the level of radiolabeled GDP-mannose was below the detection limit. The analysis of organic-solvent extracts of sugar-labeled cultures showed no evidence for the formation by the parasite of dolichol cycle intermediates, the dedicated precursors in protein N-glycosylation. Consistently, the amount of UDP-N-acetylglucosamine formed did not seem to be affected by the presence of tunicamycin in the culture. Oligosaccharyl-transferase activity was not detectable in a lysate of P. falciparum, using exogenous glycosyl donors and acceptors. Our studies show that O-glycosylation is the major form of protein glycosylation in intraerythrocytic P. falciparum, whereas there is little or no protein N-glycosylation. A part of these studies has been published in abstract form [Dieckmann-Schuppert, A., Hensel, J. and Schwarz, R. T. (1991) Biol. Chem. Hoppe-Seyler 372, 645].     

New approaches to the serotypic analysis of the epidemiology of Plasmodium falciparum

Considerable antigenic heterogeneity of Plasmodium falciparum has been demonstrated in natural parasite populations. However, very little is known about the relative virulence, transmission efficiency and prevalence over space and time of parasites expressing different serotypes of variant antigens. The recent application of recombinant DNA techniques to express a wide range of P. falciparum antigens in Escherichia coli has led to a better understanding of the molecular basis of antigenic diversity of a number of parasite proteins including the precursor to the major merozoite surface antigen (PMMSA) and the heat-stable S-antigens. Highly specific reagents such as DNA probes, monoclonal antibodies and polyclonal antisera to either cloned antigens or synthetic peptides have become available for serotypic analysis of natural parasite populations. With these reagents important epidemiological questions can now be asked concerning the population biology of different serotypes of P. falciparum. The use of the polymorphic S-antigen system as a serotypic marker to analyse the transmission dynamics of P. falciparum in Madang, Papua New Guinea (PNG) is discussed. Results of serotyping studies with the S-antigen system highlight the complexities of malaria transmission, which require consideration in the design of malaria vaccine trials.     

Protein targeting to destinations of the secretory pathway in the malaria parasite Plasmodium falciparum

The secretory pathway in the malaria parasite Plasmodium falciparum has many unique aspects in terms of protein destinations and trafficking mechanisms. Recently, several exciting insights into protein trafficking within this intracellular parasite have been unveiled: these include signals that are required for targeting of proteins to the red blood cell and the relict plastid (known as the apicoplast); and the elucidation of the pathways of the haemoglobin proteases targeted to the food vacuole. Protein-targeting to the apical organelles in P. falciparum, however, is still not very well understood, but available research offers a tantalising glimpse of the system.     

Plasmodium falciparum infection and exoerythrocytic development in mice with chimeric human livers

The exoerythrocytic stage of Plasmodium falciparum has remained a difficult phase of the parasite life-cycle to study. The host and tissue specificity of the parasite requires the experimental infection of humans or non-human primates and subsequent surgical recovery of parasite-infected liver tissue to analyze this stage of the parasites development. This type of study is impossible in humans due to obvious ethical considerations and the cost and complexity in working with primate models has precluded their use for extensive studies of the exoerythrocytic stage. In this study we assessed, for the first time, the use of transgenic, chimeric mice containing functioning human hepatocytes as an alternative for modeling the in vivo interaction of P. falciparum parasites and human hepatocytes. Infection of these mice with P. falciparum sporozoites produced morphologically and antigenically mature liver stage schizonts containing merozoites capable of invading human red blood cells. Additionally, using microdissection, highly enriched P. falciparum liver stage parasites essentially free of hepatocyte contamination, were recovered for molecular studies. Our results establish a stable murine model for P. falciparum that will have a wide utility for assessing the biology of the parasite, potential anti-malarial chemotherapeutic agents and vaccine design.     

二氢乳清酸脱氢酶靶向抗疟药研究进展

疟疾是全球危害最严重的传染性疾病之一,尤其是在非洲,发病率与死亡率仍居高不下。抗药性的出现和发展使大多数现有抗疟药在临床上失去了效用,研究和开发新型抗疟药已成为当前疟疾防治研究的迫切需求。随着恶性疟原虫基因组测序的完成和对疟原虫生物学认知的不断深入,寻找抗疟新靶点的研究得以快速发展。嘧啶生物合成途径是经临床确证有效的抗疟靶点的典范。我们简要综述了近年来以恶性疟原虫嘧啶从头合成途径第四步关键酶——二氢乳清酸脱氢酶（DHODH）为靶点的抗疟新药研究。高通量筛选、药物化学等研究已获得若干对恶性疟原虫DHODH有选择性抑制作用的化合物结构,其中有些在恶性疟原虫体外培养试验中表现出了较强的抗疟作用,且其酶抑制活性与抗疟活性间具有良好的相关性。通过三唑并嘧啶类系列先导化合物的优化研究,已获得了具有良好代谢稳定性、对鼠疟模型有效的类似物。已有大量研究表明DHODH靶向抗疟药的研发具有广阔前景。     

Assessing the impact of Plasmodium falciparum genome sequencing

With the publication of the complete sequences for chromosomes 2 and 3 and the increasing availability of shotgun sequence covering most of its genome, Plasmodium falciparum biology is entering its post-genomic era. Analysis of the results generated to date has identified higher-order organisation of gene families involved in parasite pathology, provided information regarding the unique biology of this parasite and allowed the identification of potential chemotherapeutic drug targets. Continuing efforts to complete the P. falciparum genome and the availability of sequences from other protozoan parasites will facilitate a broader understanding of their biology, particularly with respect to their pathogenicity.