Plasmodium Falciparum Genome Research Articles

A multidomain adhesion protein family expressed in Plasmodium falciparum is essential for transmission to the mosquito.

The recent sequencing of several apicomplexan genomes has provided the opportunity to characterize novel antigens essential for the parasite life cycle that might lead to the development of new diagnostic and therapeutic markers. Here we have screened the Plasmodium falciparum genome sequence for genes encoding extracellular multidomain putative adhesive proteins. Three of these identified genes, named PfCCp1, PfCCp2, and PfCCp3, have multiple adhesive modules including a common Limulus coagulation factor C domain also found in two additional Plasmodium genes. Orthologues were identified in the Cryptosporidium parvum genome sequence, indicating an evolutionary conserved function. Transcript and protein expression analysis shows sexual stage–specific expression of PfCCp1, PfCCp2, and PfCCp3, and cellular localization studies revealed plasma membrane–associated expression in mature gametocytes. During gametogenesis, PfCCps are released and localize surrounding complexes of newly emerged microgametes and macrogametes. PfCCp expression markedly decreased after formation of zygotes. To begin to address PfCCp function, the PfCCp2 and PfCCp3 gene loci were disrupted by homologous recombination, resulting in parasites capable of forming oocyst sporozoites but blocked in the salivary gland transition. Our results describe members of a conserved apicomplexan protein family expressed in sexual stage Plasmodium parasites that may represent candidates for subunits of a transmission-blocking vaccine.

Targeting a DBL3gamma domain of the Plasmodium falciparum erythrocyte membrane protein 1 to the surface of Saccharomyces cerevisiae.

The availability of the full genomes of the malarial parasite Plasmodium falciparum and its two hosts, man and Anopheles gambiae, has dramatically increased the demand for protein display systems to study host/parasite interactions at the molecular level. Here, we explored the potential of a Saccharomyces cerevisiae expression and display system that allows proteins of interest to be targeted to the yeast surface. As proof of this principle, we used a P. falciparum erythrocyte membrane protein 1 DBL3gamma domain which mediates the binding of P. falciparum-infected erythrocytes to chondroitin-4-sulfate, a host receptor involved in parasite sequestration in the placenta. Our data revealed localization of the DBL3gamma domain to the yeast surface, demonstrating the value of the yeast system as a tool for displaying P. falciparum protein fragments. However, binding of the respective yeast strains to chondroitin-4-sulfate could not be demonstrated.

Insect–malaria parasites interactions: the salivary gland

Mosquito salivary glands are organs specialized in the production of a complex mix of molecules that digest carbohydrates from plant nectars, and facilitate blood feeding by the lubrication of mouthparts and the inhibition of homeostasis. Malaria sporozoites invade salivary glands and are injected with the saliva into vertebrate hosts during blood feeding. Sporozoites utilize molecules on their surface coat and outer pellicle membrane to adhere and invade specific regions of the salivary gland lobes. They transverse the secretory cells and are stored in the salivary duct, where transcription of new genes prepares them for vertebrate host invasion. Although it is probably that specific carbohydrate molecules on the surface of salivary glands function as parasites receptors, these have not been identified, neither other molecules nor mechanisms used by the parasite to invade, survive and mature within these organs. The recent advances in the sequence of the genomes of Anopheles gambiae and Plasmodium falciparum, and new developments in genomics and proteomics may help to elucidate the participating molecules, their regulation and interactions.

Insect?malaria parasites interactions: the salivary gland

Mosquito salivary glands are organs specialized in the production of a complex mix of molecules that digest carbohydrates from plant nectars , and facilitate blood feeding by the lubrication of mouthparts and the inhibition of homeostasis . Malaria sporozoites invade salivary glands and are injected with the saliva into vertebrate hosts during blood feeding. Sporozoites utilize molecules on their surface coat and outer pellicle membrane to adhere and invade specific regions of the salivary gland lobes. They transverse the secretory cells and are stored in the salivary duct , where transcription of new genes prepares them for vertebrate host invasion. Although it is probably that specific carbohydrate molecules on the surface of salivary glands function as parasites receptors, these have not been identified, neither other molecules nor mechanisms used by the parasite to invade, survive and mature within these organs. The recent advances in the sequence of the genomes of Anopheles gambiae and Plasmodium falciparum , and new developments in genomics and proteomics may help to elucidate the participating molecules, their regulation and interactions.

Detecting selection using a single genome sequence of M. tuberculosis and P. falciparum.

Selective pressures on proteins are usually measured by comparing nucleotide sequences. Here we introduce a method to detect selection on the basis of a single genome sequence. We catalogue the relative strength of selection on each gene in the entire genomes of Mycobacterium tuberculosis and Plasmodium falciparum. Our analysis confirms that most antigens are under strong selection for amino-acid substitutions, particularly the PE/PPE family of putative surface proteins in M. tuberculosis and the EMP1 family of cytoadhering surface proteins in P. falciparum. We also identify many uncharacterized proteins that are under strong selection in each pathogen. We provide a genome-wide analysis of natural selection acting on different stages of an organism's life cycle: genes expressed in the ring stage of P. falciparum are under stronger positive selection than those expressed in other stages of the parasite's life cycle. Our method of estimating selective pressures requires far fewer data than comparative sequence analysis, and it measures selection across an entire genome; the method can readily be applied to a large range of sequenced organisms.

Malaria and the red blood cell membrane

Malaria is the most serious and widespread parasitic disease of humans and is arguably the commonest disease of red blood cells (RBCs). Malaria has exerted a powerful effect on human evolution and selection for resistance has led to the appearance and persistence of a number of inherited diseases. After parasite invasion, RBCs are progressively and dramatically modified. New structures appear inside the RBC and novel parasite proteins are exported to the erythrocyte cytoplasm and membrane skeleton. Radical biochemical, morphological, and rheological alterations manifest as increased membrane rigidity, reduced cell deformability, and greater adhesiveness for the vascular endothelium and other blood cells. Numerous protein-protein interactions between the malaria-parasite and the host RBC are important for many aspects of parasite biology and the pathogenesis of malaria. In addition, there are many other parasite proteins located within the infected red cell and at the membrane skeleton, for which no precise functional roles have yet been elucidated. Sequencing and annotation of the complete genome of Plasmodium falciparum, the production of proteomic and transcriptomic profiles of parasites, and the development of a transfection system for the asexual stage of the parasite are all recent achievements that should advance understanding of the molecular mechanisms that underlie the parasite-induced functional alterations in red cells.

Enzymes for Heme Biosynthesis are Found in Both the Mitochondrion and Plastid of the Malaria Parasite Plasmodium falciparum

All eight enzymes required for de novo heme biosynthesis have been predicted from the nuclear genome of the human malaria parasite Plasmodium falciparum. We have studied the subcellular localization of three of these using a GFP reporter in live transfected parasites. The first enzyme in the pathway delta-aminolevulinic acid synthase (ALAS) is targeted to the mitochondrion, but the next two enzymes porphobilinogen synthase (PBGS) and hydroxymethylbilane synthase (HMBS) are targeted to the plastid. An enzymatically active recombinant version of PBGS from P. falciparum was over-expressed and its activity found to be stimulated by Mg2+ (and enhanced by Mn2+) but not by Zn2+. A hypothetical scheme for the exchange of intermediates in heme biosynthesis between the mitochondrion and plastid organelle, as well as organelle attachment is discussed.

QTL analysis for discovery of genes involved in drug responses.

The completed Plasmodium falciparum genome sequence poses a significant challenge: how do we bring this wealth of data to bear against the steady march of malaria parasites towards multiple-drug resistance? Studies of parasite drug resistance have until now focused on a qualitative, single-gene concept of resistance determination; however, the emergence of powerful genomics tools insists that these questions be rephrased. It is now possible to address the true genetic complexity underlying quantitative drug sensitivities. Quantitative trait loci (QTL) mapping is an effective tool for tracking multi-gene traits by partitioning genetic effects that influence these traits into specific genomic regions. A cross between two parasite clones captures allele combinations that have segregated into progeny clones that display varying sensitivity to drugs. The specific allele forms and their combinations contributed by each parent are, in effect, genetic signatures of their unique evolutionary histories. In addition to resistance genes, per se, a drug resistant parasite carries co-evolved gene combinations comprising a genetic background of drug resistance. Research into drug resistance necessarily has been directed at specific genes and mechanisms favored by a priori knowledge and assumptions about how resistance works. QTL mapping, by superimposing real biological phenotypes on genome sequence, structural polymorphisms, and gene expression data, can provide an alternative, unbiased view of the network of gene actions that build a complex phenotype. Through an integrated approach, studies can move beyond the search for markers of resistance to instead characterize the predisposition of parasites to develop new resistances and cross-resistances.

Proteomics in malaria.

The recent completion of human, Anopheles gambiae, and Plasmodium falciparum genomes relevant to the study of human malaria allows the application of modern proteomic technologies to complement previously implemented conventional approaches. Proteomic analysis has been employed to elucidate global protein expression profiles, subcellular localization of gene products, and host-pathogen interactions that are central to disease pathogenesis and treatment. The high-throughput nature of these techniques is in accord with the pace of drug and vaccine development that have the potential to directly reduce the morbidity and mortality of disease.

δ-Aminolevulinic Acid Dehydratase from Plasmodium falciparum: INDIGENOUS VERSUS IMPORTED

The heme biosynthetic pathway of the malaria parasite is a drug target and the import of host delta-aminolevulinate dehydratase (ALAD), the second enzyme of the pathway, from the red cell cytoplasm by the intra erythrocytic malaria parasite has been demonstrated earlier in this laboratory. In this study, ALAD encoded by the Plasmodium falciparum genome (PfALAD) has been cloned, the protein overexpressed in Escherichia coli, and then characterized. The mature recombinant enzyme (rPfALAD) is enzymatically active and behaves as an octamer with a subunit Mr of 46,000. The enzyme has an alkaline pH optimum of 8.0 to 9.0. rPfALAD does not require any metal ion for activity, although it is stimulated by 20-30% upon addition of Mg2+. The enzyme is inhibited by Zn2+ and succinylacetone. The presence of PfALAD in P. falciparum can be demonstrated by Western blot analysis and immunoelectron microscopy. The enzyme has been localized to the apicoplast of the malaria parasite. Homology modeling studies reveal that PfALAD is very similar to the enzyme species from Pseudomonas aeruginosa, but manifests features that are unique and different from plant ALADs as well as from those of the bacterium. It is concluded that PfALAD, while resembling plant ALADs in terms of its alkaline pH optimum and apicoplast localization, differs in its Mg2+ independence for catalytic activity or octamer stabilization. Expression levels of PfALAD in P. falciparum, based on Western blot analysis, immunoelectron microscopy, and EDTA-resistant enzyme activity assay reveals that it may account for about 10% of the total ALAD activity in the parasite, the rest being accounted for by the host enzyme imported by the parasite. It is proposed that the role of PfALAD may be confined to heme synthesis in the apicoplast that may not account for the total de novo heme biosynthesis in the parasite.

New Antimalarial Drugs

AbstractFor Abstract see ChemInform Abstract in Full Text.

Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli

BackgroundThe expression of recombinant proteins in Escherichia coli is an important and frequently used tool within malaria research, however, this method remains problematic. High A/T versus C/G content and frequent lysine and arginine repeats in the Plasmodium falciparum genome are thought to be the main reason for early termination in the mRNA translation process. Therefore, the majority of P. falciparum derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells.MethodsSeveral domains of PfEMP1 genes obtained from different P. falciparum strains were expressed in E. coli as GST-fusion proteins. Expression was carried out under various culture conditions with a main focus on the time point of induction in relation to the bacterial growth stage.Results and conclusionsWhen expressed in E. coli recombinant proteins derived from P. falciparum sequences are often truncated and tend to aggregate what in turn leads to the formation of insoluble inclusion bodies. The analysis of various factors influencing the expression revealed that the time point of induction plays a key role in successful expression of A/T rich sequences into their native conformation. Contrary to recommended procedures, initiation of expression at post-log instead of mid-log growth phase generated significantly increased amounts of soluble protein of a high quality. Furthermore, these proteins were shown to be functionally active. Other factors such as temperature, pH, bacterial proteases or the codon optimization for E. coli had little or no effect on the quality of the recombinant protein, nevertheless, optimizing these factors might be beneficial for each individual construct. In conclusion, changing the timepoint of induction and conducting expression at the post-log stage where the bacteria have entered a decelerated growth phase, greatly facilitates and improves the expression of sequences containing rare codons.

Rapid evolution of an erythrocyte invasion gene family: the Plasmodium reichenowi Reticulocyte Binding Like (RBL) genes.

Malarial merozoites use an array of ligands, including members of the Reticulocyte Binding Like (RBL) super-family of invasion proteins, to identify and invade erythrocytes. RBL family members are large Type I membrane anchored proteins expressed at the invasive end of merozoites that share homology with the Reticulocyte Binding Proteins 1 and 2 (PvRBP1 and 2) of Plasmodium vivax. Plasmodium species vary widely both in the number and sequence of their RBL genes, with the recently completed Plasmodium falciparum genome containing five RBL genes. Of these, three encode proteins shown to be involved in erythrocyte invasion, a fourth is a pseudogene, and the role of the fifth is as yet unclear. In order to identify sequence similarities and differences that may have functional implications for erythrocyte invasion as well as to gain insights into the recent evolutionary history of the P. falciparum RBL genes, we have sequenced all five corresponding RBL genes from the chimpanzee parasite Plasmodium reichenowi, which is the closest phylogenetic relative of P. falciparum, yet is unable to invade human erythrocytes. Two of the five P. falciparum RBL genes have highly conserved complete open reading frames in both species, while the other three genes show evidence of gene conversion and rapid evolution. The RBL super-family, therefore, appears to be surprisingly dynamic and divergent, implying that it is involved in species-specific aspects of erythrocyte recognition and invasion.

Annotating the Plasmodium genome and the enigma of the shikimate pathway.

The completion of the Plasmodium falciparum genome sequence heralds a new era in the effort to identify all the parasite's genes along with their cellular functions. A combination of bioinformatics and experimental proof will facilitate this process. Many enzymes in metabolic processes have been identified, but several examples exist of incomplete pathways, such as the shikimate pathway. This review uses the example of the shikimate pathway to examine the application of bioinformatics to lead experimental design in post-genomic biology.

Entering the post-genomic era of malaria research.

The sequencing of the genome of Plasmodium falciparum promises to revolutionize the way in which malaria research will be carried out. Beyond simple gene discovery, the genome sequence will facilitate the comprehensive determination of the parasite's gene expression during its developmental phases, pathology, and in response to environmental variables, such as drug treatment and host genetic background. This article reviews the current status of the P. falciparum genome sequencing project and the unique insights it has generated. We also summarize the application of bioinformatics and analytical tools that have been developed for functional genomics. The aim of these activities is the rational, information-based identification of new therapeutic strategies and targets, based on a thorough insight into the biology of Plasmodium spp.

New antimalarial drugs.

Approximately 40% of the world population live in areas with the risk of malaria. Each year, 300-500 million people suffer from acute malaria, and 0.5-2.5 million die from the disease. Although malaria has been widely eradicated in many parts of the world, the global number of cases continues to rise. The most important reason for this alarming situation is the rapid spread of malaria parasites that are resistant to antimalarial drugs, especially chloroquine, which is by far the most frequently used. The development of new antimalarial drugs has been neglected since the 1970s owing to the end colonialism, changes in the areas of military engagement, and the restricted market potential. Only in recent years, in part supported by public funding programs, has interest in the development of antimalarial drugs been renewed. New data available from the recently sequenced genome of the malaria parasite Plasmodium falciparum and the application of methods of modern drug design promise to bring significant development in the fight against this disease.

Utilization of genomic sequence information to develop malaria vaccines.

Recent advances in the fields of genomics, proteomics and molecular immunology offer tremendous opportunities for the development of novel interventions against public health threats, including malaria. However, there is currently no algorithm that can effectively identify the targets of protective T cell or antibody responses from genomic data. Furthermore, the identification of antigens that will stimulate the most effective immunity against the target pathogen is problematic, particularly if the genome is large. Malaria is an attractive model for the development and validation of approaches to translate genomic information to vaccine development because of the critical need for effective anti-malarial interventions and because the Plasmodium parasite is a complex multistage pathogen targeted by multiple immune responses. Sterile protective immunity can be achieved by immunization with radiation-attenuated sporozoites, and anti-disease immunity can be induced in residents in malaria-endemic areas. However, the 23 Mb Plasmodium falciparum genome encodes more than 5,300 proteins, each of which is a potential target of protective immune responses. The current generation of subunit vaccines is based on a single or few antigens and therefore might elicit too narrow a breadth of response. We are working towards the development of a new generation vaccine based on the presumption that duplicating the protection induced by the whole organism may require a vaccine nearly as complex as the organism itself. Here, we present our strategy to exploit the genomic sequence of P. falciparum for malaria vaccine development.

Malaria in antiquity: a genetics perspective

Historical texts provide credible evidence regarding the occurrence of malaria in recent human history but how far this antiquity stretches back through our hominid ancestors is questionable. Genetic analysis of the Plasmodium falciparum genome sequence has thus far failed to clarify the issue, but available evidence suggests a recent expansion of this Plasmodium species within the last 6,000 years. This coincides with an expansion of both human and mosquito populations brought about by the advent of agriculture. The shift from small groups of hunter-gatherers to larger settled populations was crucial in sustaining P. falciparum transmission and it is noteworthy that a number of malaria-protective polymorphisms also have origins in this timeframe. The antiquity of other Plasmodium species is more uncertain, but preliminary studies of Plasmodium vivax suggest it is considerably older than its more deadly relative.

Malaria transfection and transfection vectors.

Malaria remains a leading cause of death due to infectious disease. The completion of the Plasmodium falciparum genome sequencing project and release of preliminary proteomics data have significantly increased our understanding of the biology of this organism. Nonetheless, additional tools for functional analysis of this massive amount of information are now indispensable to further understand the basic biology of this parasite. The genetic manipulation of specific genes via the use of plasmid constructs and transfection represents one such tool.

The Plasmodium vivax genome sequencing project.

With the successful completion of the project to sequence the Plasmodium falciparum genome, researchers are now turning their attention to other malaria parasite species. Here, an update on the Plasmodium vivax genome sequencing project is presented, as part of the Trends in Parasitology series of reviews expanding on various aspects of P. vivax research.