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.     

Functional analysis of proteins involved in Plasmodium falciparum merozoite invasion of red blood cells

Plasmodium falciparum causes the most lethal form of malaria in humans and is responsible for over two million deaths per year. The development of a vaccine against this parasite is an urgent priority and potential protein targets include those on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to transfect P. falciparum has enabled the construction of gain-of-function and loss-of-function mutants and provided new strategies to analyse the role of parasite proteins. In this review, we describe the use of this technology to examine the role of merozoite antigens in erythrocyte invasion and to address their potential as vaccine candidates.     

Anopheles gambiae genome: perspectives for malaria control

Malaria, a disease that infects 300 million people throughout the world and kills more than a million people, mostly children in sub-Saharan Africa, involves three organisms. The human host where the disease is seen, the protozoan Plasmodium parasite and the mosquito. The parasite is transmitted to humans only by the mosquito vector, which in sub-Saharan regions is generally Anopheles gambiae. Malaria along with AIDS and tuberculosis are killing large numbers of people and crippling the economies of the affected African countries. Though an enormous effort has been made during the past twenty years to develop vaccines to block malaria in humans, the incidence of the disease is increasing in Africa. The reasons for this development include a breakdown in mosquito control related to increased insecticide resistance, as well as increased parasite resistance to antimalarial drugs. It is clear that new methods of Anopheles mosquito control are needed to ameliorate the medical and economic situation in sub-Saharan Africa. As a step toward new malaria control methods, the international Plasmodium falciparum and Anopheles gambiae consortia have carried out the full genome sequencing of the most deadly malaria parasite and the most efficient vector. These, combined with the human genome sequence, provide the genomic infrastructure for a better understanding of the complex interactions within the malaria triad. This essay discusses possible strategies as to how the Anopheles genome can contribute to malaria control.     

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.     

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.     

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.     

Recent Progress in Functional Genomic Research in Plasmodium falciparum

With the completion and near completion of many malaria parasite genome-sequencing projects, efforts are now being directed to a better understanding of gene functions and to the discovery of vaccine and drug targets. Inter- and intraspecies comparisons of the parasite genomes will provide invaluable insights into parasite evolution, virulence, drug resistance, and immune invasion. Genome-wide searches for loci under various selection pressures may lead to discovery of genes conferring drug resistance or encoding for protective antigens. In addition, the Plasmodium falciparum genome sequence provides the basis for the development of various microarrays to monitor gene expression and to detect nucleotide substitution and deletion/amplification. Genome-wide profiling of the parasite proteome, chromatin modification, and nucleosome position also depend on availability of the parasite genome. In this brief review, we will highlight some recent advances and studies in characterizing gene function and related phenotype in P. falciparum that were made possible by the genome sequence, particularly the development of a genome-wide diversity map and various high-throughput genotyping methods for genome-wide association studies (GWAS).     

Genetic mapping in the human malaria parasite Plasmodium falciparum

The Plasmodium falciparum genome sequence has boosted hopes for a new era of malaria research and for the application of comprehensive molecular knowledge to disease control, but formidable obstacles remain: approximately 60% of the predicted P. falciparum proteins have no known functions or homologues, and most life cycle stages of this haploid eukaryotic parasite are relatively intractable to cultivation and biochemical manipulation. Genetic mapping based on high-resolution maps saturated with single-nucleotide polymorphisms or microsatellites is now providing effective strategies for discovering candidate genes determining important parasite phenotypes. Here we review classical linkage studies using laboratory crosses and population associations that are now amenable to genome-wide approaches and are revealing multiple candidate genes involved in complex drug responses. Moreover, mapping by linkage disequilibrium is practicable in cases where chromosomal segments flanking drug-selected genes have been preserved in populations during relatively recent P. falciparum evolution. We discuss the advantages and limitations of these various genetic mapping strategies, results from which offer complementary insights to those emerging from gene knockout experiments and/or high-throughput genomic technologies.     

Interpolated Markov models for eukaryotic gene finding.

Computational gene finding research has emphasized the development of gene finders for bacterial and human DNA. This has left genome projects for some small eukaryotes without a system that addresses their needs. This paper reports on a new system, GlimmerM, that was developed to find genes in the malaria parasite Plasmodium falciparum. Because the gene density in P. falciparum is relatively high, the system design was based on a successful bacterial gene finder, Glimmer. The system was augmented with specially trained modules to find splice sites and was trained on all available data from the P. falciparum genome. Although a precise evaluation of its accuracy is impossible at this time, laboratory tests (using RT-PCR) on a small selection of predicted genes confirmed all of those predictions. With the rapid progress in sequencing the genome of P. falciparum, the availability of this new gene finder will greatly facilitate the annotation process.     

The mosquito genome: perspectives and possibilities

Anopheles gambiae is the mosquito vector responsible for transmitting Plasmodium falciparum, a malaria parasite of humans. With the emergence of genome projects for a variety of prokaryotic and eukaryotic microorganisms, there has been a long-standing interest in sequencing the genomes of the malaria parasite and its insect vector. This tour de force effort has now been completed and reported. The alignment of putative orthologs in An. gambiae with those of Drosophila melanogaster highlights several similarities and differences. These findings could have implications in: (1) identifying new targets for insecticide development; (2) strengthening our understanding of the developmental biology of mosquitoes; and (3) possibly controlling pathogen transmission. A brief overview of these interesting findings and the implications for further studies will be discussed here.     

Microsatellite markers and genetic mapping in Plasmodium falciparum

Whole-genome methods are changing the scope of biological questions that can be addressed in malaria research. In the rich context provided by Plasmodium falciparum genome sequencing, genetic mapping is a powerful tool for identifying genes involved in parasite development, invasion, transmission and drug resistance. The recent development of a high-resolution P. falciparum linkage map consisting of hundreds of microsatellite markers will facilitate an integrated genomic approach to understanding the relationship between genetic variations and biological phenotypes. Here, Michael Ferdig and Xin-zhuan Su provide an overview for applying microsatellite markers and genetic maps to gene mapping, parasite typing and studies of parasite population changes.     

Curation of the Plasmodium falciparum genome

The malaria genome has proved invaluable to researchers worldwide in the continuing fight against malaria by stimulating and underpinning molecular approaches in gene expression studies, vaccine and drug discovery research, and by providing data to facilitate hypothesis-driven research. The combination of in silico and experimental investigations has already yielded dividends by strengthening our understanding of the many facets of the malaria parasite Plasmodium falciparum. The recently initiated curation of the genome resource is a vital investment for maintaining and enhancing the use of this genomic information in the post-genomic era.     

Functional analysis of Plasmodium falciparum merozoite antigens: implications for erythrocyte invasion and vaccine development

Malaria is a major human health problem and is responsible for over 2 million deaths per year. It is caused by a number of species of the genus Plasmodium, and Plasmodium falciparum is the causative agent of the most lethal form. Consequently, the development of a vaccine against this parasite is a priority. There are a number of stages of the parasite life cycle that are being targeted for the development of vaccines. Important candidate antigens include proteins on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to manipulate the genome of Plasmodium species has enabled the construction of gain-of-function and loss-of-function mutants and provided new strategies to analyse the role of parasite proteins. This has provided new information on the role of merozoite antigens in erythrocyte invasion and also allows new approaches to address their potential as vaccine candidates.     

A new single-step PCR assay for the detection of the zoonotic malaria parasite Plasmodium knowlesi

BACKGROUND: Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection. METHODOLOGY AND SIGNIFICANT FINDINGS: We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites. CONCLUSIONS: The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi.     

A systematic map of genetic variation in Plasmodium falciparum

Discovering novel genes involved in immune evasion and drug resistance in the human malaria parasite, Plasmodium falciparum, is of critical importance to global health. Such knowledge may assist in the development of new effective vaccines and in the appropriate use of antimalarial drugs. By performing a full-genome scan of allelic variability in 14 field and laboratory strains of P. falciparum, we comprehensively identified approximately 500 genes evolving at higher than neutral rates. The majority of the most variable genes have paralogs within the P. falciparum genome and may be subject to a different evolutionary clock than those without. The group of 211 variable genes without paralogs contains most known immunogens and a few drug targets, consistent with the idea that the human immune system and drug use is driving parasite evolution. We also reveal gene-amplification events including one surrounding pfmdr1, the P. falciparum multidrug-resistance gene, and a previously uncharacterized amplification centered around the P. falciparum GTP cyclohydrolase gene, the first enzyme in the folate biosynthesis pathway. Although GTP cyclohydrolase is not the known target of any current drugs, downstream members of the pathway are targeted by several widely used antimalarials. We speculate that an amplification of the GTP cyclohydrolase enzyme in the folate biosynthesis pathway may increase flux through this pathway and facilitate parasite resistance to antifolate drugs.     

Plasmodium falciparum gametocytes: still many secrets of a hidden life

Sexual differentiation and parasite transmission are intimately linked in the life cycle of malaria parasites. The specialized cells providing this crucial link are the Plasmodium gametocytes. These are formed in the vertebrate host and are programmed to mature into gametes emerging from the erythrocytes in the midgut of a blood-feeding mosquito. The ensuing fusion into a zygote establishes parasite infection in the insect vector. Although key mechanisms of gametogenesis and fertilization are becoming progressively clear, the fundamental biology of gametocyte formation still presents open questions, some of which are specific to the human malaria parasite Plasmodium falciparum. Developmental commitment to sexual differentiation, regulation of stage-specific gene expression, the profound molecular and cellular changes accompanying gametocyte specialization, the requirement for tissue-specific sequestration in P. falciparum gametocytogenesis are proposed here as areas for future investigation. The epidemiological relevance of parasite transmission from humans to mosquito in the spread of malaria and of Plasmodium drug resistance genes indicates that understanding molecular mechanisms of gametocyte formation is highly relevant to design strategies able to interfere with the transmission of this disease.     

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.