Gametogenesis in malaria parasites is mediated by the cGMP-dependent protein kinase.

Malaria parasite transmission requires differentiation of male and female gametocytes into gametes within a mosquito following a blood meal. A mosquito-derived molecule, xanthurenic acid (XA), can trigger gametogenesis, but the signalling events controlling this process in the human malaria parasite Plasmodium falciparum remain unknown. A role for cGMP was revealed by our observation that zaprinast (an inhibitor of phosphodiesterases that hydrolyse cGMP) stimulates gametogenesis in the absence of XA. Using cGMP-dependent protein kinase (PKG) inhibitors in conjunction with transgenic parasites expressing an inhibitor-insensitive mutant PKG enzyme, we demonstrate that PKG is essential for XA- and zaprinast-induced gametogenesis. Furthermore, we show that intracellular calcium (Ca2+) is required for differentiation and acts downstream of or in parallel with PKG activation. This work defines a key role for PKG in gametogenesis, elucidates the hierarchy of signalling events governing this process in P. falciparum, and demonstrates the feasibility of selective inhibition of a crucial regulator of the malaria parasite life cycle.

The malaria parasite cyclic GMP-dependent protein kinase plays a central role in blood-stage schizogony.

A role for the Plasmodium falciparum cyclic GMP (cGMP)-dependent protein kinase (PfPKG) in gametogenesis in the malaria parasite was elucidated previously. In the present study we examined the role of PfPKG in the asexual blood-stage of the parasite life cycle, the stage that causes malaria pathology. A specific PKG inhibitor (compound 1, a trisubstituted pyrrole) prevented the progression of P. falciparum schizonts through to ring stages in erythrocyte invasion assays. Addition of compound 1 to ring-stage parasites allowed normal development up to 30 h postinvasion, and segmented schizonts were able to form. However, synchronized schizonts treated with compound 1 for > or =6 h became large and dysmorphic and were unable to rupture or liberate merozoites. To conclusively demonstrate that the effect of compound 1 on schizogony was due to its selective action on PfPKG, we utilized genetically manipulated P. falciparum parasites expressing a compound 1-insensitive PfPKG. The mutant parasites were able to complete schizogony in the presence of compound 1 but not in the presence of the broad-spectrum protein kinase inhibitor staurosporine. This shows that PfPKG is the primary target of compound 1 during schizogony and provides direct evidence of a role for PfPKG in this process. Discovery of essential roles for the P. falciparum PKG in both asexual and sexual development demonstrates that cGMP signaling is a key regulator of both of these crucial life cycle phases and defines this molecule as an exciting potential drug target for both therapeutic and transmission blocking action against malaria.

Disruption of a Plasmodium falciparum cyclic nucleotide phosphodiesterase gene causes aberrant gametogenesis.

Phosphodiesterase (PDE) and guanylyl cyclase (GC) enzymes are key components of the cGMP signalling pathway and are encoded in the genome of Plasmodium falciparum. Here we investigate the role of specific GC and PDE isoforms in gamete formation--a process that is essential for malaria transmission and occurs in the Anopheles mosquito midgut following feeding on an infected individual. Details of the intracellular signalling events controlling development of the male and female gametes from their precursors (gametocytes) remain sparse in P. falciparum. Previous work involving the addition of pharmacological agents to gametocytes implicated cGMP in exflagellation--the emergence of highly motile, flagellated male gametes from the host red blood cell. In this study we show that decreased GC activity in parasites having undergone disruption of the PfGCbeta gene had no significant effect on gametogenesis. By contrast, decreased cGMP-PDE activity during gametocyte development owing to disruption of the PfPDEdelta gene, led to a severely reduced ability to undergo gametogenesis. This suggests that the concentration of cGMP must be maintained below a threshold in the developing gametocyte to allow subsequent differentiation to proceed normally. The data indicate that PfPDEdelta plays a crucial role in regulating cGMP levels during sexual development.

Improved synchronous production of Plasmodium falciparum gametocytes in vitro.

The sexual stages of the Plasmodium falciparum life cycle are attractive targets for vaccines and transmission blocking drugs. Difficulties in culturing and obtaining large amounts of sexual stage P. falciparum parasites, particularly early stages, have often limited research progress in this area. We present a new protocol which simplifies the process of stimulating gametocytogenesis leading to improved synchronous gametocyte production. This new method can be adapted to enrich for early stage gametocytes (I and II) with a higher degree of purity than has previously been achieved, using MACS magnetic affinity columns. The protocol described lends itself to large scale culturing and harvesting of synchronous parasites suitable for biochemical assays, northern blots, flow cytometry, microarrays and proteomic analysis.

Vaccination for vivax malaria: targeting the invaders.

Among the surface-exposed antigens of the malaria parasite, those with known essential functions that can be disrupted by antibodies represent the most promising candidates for development as malaria vaccines. Two recombinant protein subunits of the Plasmodium vivax merozoite surface protein 1 have been shown to bind to reticulocytes in enzyme-linked immunosorbent assays. This article discusses the importance of such pre-clinical analyses in the validation of candidate vaccine molecules for P. vivax, given the constraints imposed by the use of primate models and the cost of producing suitable material for human trials.

RNA interference (RNAi) inhibits growth of Plasmodium falciparum.

RNA interference (RNAi) causes degradation of targeted endogenous RNA in many diverse organisms. Erythrocyte-infecting stages of the malaria parasite Plasmodium falciparum were treated with double-stranded RNA (dsRNA) encoding a segment of the gene encoding dihydroorotate dehydrogenase (DHODH). DHODH is an enzyme in pyrimidine biosynthesis, essential for parasite growth. A decrease in parasite growth (P<0.0005) correlated with a decrease in levels of DHODH mRNA. Control treatments with single-stranded RNA, dsRNA encoding the circumsporozoite protein (a stage-specific protein not expressed in the asexual blood stage) and dsRNA encoding a gene from the related organism Toxoplasma gondii did not inhibit growth. As a test for the RNAi assay, parasites were treated with dsRNA encoding chorismate synthase (CS), an enzyme thought to be involved in folate synthesis, to examine the requirement for this enzyme for parasite growth. Growth decreased (P<0.001) though less markedly than by dsRNA encoding DHODH. These results demonstrate the utility of this assay in assessing requirements for gene products, and their potential as chemotherapeutic targets.

Genetic complexity of Plasmodium falciparum gametocytes isolated from the peripheral blood of treated Gambian children.

The genetic complexity of Plasmodium falciparum gametocytes isolated from Gambian children participating in a controlled trial of anti-malarial therapy was investigated. RNA and DNA were prepared from gametocyte-positive blood, which was also used in transmission experiments with Anopheles gambiae mosquitoes. Amplification by a reverse transcriptase-polymerase chain reaction (RT-PCR) of transcripts from the genes for the ring-infected erythrocyte surface antigen and the 16-kD antigen, which exhibit asexual and sexual stage-specific expression, was used to identify 30 post-treatment gametocyte isolates in which trophozoites persisted below the threshold of detection by microscopy. These included isolates from children who received sulfadoxine/pyrimethamine plus artesunate. Twenty-nine gametocyte-positive isolates that were free of subpatent trophozoites were examined further by PCR amplification of polymorphic genomic loci. We estimate that an average minimum of 2.3 genotypes occurred in these gametocyte-only isolates, and many of these were shown to be infective to mosquitoes. Thus, meiotic recombination between different genotypes is predicted to be a common event in this study area.

Evidence on the chromosomal location of centromeric DNA in Plasmodium falciparum from etoposide-mediated topoisomerase-II cleavage.

Centromeres are the chromosomal loci that facilitate segregation, and, in most eukaryotes, they encompass extensive regions of genomic DNA. Topoisomerase-II has been identified as a crucial regulator of segregation in a wide range of organisms and exhibits premitotic accumulation at centromeres. Consistent with this property, treatment of cells with the topoisomerase-II inhibitor etoposide promotes chromosomal cleavage at sites within centromeric DNA. In the case of the human malaria parasite Plasmodium falciparum, despite a completed genome sequence, there are no experimental data on the nature of centromeres. To address this issue, we have used etoposide-mediated topoisomerase-II cleavage as a biochemical marker to map centromeric DNA on all 14 parasite chromosomes. We find that topoisomerase-II activity is concentrated at single chromosomal loci and that cleavage sites extend over approximately 10 kb. A shared feature of these topoisomerase-II cleavage sites is the presence of an extremely AT-rich ( approximately 97%) domain with a strictly defined size limit of 2.3-2.5 kb. Repetitive arrays identified within the domains do not display interchromosomal conservation in terms of length, copy number, or sequence. These unusual properties suggest that P. falciparum chromosomes contain a class of "regional" centromere distinct from those described in other eukaryotes, including the human host.

Programmed transcription of the var gene family, but not of stevor, in Plasmodium falciparum gametocytes.

The var genes encode Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins, a set of highly diverse surface-expressed proteins that mediate adhesion of erythrocytes infected with asexual blood-stage parasites to host endothelium. Switching among expressed PfEMP1 variants in the course of a blood-stage infection is a key component of antigenic variation, and thus immune evasion, by the parasite. The majority of var loci are found in the subtelomeric regions of P. falciparum chromosomes associated with members of other multigene families, including stevor. Both PfEMP1 and STEVOR are expressed in gametocytes, the transmissible parasite stage, but the role of these proteins in the biology of sexual-stage parasites remains unknown. PfEMP1 may continue to mediate antigenic variation in gametocytes, which need to persist in the host for many days before reaching maturity. Using quantitative reverse transcription-PCR and Northern hybridization, we demonstrate that transcription of a defined subset of type C var loci occurs during gametocyte development in vitro. This transcriptional program occurs in gametocytes regardless of the var expression phenotype of their asexual progenitors and therefore is subject to regulatory processes distinct from those that manage antigenic variation in the asexual parasite. In contrast, the same stevor variants are transcribed in both gametocytes and their asexual progenitors. We also provide evidence that for both asexual parasites and gametocytes, var and stevor transcription patterns are not linked to each other.

Plasmodium falciparum: interaction of shikimate analogues with antimalarial drugs.

The shikimate pathway for aromatic biosynthesis presents a target for antimalarial drug development as this pathway is absent from animals. This study extends previous work on inhibitors of the shikimate pathway, by examining their interaction with the antimalarial drugs pyrimethamine and atovaquone. Combinations of atovaquone with several shikimate analogues exhibited synergistic effects. These findings highlight potential use of shikimate pathway inhibitors in combination therapy.

Distinct trafficking and localization of STEVOR proteins in three stages of the Plasmodium falciparum life cycle.

The genome of Plasmodium falciparum harbors three extensive multigene families, var, rif, and stevor (for subtelomeric variable open reading frame), located mainly in the subtelomeric regions of the parasite's 14 chromosomes. STEVOR variants are known to be expressed in asexual parasites, but no function has as yet been ascribed to this protein family. We have examined the expression of STEVOR proteins in intraerythrocytic sexual stages, gametocytes, and extracellular sporozoites isolated from infected Anopheles mosquitoes. In gametocytes, stevor transcripts appear transiently early in development but STEVOR proteins persist for several days and are transported out of the parasite, travel through the host cell cytoplasm, and localize to the erythrocyte plasma membrane. In contrast to asexual parasites, gametocytes move STEVOR to the periphery via a trafficking pathway independent of Maurer's clefts. In sporozoites, STEVOR appear dispersed throughout the cytoplasm in vesicle-like structures. The pattern of STEVOR localization we have observed in gametocytes and sporozoites differs significantly from that in asexual parasite stages. STEVOR variants are therefore likely to perform different functions in each stage of the parasites life cycle in which they occur.