Simple and sensitive antimalarial drug screening in vitro and in vivo using transgenic luciferase expressing Plasmodium berghei parasites.

We report two improved assays for in vitro and in vivo screening of chemicals with potential anti-malarial activity against the blood stages of the rodent malaria parasite Plasmodiumberghei. These assays are based on the determination of luciferase activity (luminescence) in small blood samples containing transgenic blood stage parasites that express luciferase under the control of a promoter that is either schizont-specific (ama-1) or constitutive (eef1alphaa). Assay 1, the in vitro drug luminescence (ITDL) assay, measured the success of schizont maturation in the presence of candidate drugs quantifying luciferase activity in mature schizonts only (ama-1 promoter). The ITDL assay generated drug-inhibition curves and EC(50) values comparable to those obtained with standard in vitro drug-susceptibility assays. The second assay, the in vivo drug-luminescence (IVDL) assay, measured parasite growth in vivo in a standard 4-day suppressive drug test, monitored by measuring the constitutive luciferase activity of circulating parasites (eef1alphaa promoter). The IVDL assay generates growth-curves that are identical to those obtained by manual counting of parasites in Giemsa-stained smears. The reading of luminescence assays is rapid, requires a minimal number of handling steps and no experience with parasite morphology or handling fluorescence-activated cell sorters, produces no radioactive waste and test-plates can be stored for prolonged periods before processing. Both tests are suitable for use in larger-scale in vitro and in vivo screening of drugs. The standard methodology of anti-malarial drug screening and validation, which includes testing in rodent models of malaria, can be improved by the incorporation of such assays.

The dynamics of interactions between Plasmodium and the mosquito: a study of the infectivity of Plasmodium berghei and Plasmodium gallinaceum, and their transmission by Anopheles stephensi, Anopheles gambiae and Aedes aegypti.

Knowledge of parasite-mosquito interactions is essential to develop strategies that will reduce malaria transmission through the mosquito vector. In this study we investigated the development of two model malaria parasites, Plasmodium berghei and Plasmodium gallinaceum, in three mosquito species Anopheles stephensi, Anopheles gambiae and Aedes aegypti. New methods to study gamete production in vivo in combination with GFP-expressing ookinetes were employed to measure the large losses incurred by the parasites during infection of mosquitoes. All three mosquito species transmitted P. gallinaceum; P. berghei was only transmitted by Anopheles spp. Plasmodium gallinaceum initiates gamete production with high efficiency equally in the three mosquito species. By contrast P. berghei is less efficiently activated to produce gametes, and in Ae. aegypti microgamete formation is almost totally suppressed. In all parasite/vector combinations ookinete development is inefficient, 500-100,000-fold losses were encountered. Losses during ookinete-to-oocyst transformation range from fivefold in compatible vector parasite combinations (P. berghei/An. stephensi), through >100-fold in poor vector/parasite combinations (P. gallinaceum/An. stephensi), to complete blockade (>1,500 fold) in others (P. berghei/Ae. aegypti). Plasmodium berghei ookinetes survive poorly in the bloodmeal of Ae. aegypti and are unable to invade the midgut epithelium. Cultured mature ookinetes of P. berghei injected directly into the mosquito haemocoele produced salivary gland sporozoites in An. stephensi, but not in Ae. aegypti, suggesting that further species-specific incompatibilities occur downstream of the midgut epithelium in Ae. aegypti. These results show that in these parasite-mosquito combinations the susceptibility to malarial infection is regulated at multiple steps during the development of the parasites. Understanding these at the molecular level may contribute to the development of rational strategies to reduce the vector competence of malarial vectors.

Comparative genomics in Plasmodium: a tool for the identification of genes and functional analysis.

Comparative genomics allows inferences to be drawn about the coding potential of related genomes, and the evolutionary forces that have influenced genome organisation. Early comparisons have indicated that there is significant synteny (conserved physical association of genes) between the human parasite Plasmodium falciparum and the malaria parasites of rodents, such as Plasmodium berghei. The various Plasmodium genome initiatives have now provided the opportunity to perform comparative genomics within different species of malaria parasites in more detail, allowing the discovery of orthologues and paralogues of less well conserved genes and addressing questions of conservation, evolution and structure of multi-gene families. A remarkable level of conservation is being revealed, illustrated here by a comparison of members of one of the first conserved gene families to emerge from the sequencing initiatives, the P48/45 gene family. We have identified two additional members in this family, Pf36p and Pfs38, and shown that all members are conserved in P. falciparum and P. berghei, opening the way for functional analyses in the latter more accessible rodent malaria model. In addition, it has been shown that direct comparison of a 13.6 kb contig of a chromosome of P. berghei and the orthologous region in P. falciparum reveals an unexpected high level of conservation of gene organisation and complexity. The results of this comparison highlight the value of a comparative approach to elucidate the gene content of complex loci and improve its annotation

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.

Interspecies conservation of gene order and intron-exon structure in a genomic locus of high gene density and complexity in Plasmodium.

A 13.6 kb contig of chromosome 5 of Plasmodium berghei, a rodent malaria parasite, has been sequenced and analysed for its coding potential. Assembly and comparison of this genomic locus with the orthologous locus on chromosome 10 of the human malaria Plasmodium falciparum revealed an unexpectedly high level of conservation of the gene organisation and complexity, only partially predicted by current gene-finder algorithms. Adjacent putative genes, transcribed from complementary strands, overlap in their untranslated regions, introns and exons, resulting in a tight clustering of both regulatory and coding sequences, which is unprecedented for genome organisation of PLASMODIUM: In total, six putative genes were identified, three of which are transcribed in gametocytes, the precursor cells of gametes. At least in the case of two multiple exon genes, alternative splicing and alternative transcription initiation sites contribute to a flexible use of the dense information content of this locus. The data of the small sample presented here indicate the value of a comparative approach for Plasmodium to elucidate structure, organisation and gene content of complex genomic loci and emphasise the need to integrate biological data of all Plasmodium species into the P.falciparum genome database and associated projects such as PlasmodB to further improve their annotation.

Functional equivalence of structurally distinct ribosomes in the malaria parasite, Plasmodium berghei.

Unlike most eukaryotes, many apicomplexan parasites contain only a few unlinked copies of ribosomal RNA (rRNA) genes. Based on stage-specific expression of these genes and structural differences among the rRNA molecules it has been suggested that Plasmodium spp. produce functionally different ribosomes in different developmental stages. This hypothesis was investigated through comparison of the structure of the large subunit rRNA molecules of the rodent malaria parasite, Plasmodium berghei, and by disruption of both of the rRNA gene units that are transcribed exclusively during development of this parasite in the mosquito (S-type rRNA gene units). In contrast to the human parasite, Plasmodium falciparum, we did not find evidence of structural differences in core regions of the distinct large subunit rRNAs which are known to be associated with catalytic activity including the GTPase site that varies in P. falciparum. Knockout P. berghei parasites lacking either of the S-type gene units were able to complete development in both the vertebrate and mosquito hosts. These results formally exclude the hypothesis that two functionally different ribosome types distinct from the predominantly blood stage-expressed A-type ribosomes, are required for development of all Plasmodium species in the mosquito. The maintenance of two functionally equivalent rRNA genes might now be explained as a gene dosage phenomenon.

Primary structure of the Plasmodium vivax crk2 gene and interference of the yeast cell cycle upon its conditional expression.

The cdc2 gene product, a 34-kDa protein kinase, plays a universal role in the M phase of the eukaryotic cell cycle. To study the cell cycle regulation in malarial parasites, we have characterized a cdc2-related gene from the most widely distributed human malaria, Plasmodium vivax (Pvcrk2). The full-length Pvcrk2 revealed 90--99% homology with Crk2 proteins from other Plasmodium species and approximately 60% homology with p34(cdc2) proteins from higher eukaryotes. We used the temperature-sensitive Schizosaccharomyces pombe cdc2 mutant (cdc2-33(ts)) for gene complementation studies. Expression of the full-length 33-kDa PvCrk2 protein, a truncated 27-kDa version, and two chimeric proteins in which we exchanged the N- and C-terminal regions of PvCrk2 with their S. pombe counterparts at the restrictive temperature in the mutant cdc2-33(ts) did not complement the cell cycle defect. However, conditional expression of the Pvcrk2 genes or the chimera containing the C terminus from Spcdc2 in mutant cdc2-33(ts) cells produced cell-cycle-arrested phenotypes only in the induced state and at the permissive temperature. Our results thus provide the first compelling genetic evidence that the plasmodial Crk2 gene product(s) is capable of interfering with the well-conserved eukaryotic cell cycle machinery.

A central role for P48/45 in malaria parasite male gamete fertility.

Fertilization and zygote development are obligate features of the malaria parasite life cycle and occur during parasite transmission to mosquitoes. The surface protein PFS48/45 is expressed by male and female gametes of Plasmodium falciparum and PFS48/45 antibodies prevent zygote development and transmission. Here, gene disruption was used to show that Pfs48/45 and the ortholog Pbs48/45 from a rodent malaria parasite P. berghei play a conserved and important role in fertilization. p48/45- parasites had a reduced capacity to produce oocysts in mosquitoes due to greatly reduced zygote formation. Unexpectedly, only male gamete fertility of p48/45- parasites was affected, failing to penetrate otherwise fertile female gametes. P48/45 is shown to be a surface protein of malaria parasites with a demonstrable role in fertilization.

The development of genetic tools for dissecting the biology of malaria parasites.

Plasmodium parasites are haploid unicellular organisms that cause malaria. In the last decade, transfection systems have been developed for both human and animal model species of Plasmodium, providing a broad range of genetic tools for the study of malaria parasite biology. Transient transfection has been used to provide insight into the regulation of gene expression by Plasmodium spp. The development of stable transfection technologies has provided the opportunity to express transgenes in Plasmodium spp., as well as elucidate the function of proteins by disrupting, modifying, or replacing the genes encoding them. These genetic tools represent an important breakthrough for malaria research and will significantly contribute to our understanding of the biology of the parasite. However, further developments in this technology are still required, especially because the full genome sequence of the major human malaria parasite Plasmodium falciparum will shortly be available. Ultimately, the biological information obtained through genetic manipulation of Plasmodium spp. will facilitate a more rational approach to vaccine and drug design.

Targeted terminal deletions as a tool for functional genomics studies in Plasmodium.

We describe a transfection system that induces terminal deletions at specific chromosome ends in malaria parasites using a linear construct containing telomeric repeats at one end and plasmodial sequences able to drive homologous recombination at the other. A site-specific deletion was generated at one extremity of chromosome 5 of Plasmodium berghei, which was stably maintained in the parasite population selected after transfection. The telomeric repeat array introduced with the construct reached the average length observed in natural telomeres of Plasmodium, indicating that in vivo telomere addition occurred at the newly formed extremity. The expression of a mutant dhfr/ts gene conferring pyrimethamine resistance, used as a selectable marker, was not affected by the proximity to the telomeric sequences, either in the presence or absence of drug pressure. In addition, no transcriptional silencing was observed on insertion of the mutant dhfr/ts gene either in subtelomeric or internal positions that are transcriptionally silent in blood-stage parasites. This suggests that the activity of its promoter is not affected by the chromatin organization of the chromosomal context.

The selectable marker human dihydrofolate reductase enables sequential genetic manipulation of the Plasmodium berghei genome.

Genetic transformation of malaria parasites has been limited by the number of selectable markers available. For the rodent malaria parasite, Plasmodium berghei, only a single selection marker has been at hand, utilising the dihydrofolate reductase-thymidylate synthase gene from either P. berghei or Toxoplasma gondii to confer resistance to the anti-malarial drug pyrimethamine. Here we report the use of the human dihydrofolate reductase (hDHFR) gene as a new selectable marker, which confers resistance to the antifolate inhibitor WR99210 upon both pyrimethamine sensitive and resistant isolates of P. berghei. Transfection with circular constructs containing the hDHFR gene resulted in the generation of highly resistant parasites containing multiple copies of episomally-maintained plasmids. These parasites showed around a 1000-fold increase in resistance to WR99210 compared to the parental parasites. We were also able to generate and select transgenic parasites harbouring only a single copy of hDHFR targeted into their genome, despite the fact that these parasites showed only a fivefold increase in resistance to WR99210 compared to the parental parasites. Importantly, and for the first time with malaria parasites, the hDHFR gene could be used in conjunction with the existing pyrimethamine selectable markers. This was demonstrated by reintroducing the circumsporozoite (CS) gene into transgenic CS-knockout mutant parasites that contained the P. berghei DHFR-TS selectable marker. The development of hDHFR as a second selectable marker will greatly expand the use of transformation technology in Plasmodium, enabling more extensive genetic manipulation and thus facilitating more comprehensive studies on the biology of the malaria parasite.

The conserved genome organisation of non-falciparum malaria species: the need to know more.

The current knowledge on genomes of non-falciparum malaria species and the potential of model malaria parasites for functional analyses are reviewed and compared with those of the most pathogenic human parasite, Plasmodium falciparum. There are remarkable similarities in overall genome composition among the different species at the level of chromosome organisation and chromosome number, conserved order of individual genes, and even conserved functions of specific gene domains and regulatory control elements. With the initiative taken to sequence the genome of P. falciparum, a wealth of information is already becoming available to the scientific community. In order to exploit the biological information content of a complete genome sequence, simple storage of the bulk of sequence data will be inadequate. The requirement for functional analyses to determine the biological role of the open reading frames is commonly accepted and knowledge of the genomes of the animal model malaria species will facilitate these analyses. Detailed comparative genome information and sequencing of additional Plasmodium genomes will provide a deeper insight into the evolutionary history of the species, the biology of the parasite, and its interactions with the mammalian host and mosquito vector. Therefore, an extended and integrated approach will enhance our knowledge of malaria and will ultimately lead to a more rational approach that identifies and evaluates new targets for anti-malarial drug and vaccine development.

The A-domain and the thrombospondin-related motif of Plasmodium falciparum TRAP are implicated in the invasion process of mosquito salivary glands.

Sporozoites from all Plasmodium species analysed so far express the thrombospondin-related adhesive protein (TRAP), which contains two distinct adhesive domains. These domains share sequence and structural homology with von Willebrand factor type A-domain and the type I repeat of human thrombospondin (TSP). Increasing experimental evidence indicates that the adhesive domains bind to vertebrate host ligands and that TRAP is involved, through an as yet unknown mechanism, in the process of sporozoite motility and invasion of both mosquito salivary gland and host hepatocytes. We have generated transgenic P.berghei parasites in which the endogenous TRAP gene has been replaced by either P.falciparum TRAP (PfTRAP) or mutated versions of PfTRAP carrying amino acid substitutions or deletions in the adhesive domains. Plasmodium berghei sporozoites carrying the PfTRAP gene develop normally, are motile, invade mosquito salivary glands and infect the vertebrate host. A substitution in a conserved residue of the A-domain or a deletion in the TSP motif of PfTRAP impairs the sporozoites' ability to invade mosquito salivary glands. Notably, midgut sporozoites from these transgenic parasites are still able to infect mice. Midgut sporozoites carrying a mutation in the A-domain of PfTRAP are motile, while no gliding motility could be detected in sporozoites with a TSP motif deletion.

Identification of the transcription initiation site of the asexually expressed rRNA genes of the malaria parasite Plasmodium berghei.

The start site of the A-type ribosomal RNA transcription units of the rodent malaria parasite, Plasmodium berghei, has been identified. The two A-type units cannot be distinguished within the transcription unit, yet exist as single copies on different chromosomes. Gene transcription initiates 820 bp upstream of the A-type small subunit (SSU) ribosomal gene and two major processing sites were mapped 610 and 611 nucleotides upstream of the SSU in the external transcribed spacer region. Surprisingly the nucleotide sequence of the DNA region containing the putative ribosomal promoter lacked repetitive DNA sequences typical of ribosomal promoters. This region was further analysed by computer using programs designed to reveal sequence-dependent structural features. Comparison of DNA curvature, duplex stability and pattern of twist angle variation revealed a striking degree of conservation between the ribosomal promoters from Plasmodium and other eukaryotes.

Heterogeneous ribosome populations are present in Plasmodium berghei during development in its vector.

The genome of the rodent malaria parasite, Plasmodium berghei, contains two sets of variant ribosomal RNA (rRNA) genes, termed the A and S types, that are expressed predominantly during the vertebrate and mosquito stages of the parasite's development respectively. Using in situ hybridization, we have examined the transcriptional activity of the A- and S-type rRNA genes, and the switch in expression of the ribosome populations that occurs after parasite transmission to the mosquito. By detection of precursor rRNA molecules, we show that A-type rRNA transcription is downregulated throughout development in the mosquito, whereas the initiation of S-type rRNA transcription is linked to the proliferative phase of the oocyst. Mature A-type rRNA persists during development of the zygote into the ookinete/young oocyst. In contrast, mature S-type rRNA is first detectable in young oocysts and is subsequently present at high levels during further development of oocysts and sporozoites. These results demonstrate that the switch in transcription between the A- and S-type rRNA genes is developmentally regulated, taking place only as the parasite begins to proliferate in the mosquito. A-type ribosomes are therefore not only translationally active in the early stages of development in the mosquito, but are also crucial at this phase.

The development of and perspectives for genetic engineering of malaria parasites.

The genetic manipulation of malaria parasites is a rapidly emerging technology that offers great promise for the investigation of many aspects of infection. Currently it is possible to transform avian, rodent, primate as well as human parasites, the latter three on a stable, drug selectable basis. This review focuses on the history of the development of the technology, current abilities and future perspectives.