Malaria parasites are haploid for most of their life cycle, with zygote formation and meiosis occurring during the mosquito phase of development. The parasites can be analyzed genetically by transmitting mixtures of cloned parasites through mosquitoes to permit cross-fertilization of gametes to occur. A cross was made between two clones of Plasmodium falciparum differing in enzymes, drug sensitivity, antigens, and chromosome patterns. Parasites showing recombination between the parent clone markers were detected at a high frequency. Novel forms of certain chromosomes, detected by pulsed-field gradient gel electrophoresis, were produced readily, showing that extensive rearrangements occur in the parasite genome after cross-fertilization. Since patients are frequently infected with mixtures of genetically distinct parasites, mosquito transmission is likely to provide the principal mechanisms for generating parasites with novel genotypes.

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The gene for the circumsporozoite (CS) protein of Plasmodium falciparum has been cloned and its nucleotide sequence determined. The gene encodes a protein of 412 amino acids as deduced from the nucleotide sequence. The protein contains 41 tandem repeats of a tetrapeptide, 37 of which are Asn-Ala-Asn-Pro and four of which are Asn-Val-Asp-Pro. Monoclonal antibodies against the CS protein of Plasmodium falciparum were inhibited from binding to the protein by synthetic peptides of the repeat sequence. The CS protein of Plasmodium falciparum and the CS protein of a simian malaria parasite, Plasmodium knowlesi, have two regions of homology, one of which is present on either side of the repeat. One region contains 12 of 13 identical amino acids. Within the nucleotide sequence of this region, 25 of 27 nucleotides are conserved. The conservation of these regions in parasites widely separated in evolution suggests that they may have a function such as binding to liver cells and may represent an invariant target for immunity.

The origin of Plasmodium falciparum, the etiological agent of the most dangerous forms of human malaria, remains controversial. Although investigations of homologous parasites in African Apes are crucial to resolve this issue, studies have been restricted to a chimpanzee parasite related to P. falciparum, P. reichenowi, for which a single isolate was available until very recently. Using PCR amplification, we detected Plasmodium parasites in blood samples from 18 of 91 individuals of the genus Pan, including six chimpanzees (three Pan troglodytes troglodytes, three Pan t. schweinfurthii) and twelve bonobos (Pan paniscus). We obtained sequences of the parasites' mitochondrial genomes and/or from two nuclear genes from 14 samples. In addition to P. reichenowi, three other hitherto unknown lineages were found in the chimpanzees. One is related to P. vivax and two to P. falciparum that are likely to belong to distinct species. In the bonobos we found P. falciparum parasites whose mitochondrial genomes indicated that they were distinct from those present in humans, and another parasite lineage related to P. malariae. Phylogenetic analyses based on this diverse set of Plasmodium parasites in African Apes shed new light on the evolutionary history of P. falciparum. The data suggested that P. falciparum did not originate from P. reichenowi of chimpanzees (Pan troglodytes), but rather evolved in bonobos (Pan paniscus), from which it subsequently colonized humans by a host-switch. Finally, our data and that of others indicated that chimpanzees and bonobos maintain malaria parasites, to which humans are susceptible, a factor of some relevance to the renewed efforts to eradicate malaria.

Intermittent episodes of febrile illness are the most benign and recognized symptom of infection with malaria parasites, although the effects on parasite survival and virulence remain unclear. In this study, we identified the molecular factors altered in response to febrile temperature by measuring differential expression levels of individual genes using high-density oligonucleotide microarray technology and by performing biological assays in asexual-stage Plasmodium falciparum parasite cultures incubated at 37°C and 41°C (an elevated temperature that is equivalent to malaria-induced febrile illness in the host). Elevated temperature had a profound influence on expression of individual genes; 336 of approximately 5,300 genes (6.3% of the genome) had altered expression profiles. Of these, 163 genes (49%) were upregulated by twofold or greater, and 173 genes (51%) were downregulated by twofold or greater. In-depth sensitive sequence profile analysis revealed that febrile temperature-induced responses caused significant alterations in the major parasite biologic networks and pathways and that these changes are well coordinated and intricately linked. One of the most notable transcriptional changes occurs in genes encoding proteins containing the predicted Pexel motifs that are exported into the host cytoplasm or inserted into the host cell membrane and are likely to be associated with erythrocyte remodeling and parasite sequestration functions. Using our sensitive computational analysis, we were also able to assign biochemical or biologic functional predictions for at least 100 distinct genes previously annotated as "hypothetical." We find that cultivation of P. falciparum parasites at 41°C leads to parasite death in a time-dependent manner. The presence of the "crisis forms" and the terminal deoxynucleotidyltransferasemediated dUTP-biotin nick end labeling-positive parasites following heat treatment strongly support the notion that an apoptosis-like cell death mechanism might be induced in response to febrile temperatures. These studies enhance the possibility of designing vaccines and drugs on the basis of disruption in molecules and pathways of parasite survival and virulence activated in response to febrile temperatures.

Malaria vaccines are being developed against different stages in the parasite's life cycle, each increasing the opportunity to control malaria in its diverse settings. Sporozoite vaccines are designed to prevent mosquito-induced infection; first generation recombinant or synthetic peptide vaccines have been tested in humans. Asexual erythrocytic stage vaccines, developed to prevent or reduce the severity of disease, have been tested in animals and in humans. A third strategy is to produce sexual stage vaccines that would induce antibodies which would prevent infection of mosquitoes when ingested in a bloodmeal containing sexual stage parasites. Although not directly protective, the sexual stage vaccine combined with a sporozoite or asexual stage vaccine (protective component) could prolong the useful life of the protective component by reducing transmission of resistant vaccine-induced mutants. In areas of low endemnicity, the sexual stage vaccine could reduce transmission below the critical threshold required to maintain the infected population, thereby assisting in the control or eradication of malaria. Transmission of Plasmodium falciparum, the major human malaria, can be blocked by monoclonal antibodies against three sexual stage-specific antigens. We have cloned the gene encoding the surface protein of relative molecular mass Mr 25,000 (25K; Pfs25), expressed on zygotes and ookinetes of P. falciparum. The deduced amino-acid sequence consists of a signal sequence, a hydrophobic C-terminus, and four tandem epidermal growth factor EGF-like domains.

ATP-binding cassette transporters play an important role in drug resistance and nutrient transport. In the human malaria parasite Plasmodium falciparum, a homolog of the human p-glycoprotein (PfPgh-1) was shown to be involved in resistance to several drugs. More recently, many transporters were associated with higher IC 50 levels in responses to chloroquine (CQ) and quinine (QN) in field isolates. Subsequent studies, however, could not confirm the associations, although inaccuracy in drug tests in the later studies could contribute to the lack of associations. Here we disrupted a gene encoding a putative multidrug resistance-associated protein (PfMRP) that was previously shown to be associated with P. falciparum responses to CQ and QN. Parasites with disrupted PfMRP (W2/MRP⌬) could not grow to a parasitemia higher than 5% under normal culture conditions, possibly because of lower efficiency in removing toxic metabolites. The W2/MRP⌬ parasite also accumulated more radioactive glutathione, CQ, and QN and became more sensitive to multiple antimalarial drugs, including CQ, QN, artemisinin, piperaquine, and primaquine. PfMRP was localized on the parasite surface membrane, within membrane-bound vesicles, and along the straight side of the D-shaped stage II gametocytes. The results suggest that PfMRP plays a role in the efflux of glutathione, CQ, and QN and contributes to parasite responses to multiple antimalarial drugs, possibly by pumping drugs outside the parasite.

Chloroquine (CQ)-resistant Plasmodium vivax malaria was first reported 12 years ago, nearly 30 years after the recognition of CQ-resistant P. falciparum. Loss of CQ efficacy now poses a severe problem for the prevention and treatment of both diseases. Mutations in a digestive vacuole protein encoded by a 13-exon gene, pfcrt, were shown recently to have a central role in the CQ resistance (CQR) of P. falciparum. Whether mutations in pfcrt orthologues of other Plasmodium species are involved in CQR remains an open question. This report describes pfcrt homologues from P. vivax, P. knowlesi, P. berghei, and Dictyostelium discoideum. Synteny between the P. falciparum and P. vivax genes is demonstrated. However, a survey of patient isolates and monkey-adapted lines has shown no association between in vivo CQR and codon mutations in the P. vivax gene. This is evidence that the molecular events underlying P. vivax CQR differ from those in P. falciparum.