Short communication

Presence of three distinct ookinete surface protein genes, Pos25, Pos28-1, and Pos28-2, in Plasmodium ovale☆

Malaria in humans is caused by six species of Plasmodium parasites, of which the nuclear genome sequences for the two Plasmodium ovale spp., P. ovale curtisi and P. ovale wallikeri, and Plasmodium malariae have not yet been analyzed. Here we present an analysis of the nuclear genome sequences of these three parasites, and describe gene family expansions therein. Plasmodium ovale curtisi and P. ovale wallikeri are genetically distinct but morphologically indistinguishable and have sympatric ranges through the tropics of Africa, Asia and Oceania. Both P. ovale spp. show expansion of the surfin variant gene family, and an amplification of the Plasmodium interspersed repeat (pir) superfamily which results in an approximately 30% increase in genome size. For comparison, we have also analyzed the draft nuclear genome of P. malariae, a malaria parasite causing mild malaria symptoms with a quartan life cycle, long-term chronic infections, and wide geographic distribution. Plasmodium malariae shows only a moderate level of expansion of pir genes, and unique expansions of a highly diverged transmembrane protein family with over 550 members and the gamete P25/27 gene family. The observed diversity in the P. ovale wallikeri and P. ovale curtisi surface antigens, combined with their phylogenetic separation, supports consideration that the two parasites be given species status.

The polymorphism of Pvs25 and Pvs28 ookinete surface proteins, their association to circumsporozoite protein repeat (CSPr) genotypes (Vk210 and Vk247) and their infectivity to local Anopheles albimanus and Anopheles pseudopunctipennis were investigated in Plasmodium vivax-infected blood samples obtained from patients in Southern Mexico. The pvs25 and pvs28 complete genes were amplified, cloned and sequenced; and the CSPr genotype was determined by PCR amplification and hybridization. The amino acid Pvs25 and Pvs28 polymorphisms were mapped to their corresponding protein structure. Infected blood samples were simultaneously provided through artificial feeders to both mosquito species; the ratio of infected mosquitoes and oocyst numbers were recorded. The polymorphism of pvs25 and pvs28 was limited to few nucleotide positions, and produced three haplotypes: type A/A parasites presented Pvs25 and Pvs28 amino acid sequences identical to that of Sal I reference strain; parasites type B1 presented a mutation 130 Ile → Thr in Pvs25, while type B2 presented 87 Gln → Lys/130 Ile → Thr in the same molecule. Both types B1 and B2 parasites presented changes in Pvs28 at 87 Asn → Asp, 110 Tyr → Asn and five GSGGE/D repeat sequences between the fourth EGF-like domain and the GPI. Most P. vivax parasites from the coastal plains and the overlapping region were Pvs25/28 A/A, CSPrVk210 and were infective only to An. albimanus (p ≤ 0.0001). Parasites originating in foothills were Pvs25/28 type B1/B or B2/B and CSPrVk210 or Vk247, and were more infective to An. pseudopunctipennis than to An. albimanus (p ≤ 0.001). These results and the analysis of Pvs25/28 from other parts of the world indicated that non-synonymous variations in these proteins occur in amino acid residues exposed on the surface of the proteins, and are likely to interact with midgut mosquito ligands. We hypothesize that these molecules have been shaped by co-evolutionary adaptations of parasites to their susceptible vectors.

Completion of the complex developmental program of Plasmodium in the mosquito is essential for parasite transmission, yet this part of its life cycle is still poorly understood. In recent years, considerable progress has been made in the identification and characterization of genes expressed during parasite development in the mosquito. This line of investigation was greatly facilitated by the availability of the genome sequence of several Plasmodium, and by the application of approaches such as proteomics, microarrays, gene disruption by homologous recombination (gene knockout) and by use of subtraction libraries. Here, we review what is presently known about genes expressed in gametocytes and during the Plasmodium life cycle in the mosquito.

Malaria remains one of the leading causes of both morbidity and mortality of humans residing in tropical countries. For many malarious regions outside of Africa, development of effective transmission-blocking vaccines will require coverage against both Plasmodium falciparum and Plasmodium vivax. The genes coding for two potential P. vivax transmission-blocking antigens, Pvs25 and Pvs28, have been cloned. Mice vaccinated with yeast-produced recombinant proteins Pvs25 and Pvs28 adsorbed to aluminum hydroxide developed strong antibody responses against the immunogens. The development of oocysts in mosquitoes was completely inhibited when these antisera were ingested with the P. vivax Salvador (Sal) I strain-infected chimpanzee blood. In a large collection of P. vivax field isolates, we found only 5 nucleotide changes that would result in amino acid substitutions in Pvs25. In contrast, the Pvs28 gene had 22 nucleotide changes that would result in conservative amino acid substitutions. How the antigenic polymorphism of Pvs25 and Pvs28 would affect the efficacy of Sal I based vaccine remains to be elucidated. Clinical trials with Pvs25 and the P. falciparum ortholog Pfs25 are in preparation.