Pfs 16 pivotal role in Plasmodium falciparum gametocytogenesis: A potential antiplasmodial drug target

Mature gametocytes, the sexual stage of Plasmodium falciparum, ensure the continued transmission of malaria from the human host to the mosquito vector. Even if gametocytes are not implicated in the malaria physiopathology it is crucial to the spread of malaria. Gametocytes are to be a key target for drugs used against Plasmodium in public health. The expression levels of 4 sexual-stage specific genes, Pfs 16, Pfs 25, Pfg 27and S 18S rRNA, during gametocytogenesis of various P. falciparum strains were analyzed by a real time PCR assay. The strains showed different capacities to produce mature gametocytes and in parallel different patterns of sexual gene expression. There was a correlation only between Pfs 16 cDNA overexpression in the first 48 h of the culture and the production of mature gametocytes. Pfs 16 is an early marker of the development of mature gametocytes in cultures and is therefore a potential target for new antimalarial drugs.     

Inhibition of the SR Protein-Phosphorylating CLK Kinases of Plasmodium falciparum Impairs Blood Stage Replication and Malaria Transmission

Cyclin-dependent kinase-like kinases (CLKs) are dual specificity protein kinases that phosphorylate Serine/Arginine-rich (SR) proteins involved in pre-mRNA processing. Four CLKs, termed PfCLK-1-4, can be identified in the human malaria parasite Plasmodium falciparum, which show homology with the yeast SR protein kinase Sky1p. The four PfCLKs are present in the nucleus and cytoplasm of the asexual blood stages and of gametocytes, sexual precursor cells crucial for malaria parasite transmission from humans to mosquitoes. We identified three plasmodial SR proteins, PfSRSF12, PfSFRS4 and PfSF-1, which are predominantly present in the nucleus of blood stage trophozoites, PfSRSF12 and PfSF-1 are further detectable in the nucleus of gametocytes. We found that recombinantly expressed SR proteins comprising the Arginine/Serine (RS)-rich domains were phosphorylated by the four PfCLKs in in vitro kinase assays, while a recombinant PfSF-1 peptide lacking the RS-rich domain was not phosphorylated. Since it was hitherto not possible to knock-out the pfclk genes by conventional gene disruption, we aimed at chemical knock-outs for phenotype analysis. We identified five human CLK inhibitors, belonging to the oxo-β-carbolines and aminopyrimidines, as well as the antiseptic chlorhexidine as PfCLK-targeting compounds. The six inhibitors block P. falciparum blood stage replication in the low micromolar to nanomolar range by preventing the trophozoite-to-schizont transformation. In addition, the inhibitors impair gametocyte maturation and gametogenesis in in vitro assays. The combined data show that the four PfCLKs are involved in phosphorylation of SR proteins with essential functions for the blood and sexual stages of the malaria parasite, thus pointing to the kinases as promising targets for antimalarial and transmission blocking drugs.     

Functional characterisation of sexual stage specific proteins in Plasmodium falciparum

The various stages of the malaria parasites in the vertebrate host and in the mosquito vector offer numerous candidates for vaccine and drug development. However, the biological complexity of the parasites and the interaction with the immune system of the host continue to frustrate all such efforts thus far. While most of the targets for drug and vaccine design have focused on the asexual stages, the sexual stages of the parasite are critical for transmission and maintenance of parasites among susceptible vertebrate hosts. Sexual stage parasites undergo a series of morphological and biochemical changes during their development, accompanied by a co-ordinated cascade of a distinct expression pattern of sexual stage specific proteins. Mechanisms underlying the developmental switch from asexual parasite to sexual parasite still remain elusive. Methods that can break the malaria transmission cycle thus occupy a central place in the overall malaria control strategies. This paper provides a review of genes expressed in sexually differentiated Plasmodium. In the past few years, a molecular approach based on targeted gene disruption has revealed fascinating biological roles for many of the sexual stage gene products. In addition, we will briefly discuss other functional genomic approaches employed to study not only sexual but also other aspects of host-parasite biology.     

A Potent Malaria Transmission Blocking Vaccine Based on Codon Harmonized Full Length Pfs48/45 Expressed in Escherichia coli

Malaria caused by Plasmodium falciparum is responsible for nearly 1 million deaths annually. Although much progress has been made in the recent past, the development of a safe, effective and affordable malaria vaccine has remained a challenge. A vaccine targeting sexual stages of the parasite will not only reduce malaria transmission by female Anopheles mosquitoes, but also reduce the spread of parasites able to evade immunity elicited by vaccines targeting pre-erythrocytic and erythrocytic asexual stages. We focused our studies on Pfs48/45, a protein expressed in the sexual stages developing within an infected person and one of the most promising transmission-blocking vaccine targets. Functional immunogenicity of Pfs48/45 protein requires proper disulfide bond formation, consequently evaluation of the immunogenicity of recombinant full-length Pfs48/45 has been hampered by difficulties in expressing properly folded protein to date. Here we present a strategy involving harmonization of codons for successful recombinant expression of full length Pfs48/45 in Escherichia coli. The purified protein, designated CH-rPfs48/45, was recognized by monoclonal antibodies directed against reduction-sensitive conformational epitopes in the native protein. Immunogenicity evaluation in mice revealed potent transmission blocking activity in membrane feeding assays of antisera elicited by CH-rPfs48/45 formulated in three different adjuvants, i.e. Alum, Montanide ISA-51 and complete Freund''s adjuvant. More importantly, CH-rPfs48/45 formulated with Montanide ISA-51 when administered to nonhuman primates (Olive baboons, Papio anubis) resulted in uniformly high antibody responses (ELISA titers >2 million) in all five animals. Sera from these animals displayed greater than 93% blocking activity in membrane feeding assays after a single immunization, reaching nearly complete blocking after a booster dose of the vaccine. The relative ease of expression and induction of potent transmission blocking antibodies in mice and nonhuman primates provide a compelling rationale and basis for development of a CH-rPfs48/45 based malaria transmission blocking vaccine.