Studies on Radiation Attenuated Sporozoites and Plasmodium Falciparum Gene Expression
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Human malaria is caused by five species of the apicomplexan parasite, Plasmodium. Research into the life cycle of the parasite, especially the pre-erythrocytic stages, to obtain important molecular information to help in the disease control has been difficult. This is largely due to the scarcity of suitable hepatoma cell lines that allow the parasite’s infection and complete intra-hepatic growth. Generally, Plasmodium sporozoites obtained from laboratory strains of the parasite have been unable to infect these hepatoma cell lines, and when they do, neither full intra-hepatic maturation nor the release of infective merozoites is observed. Of the five human Plasmodium species, P. falciparum is the most virulent largely because of the genes: var, rif and stevor, which the organism employs for survival through antigenic variation and sequestration in deep tissues. Radiation Attenuated Sporozoites (RAS) of Plasmodium retain their ability to express stage-specific proteins and thus elicit immunity against malaria. Generally, RAS are unable to develop beyond the liver stage; some RAS however develop to the erythrocytic stages. The genes expressed for survival by the attenuated erythrocytic parasites were previously unknown. In this study, rather than using laboratory-adapted strains, natural (wild-type) P. falciparum obtained from hospital patients in Khartoum, Sudan, was used. Wild-type sporozoites were cultivated by feeding the gametocyte infected blood obtained to Anopheles arabiensis mosquitoes. Some of the sporozoites were then exposed to a 10Krad dose of x-irradiation from a Cobalt-60 (Co-60) radiation source (RAS) while the sporozoites that were not exposed to the x-irradiation served as the Control. The two sporozoites groups (Control and RAS) were used to infect the human hepatoma cell line, HepG2, to yield merozoites and subsequently, erythrocytic stage parasites on adding O+ RBCs into both the Control and RAS groups. Total RNA isolation was carried out on the infected erythrocytes in both groups, followed by genome-wide expression profiling, using the Agilent Custom Plasmodium falciparum Gene Expression Microarray (8X15K format, AMADID 040812, Agilent Technologies Inc. Wilmington DE, USA). Compared to the wild-type parasite, the attenuated erythrocytic parasites differentially expressed 10 genes. Thymidylate kinase (tmk), and the rif gene, PF10_0002, were upregulated (p(corr) <0.05; FC >2). Two rif genes, PFL2655w and PF10_0401, were downregulated (p(corr) <0.05; FC < -2). The genes for protein kinase and mitochondrial phosphate carrier (mpc), were also downregulated (p corr) <0.05; FC < -2). An in vitro P. falciparum life cycle was achieved in this study; also, to the best of our knowledge, the first successful infection of P. falciparum sporozoites into HepG2 cells with subsequent full intra-hepatic development and the release of infective merozoites that yielded erythrocytic stages of the parasite are reported. Prior attempts, which have used laboratory-adapted strains of P. falciparum had not been successful. Using natural isolates of Plasmodium for infection studies on already established hepatoma cell lines is postulated to give a range of important novel findings compared to what is currently reported in literature.
A Thesis Submitted to the School of Postgraduate Studies, University of Lagos
Plasmodium , Human malaria , Immunity , Disease control , Research Subject Categories::MEDICINE::Microbiology, immunology, infectious diseases
Adeyemi, O.A (2015). Studies on Radiation Attenuated Sporozoites and Plasmodium Falciparum Gene Expression. A Thesis Submitted to University of Lagos School of Postgraduate Studies Phd Thesis and Dissertation.