Of the 515 million clinical episodes of Plasmodium falciparum malaria annually, 2-3 million result in severe infections, and one million deaths occur. It is likely that both host and parasite factors contribute to a severe disease outcome. The research objective of this application is to characterize P. falciparum var genes associated with severe malaria in an urban, sub-Saharan setting. Var genes encode PfEMPI, a parasite virulence factor expressed on the infected erythrocyte surface that binds host endothelial receptors, and that facilitates immune evasion through antigenic variation. Of the ~60 distinct var genes in a single genome, one PfEMPI antigen is ultimately expressed on the erythrocyte surface. Based on previous studies, we hypothesize that of the three major var groups (A,B,C), Group A var genes are preferentially transcribed in severe malaria. The first specific aim is to determine the population structure and genomic diversity of var genes in the urban setting of Mombasa, Kenya for comparison with a nearby rural dataset. In order to examine var gene transcription in urban cases of severe malaria, it is necessary first to characterize the underlying urban var population structure. Parasite DNA will be extracted from clinical blood samples. Single genome infections will be identified by MSP2 genotyping. The var DBLa domain will be amplified by PCR using universal primers, and multiple amplicons will be cloned and sequenced. Unique var genes will be deformed through sequence alignment. Using population genetics methods, var gene repertoires from each isolate will be compared within and between the urban and rural populations for assessment of population structure.
The second aim i s to test the hypothesis that urban severe malaria is associated with the preferential transcription of Group A var genes. In a case-control study, patients will be enrolled as severe malaria cases or uncomplicated malaria controls. Parasite RNA will be isolated from patient blood samples and reverse transcriptase PCR will be performed using universal primers to the var DBLa domain. PCR amplicons will be cloned and sequenced, and through phylogenetic comparison with known var sequences, var groups will be determined. The dominant var transcripts and var group for each patient sample will be compiled, and statistical comparison between cases and controls will be performed to test for an association between severe malaria and Group A var gene transcription. Relevance: P. falciparum malaria is a global public health problem affecting half a billion people each year. Identification of parasite gene variants associated with severe malaria may further understanding of malaria pathogenesis, lead to new drug or vaccine targets, yield clinical predictors of a severe disease, and improve public health disease surveillance. ? ? ?
| Zilversmit, Martine M; Chase, Ella K; Chen, Donald S et al. (2013) Hypervariable antigen genes in malaria have ancient roots. BMC Evol Biol 13:110 |
| Artzy-Randrup, Yael; Rorick, Mary M; Day, Karen et al. (2012) Population structuring of multi-copy, antigen-encoding genes in Plasmodium falciparum. Elife 1:e00093 |
| Chen, Donald S; Barry, Alyssa E; Leliwa-Sytek, Aleksandra et al. (2011) A molecular epidemiological study of var gene diversity to characterize the reservoir of Plasmodium falciparum in humans in Africa. PLoS One 6:e16629 |
