The impact of common and rare human genetic variants on erythrocyte invasion by Plasmodium falciparum malaria parasites

Abstract

Malaria still remains a leading cause of mortality in several countries, particularly in sub-Saharan Africa. The African population is genetically diverse, and some people carry natural variants that confer varying levels of protection against the disease. In recent years, several large-scale genome-wide association studies (GWAS) have identified novel human genetic variants (e.g., variants at the GYPA/B/E gene cluster) and confirmed well-known variants (e.g., Duffy negativity) that are associated with varying degrees of protection against severe malaria. However, even for variants that have been studied for decades, the underlying mechanism of protection often remains unknown or poorly understood. Both Duffy negativity and the GYPA/B/E variants are relatively prevalent in African populations, which has led to the suggestion that both Duffy negativity and GYPA/B/E variants may be selected to protect against malaria. The Duffy gene encodes DARC (Duffy antigen receptor for chemokines), which is used by both P. vivax and P. knowlesi parasites to invade red blood cells (RBCs). A single nucleotide substitution in the promoter of the gene where erythroid-specific transcription factors bind disrupts the expression of the DARC receptor on the surface of RBCs, and individuals who are homozygous for the mutation are termed Duffy-negative. The Duffy-negative phenotype (Fya-b-) is associated with resistance to both P. vivax and P. knowlesi and is found in over 95% of West and Central Africans. By contrast, P. falciparum uses multiple RBC receptors for invasion, but whether DARC is one of these receptors has never been systematically explored. In contrast to Duffy negativity, the GYPA/B/E DEL1 variant results in a 110kb deletion that affects the coding regions of the GYPB gene but could also affect regulatory elements in the non-coding region of the GYPB unit that may affect other genes. A variant at the same locus, DUP4 (Dantu), confers a strong protective advantage against malaria by altering the biophysical properties of the RBC. However, the impact of the DEL1 and other structural variants at the locus on the RBC and P. falciparum invasion is not well understood, in part due to the lack of genotyping assays to support functional studies. The work described in this thesis explored the functional impact of Duffy negativity and GYPB DEL1 variant on the RBC (surface proteome and biomechanical characteristics) and on invasion by P. falciparum malaria parasites, using a combination of in vitro parasite assays, microscopy, and quantitative proteomics techniques. The work revealed, for the first time, that Duffy negativity alters the RBC surface proteome and inhibits RBC invasion by P. falciparum in a minor but consistent dose-dependent manner, suggesting that DARC plays either a direct or an indirect role during P. falciparum invasion. It also explored the prevalence of the GYPB DEL1 and DEL2 variants in The Gambia, and functional assays with the Gambian RBC samples showed that the DEL1 variant had no significant impact on the invasion and growth of P. falciparum 3D7, Dd2, 7G8, and GB4 strains. In conclusion, this thesis showed how both common and rare human genetic variants can alter not only the invasion preference of P. falciparum malaria parasites but also result in significant alterations of RBC properties (surface proteome and biophysical properties) and has provided insights into the molecular mechanisms by which these natural human genetic variants protect against malaria.