Abstract
ABSTRACTPlasmodium falciparum, the parasite that causes the deadliest form of malaria, has evolved multiple proteins known as invasion ligands that bind to specific erythrocyte receptors to facilitate invasion of human erythrocytes. The EBA-175/glycophorin A (GPA) and Rh5/basigin ligand-receptor interactions, referred to as invasion pathways, have been the subject of intense study. In this study, we focused on the less-characterized sialic acid-containing receptors glycophorin B (GPB) and glycophorin C (GPC). Through bioinformatic analysis, we identified extensive variation in glycophorin B (GYPB) transcript levels in individuals from Benin, suggesting selection from malaria pressure. To elucidate the importance of the GPB and GPC receptors relative to the well-described EBA-175/GPA invasion pathway, we used an ex vivo erythrocyte culture system to decrease expression of GPA, GPB, or GPC via lentiviral short hairpin RNA transduction of erythroid progenitor cells, with global surface proteomic profiling. We assessed the efficiency of parasite invasion into knockdown cells using a panel of wild-type P. falciparum laboratory strains and invasion ligand knockout lines, as well as P. falciparum Senegalese clinical isolates and a short-term-culture-adapted strain. For this, we optimized an invasion assay suitable for use with small numbers of erythrocytes. We found that all laboratory strains and the majority of field strains tested were dependent on GPB expression level for invasion. The collective data suggest that the GPA and GPB receptors are of greater importance than the GPC receptor, supporting a hierarchy of erythrocyte receptor usage in P. falciparum.
Highlights
Malaria is a disease of major global health importance that is caused by parasites of the genus Plasmodium, of which Plasmodium falciparum is the most virulent [1]
Erythrocyte receptors involved in P. falciparum invasion and their regulatory regions harbor polymorphisms, some of which are overrepresented in regions where malaria is endemic and are suggested to have arisen as a consequence of the selective force of malaria on the human genome
Of 238 erythroid cell-specific/blood group transcripts from 61 healthy children included in the analysis, we identified four genes with wide expression ranges (95 quantile to 5 quantile ratio larger than 10 and z score greater than 3): those for carbonic anhydrase 1 (CA1), hemoglobin zeta chain (HBZ), RAP1 GTPase-activating protein 1 (RAP1GAP), and, unexpectedly, glycophorin B (GYPB) (Fig. 1A)
Summary
Malaria is a disease of major global health importance that is caused by parasites of the genus Plasmodium, of which Plasmodium falciparum is the most virulent [1]. The set of dominant invasion pathways used during invasion is strain dependent and has led to a broad classification of P. falciparum strains as sialic acid dependent or independent. The virulence of P. falciparum has been partly attributed to its extensive set of invasion pathways, which enable it to efficiently invade diverse host erythrocytes harboring different receptor polymorphisms. Less characterized are the EBA-140/GPC sialic acid-dependent invasion pathway [8, 9, 28, 29] and the sialic acid-dependent parasite invasion ligand EBA-181, for which no receptor has been identified [30,31,32]. One study reported a complete block in invasion of the sialic acid-independent strain 7G8 into GPB-null (S-s-U-) erythrocytes [33], a prior study showed little inhibition of this strain [34]. Donor-to-donor blood group differences and differences in receptors may contribute to the variable invasion phenotypes of GPB-null cells, which underscores a potential weakness of comparing nonisogenic mutant and wild-type erythrocytes
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