Genetic Evidence for Erythrocyte Receptor Glycophorin B Expression Levels Defining a Dominant Plasmodium falciparum Invasion Pathway into Human Erythrocytes.

Plasmodium 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

Adaptation of Plasmodium falciparum to humans involved the loss of an ape-specific erythrocyte invasion ligand

Abstract: Plasmodium species are frequently host-specific, but little is currently known about the molecular factors restricting host switching. This is particularly relevant for P. falciparum, the only known human-infective species of the Laverania sub-genus, all other members of which infect African apes. Here we show that all tested P. falciparum isolates contain an inactivating mutation in an erythrocyte invasion associated gene, PfEBA165, the homologues of which are intact in all ape-infective Laverania species. Recombinant EBA165 proteins only bind ape, not human, erythrocytes, and this specificity is due to differences in erythrocyte surface sialic acids. Correction of PfEBA165 inactivating mutations by genome editing yields viable parasites, but is associated with down regulation of both PfEBA165 and an adjacent invasion ligand, which suggests that PfEBA165 expression is incompatible with parasite growth in human erythrocytes. Pseudogenization of PfEBA165 may represent a key step in the emergence and evolution of P. falciparum

Author Correction: Adaptation of Plasmodium falciparum to humans involved the loss of an ape-specific erythrocyte invasion ligand.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-21491-y</jats:p

Determinants of brain swelling in pediatric and adult cerebral malaria.

Cerebral malaria (CM) affects children and adults, but brain swelling is more severe in children. To investigate features associated with brain swelling in malaria, we performed blood profiling and brain MRI in a cohort of pediatric and adult patients with CM in Rourkela, India, and compared them with an African pediatric CM cohort in Malawi. We determined that higher plasma Plasmodium falciparum histidine rich protein 2 (PfHRP2) levels and elevated var transcripts that encode for binding to endothelial protein C receptor (EPCR) were linked to CM at both sites. Machine learning models trained on the African pediatric cohort could classify brain swelling in Indian children CM cases but had weaker performance for adult classification, due to overall lower parasite var transcript levels in this age group and more severe thrombocytopenia in Rourkela adults. Subgrouping of patients with CM revealed higher parasite biomass linked to severe thrombocytopenia and higher Group A-EPCR var transcripts in mild thrombocytopenia. Overall, these findings provide evidence that higher parasite biomass and a subset of Group A-EPCR binding variants are common features in children and adult CM cases, despite age differences in brain swelling

Sialic acid variation as a determinant of Plasmodium invasion of erythrocytes in malaria infection

Sialic acids are acidic sugars that terminate glycan chains on proteins or lipids on vertebrate cell surfaces. They vary greatly in structure, presentation and amount, all of which are important physiologically, but can also impact the tissue and host tropism of diverse pathogens. Parasites of the genus Plasmodium cause malaria, a disease characterized by a cyclical process of parasite invasion of host erythrocytes, growth and replication and fresh invasion of new erythrocytes. During erythrocyte invasion – an event central to malaria pathogenesis – proteins on the surface of the parasite, known as invasion ligands, bind to specific erythrocyte receptors, many of which are sialylated. In this dissertation, we determined how sialic acid variation impacts erythrocyte invasion by the zoonotic parasite, Plasmodium knowlesi and the most virulent human parasite, Plasmodium falciparum. For studies on P. knowlesi, we determined if Neu5Gc, a sialic acid that is absent in humans but present in most other primates, is a major determinant of parasite tropism. We used the recently described ex vivo erythrocyte culture system to transgenically express the CMAH enzyme, responsible for production of Neu5Gc. P. knowlesi showed significantly increased invasion of Neu5Gc-expressing human erythrocytes, providing evidence that loss of Neu5Gc in humans restricts P. knowlesi invasion of human erythrocytes. We then biochemically characterized two P. knowlesi invasion ligands of the EBL family and found they specifically bind Neu5Gc. These ligands potentially mediate Neu5Gc-dependent invasion of human and macaque erythrocytes. We finally showed that in natural human infections, P. knowlesi can adapt to infect erythrocytes independently of sialic acid. We also studied the use of sialic acid-containing erythrocyte receptors by P. falciparum using the ex vivo erythrocyte culture system. We determined the importance in invasion of glycophorin B (GPB), receptor for P. falciparum invasion ligand, EBL-1, and one of the highly sialylated receptors on the erythrocyte surface. We specifically knocked down gene expression of GPB as well as two well characterized receptors involved in P. falciparum invasion – GPA, the largest contributor to erythrocyte sialic acid and GPC, another sialylated receptor. Invasion assays using P. falciparum laboratory strains and field isolates revealed that GPB is a dominant receptor in P. falciparum invasion, of comparable importance to GPA.Biological Sciences in Public Healt

Ancient human sialic acid variant restricts an emerging zoonotic malaria parasite

Plasmodium knowlesi is a zoonotic parasite transmitted from macaques causing malaria in humans in Southeast Asia. Plasmodium parasites bind to red blood cell (RBC) surface receptors, many of which are sialylated. While macaques synthesize the sialic acid variant N-glycolylneuraminic acid (Neu5Gc), humans cannot because of a mutation in the enzyme CMAH that converts N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. Here we reconstitute CMAH in human RBCs for the reintroduction of Neu5Gc, which results in enhancement of P. knowlesi invasion. We show that two P. knowlesi invasion ligands, PkDBPβ and PkDBPγ, bind specifically to Neu5Gc-containing receptors. A human-adapted P. knowlesi line invades human RBCs independently of Neu5Gc, with duplication of the sialic acid-independent invasion ligand, PkDBPα and loss of PkDBPγ. Our results suggest that absence of Neu5Gc on human RBCs limits P. knowlesi invasion, but that parasites may evolve to invade human RBCs through the use of sialic acid-independent pathways.National Institutes of Health (U.S.) (grant AI091787)Centers for Disease Control and Prevention (U.S.) (grant (R36-CK000119-01))National Institutes of Health (U.S.) (Epidemiology of Infectious Disease and Biodefense Training Grant, 2-T32-AI007535-12

Ancient human sialic acid variant restricts an emerging zoonotic malaria parasite

Plasmodium knowlesi is a zoonotic parasite transmitted from macaques causing malaria in humans in Southeast Asia. Plasmodium parasites bind to red blood cell (RBC) surface receptors, many of which are sialylated. While macaques synthesize the sialic acid variant N-glycolylneuraminic acid (Neu5Gc), humans cannot because of a mutation in the enzyme CMAH that converts N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. Here we reconstitute CMAH in human RBCs for the reintroduction of Neu5Gc, which results in enhancement of P. knowlesi invasion. We show that two P. knowlesi invasion ligands, PkDBPβ and PkDBPγ, bind specifically to Neu5Gc-containing receptors. A human-adapted P. knowlesi line invades human RBCs independently of Neu5Gc, with duplication of the sialic acid-independent invasion ligand, PkDBPα and loss of PkDBPγ. Our results suggest that absence of Neu5Gc on human RBCs limits P. knowlesi invasion, but that parasites may evolve to invade human RBCs through the use of sialic acid-independent pathways

Adaptation of Plasmodium falciparum to humans involved the loss of an ape-specific erythrocyte invasion ligand

Abstract: Plasmodium species are frequently host-specific, but little is currently known about the molecular factors restricting host switching. This is particularly relevant for P. falciparum, the only known human-infective species of the Laverania sub-genus, all other members of which infect African apes. Here we show that all tested P. falciparum isolates contain an inactivating mutation in an erythrocyte invasion associated gene, PfEBA165, the homologues of which are intact in all ape-infective Laverania species. Recombinant EBA165 proteins only bind ape, not human, erythrocytes, and this specificity is due to differences in erythrocyte surface sialic acids. Correction of PfEBA165 inactivating mutations by genome editing yields viable parasites, but is associated with down regulation of both PfEBA165 and an adjacent invasion ligand, which suggests that PfEBA165 expression is incompatible with parasite growth in human erythrocytes. Pseudogenization of PfEBA165 may represent a key step in the emergence and evolution of P. falciparum

Adaptation of Plasmodium falciparum to humans involved the loss of an ape-specific erythrocyte invasion ligand

Abstract: Plasmodium species are frequently host-specific, but little is currently known about the molecular factors restricting host switching. This is particularly relevant for P. falciparum, the only known human-infective species of the Laverania sub-genus, all other members of which infect African apes. Here we show that all tested P. falciparum isolates contain an inactivating mutation in an erythrocyte invasion associated gene, PfEBA165, the homologues of which are intact in all ape-infective Laverania species. Recombinant EBA165 proteins only bind ape, not human, erythrocytes, and this specificity is due to differences in erythrocyte surface sialic acids. Correction of PfEBA165 inactivating mutations by genome editing yields viable parasites, but is associated with down regulation of both PfEBA165 and an adjacent invasion ligand, which suggests that PfEBA165 expression is incompatible with parasite growth in human erythrocytes. Pseudogenization of PfEBA165 may represent a key step in the emergence and evolution of P. falciparum