Person:

Dankwa, Selasi

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.

The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs) that are structurally related to eukaryotic kinases. To gain insight into the role of Ser/Thr phosphorylation in this major global pathogen, we used a phosphoproteomic approach to carry out an extensive analysis of protein phosphorylation in M. tuberculosis. We identified more than 500 phosphorylation events in 301 proteins that are involved in a broad range of functions. Bioinformatic analysis of quantitative in vitro kinase assays on peptides containing a subset of these phosphorylation sites revealed a dominant motif shared by six of the M. tuberculosis STPKs. Kinase assays on a second set of peptides incorporating targeted substitutions surrounding the phosphoacceptor validated this motif and identified additional residues preferred by individual kinases. Our data provide insight into processes regulated by STPKs in M. tuberculosis and create a resource for understanding how specific phosphorylation events modulate protein activity. The results further provide the potential to predict likely cognate STPKs for newly identified phosphoproteins.