Understanding the Determinants of Human Red Blood Cell Tropism of Understudied Plasmodium Species

Malaria control programs in the past decade have been successful in decreasing the global burden of Plasmodium falciparum, the more virulent of the Plasmodium species causing malaria in humans. However, as the public health community gears towards elimination and eradication of malaria, there is need for an attention shift towards research of neglected Plasmodium species. A central event in malaria pathogenesis is the invasion of host red blood cells (RBCs) mediated by specific interactions between parasite ligands and RBC receptors. These interactions, also called invasion pathways, can be major determinants of host tropism. Restriction in invasion due to tropism is attributed to limiting disease severity, as it limits the proliferation of the parasite in vivo. In this dissertation, we determine the host cell tropism of Plasmodium knowlesi and Plasmodium vivax, two understudied Plasmodium species. The zoonotic P. knowlesi is now the major cause of malaria in parts of South East Asia, and P. vivax is the most widespread species worldwide causing substantial morbidity. After a review of the current literature on biological and clinical features of these species and their invasion pathways in Chapter 1, we determined the tropism of P. knowlesi in human RBCs in Chapter 2. We used density-based enrichment methods to test the invasion of P. knowlesi into human RBCs of varying age. Incorporating mathematical modeling and experimental adaptation, we demonstrated that an expansion of host cell niche is required for the parasite to reach densities observed clinically. We also obtained parasite lines adapted to efficient proliferation in human RBCs. In Chapter 3, we investigated the molecular basis of increased invasion of human RBCs through whole genome sequencing analysis of newly human-adapted lines, as well as historical strains we adapted to in vitro culture. We showed that different genetic changes in P. knowlesi can lead to the upregulation of PkDBPα-DARC pathway, and have provided evidence of major divergence in invasion ligands in recent field isolates. Finally, in Chapter 4, we studied the variation in human RBC preference of patient isolates for P. knowlesi and P. vivax. We confirmed that recent human P knowlesi isolates also vary in the niche of susceptible RBCs, with a subset exhibiting a lack of restriction in invading human RBCs. We also evaluated ex vivo P. vivax patient isolates for their host RBC preference. We determined that P. vivax field isolates differ in their level of reticulocyte preference. We further found an association between increased reticulocyte preference and schizont maturation. This body of work aims to contribute to our overall understanding of human RBC tropism of Plasmodium species of public health importance and the implication this has for parasite adaptation to humans.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

Infection of laboratory colonies of Anopheles mosquitoes with Plasmodium vivax from cryopreserved clinical isolates

© 2016 Australian Society for Parasitology Plasmodium vivax is the most geographically widespread malaria parasite. Unique features of transmission biology complicate P. vivax control. Interventions targeting transmission are required for malaria eradication. In the absence of an in vitro culture, transmission studies rely on live isolates from non-human primates or endemic regions. Here, we demonstrate P. vivax gametocytes from both India and Brazil are stable during cryopreservation. Importantly, cryopreserved gametocytes from Brazil were capable of infecting three anopheline mosquito species in feedings done in the United States. These findings create new opportunities for transmission studies in diverse locales

Insights into an Optimization of <i>Plasmodium vivax</i> Sal-1 <i>In Vitro</i> Culture: The <i>Aotus</i> Primate Model

<div><p>Malaria is one of the most significant tropical diseases, and of the <i>Plasmodium</i> species that cause human malaria, <i>P</i>. <i>vivax</i> is the most geographically widespread. However, <i>P</i>. <i>vivax</i> remains a relatively neglected human parasite since research is typically limited to laboratories with direct access to parasite isolates from endemic field settings or from non-human primate models. This restricted research capacity is in large part due to the lack of a continuous <i>P</i>. <i>vivax in vitro</i> culture system, which has hampered the ability for experimental research needed to gain biological knowledge and develop new therapies. Consequently, efforts to establish a long-term <i>P</i>. <i>vivax</i> culture system are confounded by our poor knowledge of the preferred host cell and essential nutrients needed for <i>in vitro</i> propagation. Reliance on very heterogeneous <i>P</i>. <i>vivax</i> field isolates makes it difficult to benchmark parasite characteristics and further complicates development of a robust and reliable culture method. In an effort to eliminate parasite variability as a complication, we used a well-defined <i>Aotus</i>-adapted <i>P</i>. <i>vivax</i> Sal-1 strain to empirically evaluate different short-term <i>in vitro</i> culture conditions and compare them with previous reported attempts at <i>P</i>. <i>vivax in vitro</i> culture Most importantly, we suggest that reticulocyte enrichment methods affect invasion efficiency and we identify stabilized forms of nutrients that appear beneficial for parasite growth, indicating that <i>P</i>. <i>vivax</i> may be extremely sensitive to waste products. Leuko-depletion methods did not significantly affect parasite development. Formatting changes such as shaking and static cultures did not seem to have a major impact while; in contrast, the starting haematocrit affected both parasite invasion and growth. These results support the continued use of <i>Aotus</i>-adapted Sal-1 for development of <i>P</i>. <i>vivax</i> laboratory methods; however, further experiments are needed to optimize culture conditions to support long-term parasite development.</p></div

Media supplementation can influence the health and growth of the parasite.

<p>(<b>A</b>) Parasitemia graph showing the effect of GlutaMAX on parasitemia at different time points using three independent biologicals <i>P</i>. <i>vivax</i> Sal-1 parasites from MN23062a and b and MN23009a. Reticulocytes from hemochromatosis patients enriched by density were used for these experiments. (<b>B</b>) Histogram comparing parasite stages at different time points from the parasites in A: (<b>C</b>) Conversion percentages of rings to trophozoites and trophozoites to schizonts in media supplemented with or without GlutaMAX. Data from the three independent biological replicates in A. Error bars represent the standard error. While the parasitemia differences are not statically significant, we did observed a longer persistence of parasites in the cultures supplemented with GlutaMAX and we did observed reinvasion only in the GlutaMAX culture.</p

Experimental design using <i>Aotus lemurinus lemurinus</i> monkeys.

<p>Diagram showing the inoculations of the <i>Aotus</i> monkeys with <i>P</i>. <i>vivax</i> Sal-1. The percentages shown are for staging of the parasites at bleed (R, rings; T, trophozoites; S, schizonts; G, gametocytes). Table shows the experiments that each bleed was used for.</p