Severe malaria due to Plasmodium falciparum is a parasitic disease spread by mosquitoes that causes immense morbidity and mortality in the developing world. Approximately 200 million people contract malaria each year, and there are ~500,000 deaths annually, primarily among children under the age of 5. The symptoms of severe malaria occur when P. falciparum enters the blood stage of its life cycle, where it invades and grows exponentially in erythrocytes. While antimalarial drugs are in widespread use, their efficacy is threatened by the rapid development of resistance among the parasites. As these drugs all target parasite factors, an alternative approach could be to target host factors that the parasite needs for survival. Although long- standing epidemiological data indicate that certain erythrocyte traits have been under natural selection due to malaria, it has historically been challenging to study mature erythrocytes at a molecular level as these unusual cells lack a genome and nucleus, making them genetically intractable. Here, we propose a proteome-wide forward genetic screen to identify host factors critical for malaria using enucleated erythrocytes derived from human hematopoietic stem cells (HSCs). We recently reported a screen of human blood group genes using erythroblasts, which identified a new host factor for parasite invasion. We hypothesize that comprehensive screening in erythrocytes (the cell the parasite invades in vivo) will identify new classes of host determinants. We will use RNAi- and CRISPR-based genetic perturbations in HSCs and immortalized CD34+ cells to generate enucleated erythrocytes with gene depletion or disruption, and infect the cells with fluorescent P. falciparum. Using deep, quantitative RNA-sequencing, we will correlate shRNA/gRNA abundance with parasite survival and host cell enucleation, thereby generating a list of candidate host factors for P. falciparum as well as enucleation determinants. Candidates will be validated using complementary approaches including reverse genetics, live cell microscopy, in vitro parasite assays and hematological characterization. We will use the candidates to further investigate host-pathogen interactions using small molecules and parasite resistance selection. We anticipate that this project will lead to the discovery and characterization of critical host factors for P. falciparum, laying the foundation for the rational development of host-directed therapeutics for malaria.
Egan, Elizabeth S. Public Health Relevancy Statement Severe malaria due to infection with the parasite Plasmodium falciparum is a leading cause of morbidity and mortality in the developing world, particularly among young children and pregnant women. Parasites have rapidly evolved resistance to available antimalarial agents, and new strategies are needed to control the disease. Here we propose to identify and characterize critical host factors for P. falciparum, laying the foundation for the potential development of host-directed therapeutics for malaria.
| Egan, Elizabeth S; Weekes, Michael P; Kanjee, Usheer et al. (2018) Erythrocytes lacking the Langereis blood group protein ABCB6 are resistant to the malaria parasite Plasmodium falciparum. Commun Biol 1:45 |
| Egan, Elizabeth S (2018) Beyond Hemoglobin: Screening for Malaria Host Factors. Trends Genet 34:133-141 |
