The malaria parasite Plasmodium falciparum kills over 650,000 people a year and causes widespread morbidity. Human infection commences with a silent phase in the liver, after which the parasites emerge into the blood to infect red blood cells. There, the parasites develop in an ordered, yet graded, progression from rings to trophozoites to schizonts before escaping as merozites to start another erythrocytic cycle and disease. During the erythrocytic cycle, a structured cycle of mRNA abundance is evident, but the correlation between protein and mRNA levels is, at best, modest. This project explores the hypothesis that translational regulation tunes the proteome in the face of a regimented program for mRNA expression. Recently work has shown that translational controls are likely to be important in the erythrocytic cycle, as well as in the transition from mosquito sporozoites to live stages and in the development of the sexual stages essential to transmission. A corollary of these studies is that gene products that are highly regulated at the level of translation are likel important modulators of developmental change and the parasite's response to environmental stress. The project goals are to globally and quantitatively assess the rate at which each mRNA is actively translated in specific erythrocytic stages by coupling the ability to isolate the speciic "footprints" of mRNAs that are occupied by ribosomes (an indicator of translation) with the depth and breadth of next generation sequencing.
Aim 1 will establish the ribosome footprinting technology in P. falciparum using trophozoite stage parasites. It will optimize conditions for nuclease treatment to generate the mRNA footprints protected by ribosomes and for the generation of unbiased libraries from the RNA footprints for Illumina deep sequencing. It will also include maturation of the bioinformatics pipeline to analyze resulting sequence data, adapting existing pipelines that are used for RNA-Seq in P. falciparum and for ribosome footprinting of trypanosomes.
Aim 2 will expand into other stages of the asexual erythrocytic cycle, identifying genes that are differentially translated across these pathogenic stages. It will also examine gametocytes, which are poised for an abrupt environmental change upon ingestion by the mosquito. The proposed work will yield both an overview of the extent of translational control in P. falciparum blood stages, as well as a quantification of the translationl control of individual gene products. From such data, additional hypotheses on mechanisms of control and functions of regulated proteins can be generated and tested. The project will also improve genome annotation by determining whether proteins are translated from mRNAs corresponding to individual open reading frames, particularly those annotated as hypothetical proteins and those encoded by non-canonical protein-coding open reading frames, to provide a comprehensive view of the translational landscape of P. falciparum erythrocytic stages.
The malaria parasite Plasmodium falciparum kills between 650,000 and 1.2 million people per year, and impacts travelers and the U.S. military due to lack of an effective vaccine and resistance to drugs. This project will apply a new technology to measure the synthesis of each protein as the parasite develops in the blood, thereby causing disease. This knowledge should accelerate the development of new drugs and vaccines to fight malaria.