Characterizing the Plasmodium falciparum Apicomplexan amino acid transporter 2 and its proline transport capability

PROJECT SUMMARY Malaria remains one of the leading causes of death worldwide and affects about half the global population, despite decades of public health efforts. Plasmodium falciparum, the parasitic agent responsible for malaria, undergoes a complex life cycle involving intracellular parasitism in the human host and extracellular development in the mosquito vector. We have previously shown that resistance to halofuginone (HFG), a potent antimalarial that targets the P. falciparum cytoplasmic prolyl tRNA synthetase (PfcProRS), can be achieved by the parasite through two different mechanisms: mutation in the target gene or an increase in intracellular proline, which competes with binding of HFG to PfcProRS. We have recently discovered that the increase in proline is due to loss of function mutations in the Apicomplexan Amino acid Transporter 2 (PfApiAT2). Parasites with premature stop codons obtained by selection with HFG or a knockout (KO) of PfApiAT2 have a up to 20-fold increase in cellular proline compared to the parental line. These KO parasites do not have a growth phenotype in the blood stages of the parasite but our preliminary data as well as published data in P. berghei suggests that ApiAT2 is essential in the mosquito stages. While blood stage parasites obtain the majority of their amino acid needs from parasite specific hemoglobin digestion, mosquito stage parasites are extracellular and need to take up all their nutrients form the hemolymph on the mosquito. The molecular mechanisms for this, however, remain largely unexplored. Our central hypothesis is that PfApiAT2 is a proline transporter which transports excess proline out of the parasite in the blood stages and imports proline into the parasite from the hemolymph in the mosquito stages. We will test this hypothesis by characterizing the proline transport capacity of PfApiAT2 in the heterologous expression system of Xenopus laevis oocytes as well as in P. falciparum parasites directly using radiolabeled proline. To better understand the biological function of PfApiAT2, we will generate KO parasites and determine their exact time of arrest in the mosquito. In addition, we will use the novel technology of ultrastructure expansion microscopy to gain high-resolution localization of PfApiAT2 in the mosquito stages. Our specific aims are as follows: Aim 1. Determine the proline transport capability of PfApiAT2 Aim 2 Assess the function and localization of PfApiAT2 in the mosquito stages of P. falciparum