Plasmodium falciparum, the causative agent of malaria, lives inside the red blood cells of its human hosts. In doing so, it uses the protein haemoglobin as its food source. The last stages of haemoglobin digestion requires aminopeptidases to release free amino acids from globin peptides through the hydrolysis of amide bonds. Both PfA-M1 and PfA-M17 are zinc aminopeptidases currently implicated in this step, both with varied substrate specificities and parasite localisations. PfA-M1 has the ability to cleave a wide range of different amino acids, while PfA-M17 predominantly cleaves leucine1. It has been reported that PfA-M1 localises to the food vacuole, the nucleus and the parasitophorous vacuole; in contrast, PfA-M17 has only been localised to the parasite cytosol1. Both have so far been immune to disruption using conventional methods, suggesting that these proteins may be essential for parasite survival. Identifying essential genes is an important step in finding drug targets for future therapeutic development.
To further understand these proteins, we will employ different molecular strategies to edit the genome material of P. falciparum parasites. Firstly, CRISPR-Cas9 will be used to knockout each gene to determine essentiality, and then knockdown lines will be created using glmS riboswitch, which also contains a C-terminal HA tag. Phenotypic analysis of these lines will provide further insight into the function of each gene. Additionally, immunofluorescence will be used to determine localisation of HA-tagged proteins. So far, all efforts to knock out PfA-M1 have been unsuccessful, suggesting that this gene is essential. We have achieved significant knockdown with the PfA-M17-glmS line and phenotypic analysis is currently underway. This project aims to tease out the function and importance of both the PfA-M1 and PfA-M17 aminopeptidases.