Protein Kinase A Is Essential for Invasion of Plasmodium falciparum into Human Erythrocytes.

Authors:
Wilde ML1,2, Triglia T1, Marapana D1, Thompson JK1, Kouzmitchev AA3, Bullen HE4, Gilson PR4,5, Cowman AF1,2, Tonkin CJ6,2.
In:
Source: mBio
Publication Date: (2019)
Issue: 10: 5
Research Area:
Parasitology
Drug Discovery
Cells used in publication:
Plasmodium falciparum
Species: unicellular
Tissue Origin:
Platform:
Nucleofector™ I/II/2b
Experiment

Methods: Transfections utilized Amaxa Basic Parasite Nucleofector kit 2 (Lonza,  Germany). A 100-ul volume of each plasmid was resuspended in 85 _l solution 2 and 15 _l solution 3. This solution was mixed with parasite PEMS and transferred to a 2-mm-gap-size cuvette (Lonza, Hilden, Germany). Transfections were performed using Amaxa 2b Nucleofector and condition U-033. Transfected cells were added to 10 ml RPMI-HEPES media at 4% hematocrit and cultured as usual. Blasticidin (BSD; 2.5 _g/ml) or WR99210 (2.5 nM) was added after 24 h to select for positive transfectants.

Abstract

Understanding the mechanisms behind host cell invasion by Plasmodium falciparum remains a major hurdle to developing antimalarial therapeutics that target the asexual cycle and the symptomatic stage of malaria. Host cell entry is enabled by a multitude of precisely timed and tightly regulated receptor-ligand interactions. Cyclic nucleotide signaling has been implicated in regulating parasite invasion, and an important downstream effector of the cAMP-signaling pathway is protein kinase A (PKA), a cAMP-dependent protein kinase. There is increasing evidence that P. falciparum PKA (PfPKA) is responsible for phosphorylation of the cytoplasmic domain of P. falciparum apical membrane antigen 1 (PfAMA1) at Ser610, a cAMP-dependent event that is crucial for successful parasite invasion. In the present study, CRISPR-Cas9 and conditional gene deletion (dimerizable cre) technologies were implemented to generate a P. falciparum parasite line in which expression of the catalytic subunit of PfPKA (PfPKAc) is under conditional control, demonstrating highly efficient dimerizable Cre recombinase (DiCre)-mediated gene excision and complete knockdown of protein expression. Parasites lacking PfPKAc show severely reduced growth after one intraerythrocytic growth cycle and are deficient in host cell invasion, as highlighted by live-imaging experiments. Furthermore, PfPKAc-deficient parasites are unable to phosphorylate PfAMA1 at Ser610. This work not only identifies an essential role for PfPKAc in the P. falciparum asexual life cycle but also confirms that PfPKAc is the kinase responsible for phosphorylating PfAMA1 Ser610.IMPORTANCE Malaria continues to present a major global health burden, particularly in low-resource countries. Plasmodium falciparum, the parasite responsible for the most severe form of malaria, causes disease through rapid and repeated rounds of invasion and replication within red blood cells. Invasion into red blood cells is essential for P. falciparum survival, and the molecular events mediating this process have gained much attention as potential therapeutic targets. With no effective vaccine available, and with the emergence of resistance to antimalarials, there is an urgent need for the development of new therapeutics. Our research has used genetic techniques to provide evidence of an essential protein kinase involved in P. falciparum invasion. Our work adds to the current understanding of parasite signaling processes required for invasion, highlighting PKA as a potential drug target to inhibit invasion for the treatment of malaria.