Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress and invasion.

Anja C Schlott,Edward W Tate,Ambrosius P Snijders,Catherine Maclachlan,Anthony A Holder,Lucy M Collinson,Abigail J Perrin,Philip Hobson,Julia Morales-Sanfrutos,Ellen Knuepfer,Aaron J Borg,Judith L Green,Michael Duffy

doi:10.1371/journal.pbio.3001408

Abstract

We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of “pseudoschizonts,” which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.

Highlights

The malarial parasite asexual blood stage is largely intraerythrocytic as the parasite invades, develops, and proliferates within red blood cells (RBCs) over a period of approximately 45 to 48 hours in the case of Plasmodium falciparum, the most lethal human parasite
The location and abundance of GAP50 were unaffected. These findings indicate that glideosome-associated protein 45 (GAP45) is important for recruiting myosin A (MyoA) and myosin tail interacting protein (MTIP) to the inner membrane complex (IMC), its N-terminal glycine and its myristoylation is not required for this activity
The consequences of N-myristoyl transferase (NMT) inhibition with IMP-1002 for P. falciparum depend on the length of incubation with the inhibitor and its concentration, as well as the stage of parasite development to which the inhibitor is added

Summary

Introduction

The malarial parasite asexual blood stage is largely intraerythrocytic as the parasite invades, develops, and proliferates within red blood cells (RBCs) over a period of approximately 45 to 48 hours in the case of Plasmodium falciparum, the most lethal human parasite. At the end of schizogony, the multinucleate coenocyte undergoes cytokinesis that draws the parasite plasma membrane (PM) around each of the developing progeny to form highly polarized merozoites, each with its own nucleus, a surface pellicle comprised of PM and IMC, and apical organelles for subsequent invasion and modification of a new RBC. Completion of this process is followed by lysis of the infected RBC and egress of the extraerythrocytic merozoites, which attach to and invade new RBCs to establish the intraerythrocytic proliferation cycle. This stage of the parasite life cycle is responsible for the disease pathology and is a principal target for the development of drugs to kill the parasite

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Conclusion