Ubiquitin activation is essential for schizont maturation in Plasmodium falciparum blood-stage development.

Judith L Green,Vesela Encheva,Vikram Sharma,Chairat Uthaipibull,Irene Pinzuti,Sebastian Bothe,Edwin Lasonder,Ngoc Truongvan,Hermann Schindelin,Benjamin Stieglitz,Yang Wu,Somdet Srichairatanakool,Adchara Prommaban,Gordon Langsley,Munira Grainger,Simone Kunzelmann,Anthony A Holder,Evangelos Christodoulou,Wei Song,Ambrosius P Snijders,Véronique Birault

doi:10.1371/journal.ppat.1008640

Abstract

Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites.

Highlights

The malaria parasite’s life cycle includes an asexual blood stage infection that is responsible for the disease
Malaria is a significant global health problem caused by a parasite that invades and multiplies within red blood cells, an invasion cycle resulting in death and disease
In this study we found that many parasite proteins are modified with a ubiquitin tag while the parasite matures within red blood cells and that even more proteins seem to be tagged in the extracellular invasive merozoite form

Summary

Introduction

The malaria parasite’s life cycle includes an asexual blood stage infection that is responsible for the disease. Following erythrocyte invasion by the merozoite, the parasite develops through haploid ring and trophozoite stages and in the schizont stage, DNA synthesis, mitosis and nuclear division lead to a multinucleate syncytium. At the end of schizogony, cytokinesis leads to the differentiation of new merozoites, which are subsequently released by rupture of the host erythrocyte (egress) and go on to invade other erythrocytes. During schizogony cellular structures are elaborated, including secretory organelles such as the micronemes, rhoptries and dense granules, and the inner membrane complex (IMC) that forms part of the merozoite surface pellicle. For each cycle of development, structures of the intracellular parasite such as the remnant body are discarded at egress, and the merozoite secretory organelles are disassembled following invasion. It is difficult to distinguish morphologically extracellular merozoites from intracellular merozoites at the final stage of schizogony, there are substantial differences in the merozoite and schizont phosphoproteomes [1, 2], and a significant increase in protein ubiquitylation in extracellular merozoites [2]

Methods

Results

Discussion

Conclusion