Plasmodium falciparum erythrocytic stage parasites require the putative autophagy protein PfAtg7 for normal growth.

Dawn M Walker,Maribeth Spangler,Mark E Drew,Viral C Patel,Katherine A Kemme,Najmus Mahfooz,Georges Snounou

doi:10.1371/journal.pone.0067047

Dawn M Walker, Maribeth Spangler + Show 5 more

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https://doi.org/10.1371/journal.pone.0067047

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Abstract

Analysis of the Plasmodium falciparum genome reveals a limited number of putative autophagy genes, specifically the four genes involved in ATG8 lipidation, an essential step in formation of autophagosomes. In yeast, Atg8 lipidation requires the E1-type ligase Atg7, an E2-type ligase Atg3, and a cysteine protease Atg4. These four putative P. falciparum ATG (PfATG) genes are transcribed during the parasite’s erythrocytic stages. PfAtg7 has relatively low identity and similarity to yeast Atg7 (14.7% and 32.2%, respectively), due primarily to long insertions typical of P. falciparum. Excluding the insertions the identity and similarity are higher (38.0% and 70.8%, respectively). This and the fact that key residues are conserved, including the catalytic cysteine and ATP binding domain, we hypothesize that PfAtg7 is the activating enzyme of PfAtg8. To assess the role of PfAtg7 we have generated two transgenic parasite lines. In one, the PfATG7 locus was modified to introduce a C-terminal hemagglutinin tag. Western blotting reveals two distinct protein species, one migrating near the predicted 150 kDa and one at approximately 65 kDa. The second transgenic line introduces an inducible degradation domain into the PfATG7 locus, allowing us to rapidly attenuate PfAtg7 protein levels. Corresponding species are also observed in this parasite line at approximately 200 kDa and 100 kDa. Upon PfATG7 attenuation parasites exhibit a slow growth phenotype indicating the essentiality of this putative enzyme for normal growth.

Highlights

IntroductionAutophagy (from Greek auto, self+phagein, eating) has recently become accepted as an important lysosome-mediated catabolic process in eukaryotes
Autophagy has recently become accepted as an important lysosome-mediated catabolic process in eukaryotes
In this study we show that the putative Atg8 lipidation pathway members PfATG3 (PF3D7_0905700.2), PfATG4 (PF3D7_1417300), PfATG7 (PF3D7_1126100) and PfATG8 (PF3D7_1019900) are transcribed in erythrocytic stage parasites

Summary

Introduction

Autophagy (from Greek auto, self+phagein, eating) has recently become accepted as an important lysosome-mediated catabolic process in eukaryotes. Despite ATG genes being highly conserved across eukaryotes, there is seemingly only a subset conserved among parasitic protozoa including Leishmania, Toxoplasma, Trypanosoma and Plasmodium [18,19,20] In common to these protozoa are members of the Atg lipidation pathway, an essential pathway for formation of autophagosomes, the double membrane vesicles that envelop cargo for delivery to the lysosome. The small ubiquitin-like modifier Atg plays an integral role in this process [21] Under certain conditions, such as nutrient starvation, autophagy becomes highly upregulated as the E1- and E2- type ligases Atg and Atg activate cytosolic Atg by conjugating it to phosphatidylethanolamine (PE) [22], with a result in an increase in autophagosome formation [23]

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