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Regulation of Atg8 membrane deconjugation by cysteine proteases in the malaria parasite Plasmodium berghei

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Abstract

During macroautophagy, the Atg8 protein is conjugated to phosphatidylethanolamine (PE) in autophagic membranes. In Apicomplexan parasites, two cysteine proteases, Atg4 and ovarian tumor unit (Otu), have been identified to delipidate Atg8 to release this protein from membranes. Here, we investigated the role of cysteine proteases in Atg8 conjugation and deconjugation and found that the Plasmodium parasite consists of both activities. We successfully disrupted the genes individually; however, simultaneously, they were refractory to deletion and essential for parasite survival. Mutants lacking Atg4 and Otu showed normal blood and mosquito stage development. All mice infected with Otu KO sporozoites became patent; however, Atg4 KO sporozoites either failed to establish blood infection or showed delayed patency. Through in vitro and in vivo analysis, we found that Atg4 KO sporozoites invade and normally develop into early liver stages. However, nuclear and organelle differentiation was severely hampered during late stages and failed to mature into hepatic merozoites. We found a higher level of Atg8 in Atg4 KO parasites, and the deconjugation of Atg8 was hampered. We confirmed Otu localization on the apicoplast; however, parasites lacking Otu showed no visible developmental defects. Our data suggest that Atg4 is the primary deconjugating enzyme and that Otu cannot replace its function completely because it cleaves the peptide bond at the N-terminal side of glycine, thereby irreversibly inactivating Atg8 during its recycling. These findings highlight a role for the Atg8 deconjugation pathway in organelle biogenesis and maintenance of the homeostatic cellular balance.

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All data are available within this manuscript, and raw data are available from the corresponding author upon reasonable request. Materials generated in this study are available from the corresponding author on request.

Abbreviations

Atg:

Autophagy-related genes

Atg4:

Autophagy-related protein 4

Otu:

Ovarion tumor unit

EEF:

Exoerythrocytic forms

HA:

Hemagglutinin

Hsp70:

Heat Shock Protein70

MSP:

Merozoite Surface Protein 1

ACP:

Acyl Carrier Protein

UIS4:

Upregulated in infectious sporozoites gene 4

GFP:

Green fluorescent protein

TRAP:

Thrombospondin-related anonymous protein

iRBC:

Infected RBC

UTR:

Untranslated region

IV:

Intravenously

IFA:

Immunofluorescence assay

hDHFR:

Human dihydrofolate reductase

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Acknowledgements

We thank Dr. Kota Arun Kumar (University of Hyderabad, India) for the pBC–3XHA–mCherry and pBC–mCherry–TgDHFR vectors and Pb mCherry parasites. We acknowledge Sanger as having made available the Materials and Lucigen as the source of the plasmid vector used to generate the PlasmoGEM resource. We thank Dr. Saman Habib (CSIR-CDRI, India), Dr. Anthony A. Holder (The Francis Crick Institute, UK), Drs. Photini Sinnis and Sean Prigge (Johns Hopkins University, USA) for anti-ICT1, anti-MSP1, anti-UIS4 and anti-ACP antibodies, respectively. We also thank Dr. Puran Singh Sijwali (CCMB, Hyderabad) for the pET32a–PfAtg8 plasmid. We acknowledge the THUNDER (BSC0102) and MOES (GAP0118) Intravital and Confocal microscopy facility of CSIR-CDRI. We thank Rima Ray Sarkar and Anil Kumar for their technical assistance with microscopy. The University Grants Commission and Council of Scientific and Industrial Research, Government of India research fellowships supported AM and AV. This manuscript is CDRI Communication No. 10683.

Funding

The work was supported by the CSIR-CDRI innovative idea grant [CII7045].

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Authors and Affiliations

  1. Division of Molecular Microbiology and Immunology, CSIR-Central Drug Research Institute, Lucknow, 226031, India

    Akancha Mishra, Aastha Varshney & Satish Mishra

  2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India

    Akancha Mishra & Satish Mishra

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  1. Akancha Mishra
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AM and SM conceived the idea, designed and performed the experiments, analyzed the data and wrote the manuscript. AV performed the experiments. All the authors have read and approved the manuscript.

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Correspondence to Satish Mishra.

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Mishra, A., Varshney, A. & Mishra, S. Regulation of Atg8 membrane deconjugation by cysteine proteases in the malaria parasite Plasmodium berghei. Cell. Mol. Life Sci. 80, 344 (2023). https://doi.org/10.1007/s00018-023-05004-2

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