Abstract
Toxoplasmosis is caused by an obligate intracellular parasite, the protozoan Toxoplasma gondii Discovery of novel drugs against T. gondii infection could circumvent the toxicity of existing drugs and T. gondii resistance to current treatments. The autophagy-related protein 8 (Atg8)-Atg3 interaction in T. gondii is a promising drug target because of its importance for regulating Atg8 lipidation. We reported previously that TgAtg8 and TgAtg3 interact directly. Here we validated that substitutions of conserved residues of TgAtg8 interacting with the Atg8 family-interacting motif (AIM) in Atg3 disrupt the TgAtg8-TgAtg3 interaction and reduce TgAtg8 lipidation and autophagosome formation. These findings were consistent with results reported previously for Plasmodium Atg8, suggesting functional conservation of Atg8 in Toxoplasma and Plasmodium. Moreover, using peptide and AlphaScreen assays, we identified the AIM sequence in TgAtg3 that binds TgAtg8. We determined that the core TgAtg3 AIM contains a Phe239-Ala240-Asp241-Ile242 (239FADI242) signature distinct from the 105WLLP108 signature in the AIM of Plasmodium Atg3. Furthermore, an alanine-scanning assay revealed that the TgAtg8-TgAtg3 interaction in T. gondii also depends strongly on several residues surrounding the core TgAtg3 AIM, such as Asn238, Asp243, and Cys244 These results indicate that distinct AIMs in Atg3 contribute to differences between Toxoplasma and Plasmodium Atg8-Atg3 interactions. By elucidating critical residues involved in the TgAtg8-TgAtg3 interaction, our work paves the way for the discovery of potential anti-toxoplasmosis drugs. The quantitative and straightforward AlphaScreen assay developed here may enable high-throughput screening for small molecules disrupting the TgAtg8-TgAtg3 interaction.
Highlights
Toxoplasmosis is caused by an obligate intracellular parasite, the protozoan Toxoplasma gondii
Using the sequence and crystal structure of PfAtg8 (PDB code 4EOY) as a reference, the bioinformatics analysis revealed that T. gondii Atg8 (TgAtg8) possesses a conserved molecular structure and sequences involved in the interaction with the Atg8 family–interacting motif (AIM), such as the W-site, L-site, and Apicomplexa-specific loop (A-loop) (Fig. 1, A and B)
Because of the high conservation of autophagy-related protein 8 (Atg8) in structure and sequence involved in the interaction with the AIM between Toxoplasma and Plasmodium, we mutated residues R27E, D44A/K45S/ K46A, and deleted residues 68 –76, which form the A-loop region, because they have been confirmed as key residues mediating the PfAtg8 –Plasmodium falciparum Atg3 (PfAtg3) interaction [28]
Summary
Toxoplasmosis is caused by an obligate intracellular parasite, the protozoan Toxoplasma gondii. We validated that substitutions of conserved residues of TgAtg interacting with the Atg family–interacting motif (AIM) in Atg disrupt the TgAtg8 – TgAtg interaction and reduce TgAtg lipidation and autophagosome formation. An alanine-scanning assay revealed that the TgAtg8 –TgAtg interaction in T. gondii depends strongly on several residues surrounding the core TgAtg AIM, such as Asn238, Asp243, and Cys244. These results indicate that distinct AIMs in Atg contribute to differences between Toxoplasma and Plasmo-. We demonstrate that TgAtg interacts directly with TgAtg through a conserved AIM sequence, 239FADI242 These results establish the importance of these residues for the TgAtg8 –TgAtg interaction, providing mechanistic insights into the TgAtg8 –PE conjugation cascade in T. gondii
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