Harnessing the Nascent Proteome in the Plasmodium Liver Stages: New Approaches for Drug Discovery and Parasite Biology.




McLellan, James Lyle

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Plasmodium parasite resistance to antimalarial drugs poses a devastating threat to the millions of lives that depend on their efficacy every year. New antimalarial drugs and novel drug targets are in critical need, along with novel assays to accelerate their identification. Given the essentiality of protein synthesis throughout the complex parasite lifecycle, parasite specific translation inhibitors are a highly promising drug class, capable of targeting the disease-causing blood stage of infection, as well as the asymptomatic liver stage, a crucial target for infection prophylaxis. To identify compounds capable of inhibiting parasite translation, we developed an assay to visualize and quantify liver stage translation in the P. berghei-HepG2 infection model. After labeling infected monolayers with o-propargyl puromycin (OPP), a functionalized analog of puromycin permitting subsequent bio-orthogonal addition of a fluorophore to each OPP-terminated nascent polypeptide, we use automated confocal feedback microscopy (ACFM) and batch image segmentation and feature extraction to visualize and quantify the nascent proteome in individual P. berghei parasites and host HepG2 cells simultaneously. After assay validation, we demonstrate specific, concentration dependent liver stage translation inhibition by both parasite-selective and pan-eukaryotic active compounds, and further show that acute pre-treatment and competition modes of the OPP assay can distinguish between direct and indirect translation inhibitors. Striking compound-specific differences in translation inhibition efficacy were found, with the presumed eEF2 inhibitor and clinical candidate, DDD107498, the least effective translation inhibitor tested. To investigate the effects of translation inhibitors during early liver stage schizogony, when we detected peak levels of translational output, we quantified translation and growth inhibition after an acute, 4-hour treatment with 5 translation inhibitors with diverse properties. While we found the initial extent of growth inhibition to be well-correlated with that of translation inhibition, subsequent characterization of parasite translation, growth, and development through to merosome release demonstrated that, surprisingly, a compound's translation inhibition efficacy does not determine the extent of its antiplasmodial effect. We then utilized our assay to characterize the translational output of P. berghei parasites throughout liver stage development and adapted our computational workflow to additionally characterize the subcellular changes in nascent proteome localization we observed. Early schizonts show a marked accumulation of OPP-labeled polypeptides which appeared surrounded by DNA, but not overlapping it, a localization which disappeared later in schizogony concomitant with striking DNA shape changes in the parasite. After implementation of expansion microscopy, we demonstrate that this DNA-dim clearing which is filled with OPP-labeled polypeptides is inside the nucleus, and likely represents the liver stage parasite nucleolus, the site of eukaryotic ribosome biogenesis.


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Drug Discovery, Plasmodium Liver stage, Quantitative Bioimaging



Molecular Microbiology and Immunology