Single-cell quantitative bioimaging of <i>P. berghei</i> liver stage translation
Bibliographic record
Abstract
ABSTRACT Plasmodium parasite resistance to existing antimalarial drugs poses a devastating threat to the lives of many who depend on their efficacy. 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, translation inhibitors are a promising drug class, capable of targeting the disease-causing blood stage of infection, as well as the asymptomatic liver stage, a crucial target for prophylaxis. To identify compounds capable of inhibiting liver stage parasite translation, we developed an assay to visualize and quantify translation in the Plasmodium berghei- HepG2 infection model. After labeling infected monolayers with o-propargyl puromycin (OPP), a functionalized analog of puromycin permitting subsequent bioorthogonal addition of a fluorophore to each OPP-terminated nascent polypeptide, we use automated confocal feedback microscopy followed by batch image segmentation and feature extraction to visualize and quantify the nascent proteome in individual P. berghei liver stage parasites and host cells simultaneously. After 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 known direct translation inhibitors and other compounds which we identify as indirect translation inhibitors. Using this framework, we identify a Malaria Box compound, MMV019266, as a direct translation inhibitor in P. berghei liver stages and confirm this potential mode of action in Plasmodium falciparum asexual blood stages. IMPORTANCE Plasmodium parasites cause malaria in humans. New multistage active antimalarial drugs are needed, and a promising class of drugs targets the core cellular process of translation, which has many potential molecular targets. During the obligate liver stage, Plasmodium parasites grow in metabolically active hepatocytes, making it challenging to study core cellular processes common to both host cells and parasites, as the signal from the host typically overwhelms that of the parasite. Here, we present and validate a flexible assay to quantify Plasmodium liver stage translation using a technique to fluorescently label the newly synthesized proteins of both host and parasite followed by computational separation of their respective nascent proteomes in confocal image sets. We use the assay to determine whether a test set of known compounds are direct or indirect liver stage translation inhibitors and show that the assay can also predict the mode of action for novel antimalarial compounds.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".