Fig. 2. Genome-wide screen under sublethal DHA concentration reveals that loss of Tmem14c increases DHA susceptibility.

a Screening workflow. The drug score is defined as the log₂-fold change in the relative gRNA abundance between the DHA-treated and vehicle populations, where lower scores are indicative of genes that enhance drug susceptibility when disrupted. b Results of a genome-wide CRISPR screen (calculated as described in a) comparing treatment with 50 nM DHA (equivalent to EC₅) to vehicle-treated parasites. Guides against one gene, Tmem14c, were significantly depleted by the sublethal DHA concentration, but were retained in the vehicle control. c ΔTmem14c parasites formed plaques normally under standard growth conditions, but their plaquing ability was attenuated in the presence of 100 nM DHA. The parental strain proliferated normally in both conditions. d Extracellular ∆Tmem14c parasites had a decreased EC₅₀ compared to the parental line (p = 0.0003, extra-sum-of-squares F-test). Results are mean ± SEM for n = 7 independent experiments. e Monolayer lysis following infection with parental or ΔTmem14c parasites and treatment with varying concentrations of DHA during hours 1–6 (top panel) or 24–29 (bottom panel) post infection. Results are mean ± SEM for n = 4 or 3 independent experiments for parental or ΔTmem14c parasites, respectively. f Overexpression of Tmem14c-Ty co-localized with mitochondrial marker mtHSP70. Scale bar is 5 μm. g Relative abundance of selected amino acids from targeted metabolomics of intracellular parental or ∆Tmem14c parasites. Results are mean ± SD for n = 3 technical replicates, with p-values calculated by one-way ANOVA.