Cellular function relies on a balance between protein synthesis and breakdown. Macromolecular breakdown through autophagy is broadly required for cellular and tissue development, function, and recovery from stress. While Caenorhabditis elegans is frequently used to explore cellular responses to development and stress, the most common assays for autophagy in this system lack tissue-level resolution. Different tissues within an organism have unique functional characteristics and likely vary in their reliance on autophagy under different conditions. To generate a tissue-specific map of autophagy in C. elegans we used a dual fluorescent protein (dFP) tag that releases monomeric fluorescent protein (mFP) upon arrival at the lysosome. Tissue-specific expression of dFP::LGG-1 revealed autophagic flux in all tissues, but mFP accumulation was most dramatic in the intestine. We also observed variable responses to stress: starvation increased autophagic mFP release in all tissues, whereas anoxia primarily increased intestinal autophagic flux. We observed autophagic flux with tagged LGG-1, LGG-2, and two autophagic cargo reporters: a soluble cytoplasmic protein, and mitochondrial TOMM-7. Finally, an increase in mFP in older worms was consistent with an age-dependent shift in proteostasis. These novel measures of autophagic flux in C. elegans reveal heterogeneity in autophagic response across tissues during stress and aging.

Isopentenyl pyrophosphate (IPP) is an essential metabolic output of the apicoplast organelle in Plasmodium falciparum malaria parasites and is required for prenylation-dependent vesicular trafficking and other cellular processes. We have elucidated a critical and previously uncharacterized role for IPP in apicoplast biogenesis. Inhibiting IPP synthesis blocks apicoplast elongation and inheritance by daughter merozoites, and apicoplast biogenesis is rescued by exogenous IPP and polyprenols. Knockout of the only known isoprenoid-dependent apicoplast pathway, tRNA prenylation by MiaA, has no effect on blood-stage parasites and thus cannot explain apicoplast reliance on IPP. However, we have localized an annotated polyprenyl synthase (PPS) to the apicoplast. PPS knockdown is lethal to parasites, rescued by IPP and long- (C50) but not short-chain (≤C20) prenyl alcohols, and blocks apicoplast biogenesis, thus explaining apicoplast dependence on isoprenoid synthesis. We hypothesize that PPS synthesizes long-chain polyprenols critical for apicoplast membrane fluidity and biogenesis. This work critically expands the paradigm for isoprenoid utilization in malaria parasites and identifies a novel essential branch of apicoplast metabolism suitable for therapeutic targeting.

Doxycycline (DOX) is a key antimalarial drug thought to kill Plasmodium parasites by blocking protein translation in the essential apicoplast organelle. Clinical use is primarily limited to prophylaxis due to delayed second-cycle parasite death at 1-3 µM serum concentrations. DOX concentrations >5 µM kill parasites with first-cycle activity but have been ascribed to off-target mechanisms outside the apicoplast. We report that 10 µM DOX blocks apicoplast biogenesis in the first cycle and is rescued by isopentenyl pyrophosphate, an essential apicoplast product, confirming an apicoplast-specific mechanism. Exogenous iron rescues parasites and apicoplast biogenesis from first- but not second-cycle effects of 10 µM DOX, revealing that first-cycle activity involves a metal-dependent mechanism distinct from the delayed-death mechanism. These results critically expand the paradigm for understanding the fundamental antiparasitic mechanisms of DOX and suggest repurposing DOX as a faster-acting antimalarial at higher dosing whose multiple mechanisms would be expected to limit parasite resistance.

Doxycycline (DOX) is a key antimalarial drug that is thought to kill Plasmodium falciparum parasites by blocking protein translation in the essential apicoplast organelle. Although parasite resistance to DOX has not emerged, clinical use is primarily limited to prophylaxis due to delayed second-cycle parasite death at 1-3 μM, the DOX concentration readily achieved in human plasma at current dosing. Slow antiparasitic activity is thought to be a fundamental limitation of DOX and other antibiotics that target apicoplast maintenance. We report that slightly higher 8-10 μM DOX concentrations kill parasites with first-cycle activity that blocks apicoplast biogenesis. This faster activity is rescued by exogenous isopentenyl pyrophosphate, an essential apicoplast product, confirming an apicoplast-specific target. Exogenous iron rescues first- but not second-cycle activity by 1 or 10 μM DOX, revealing that first-cycle activity involves a metal-dependent mechanism that is distinct from the canonical delayed-death mechanism. These results provide a new paradigm for understanding fundamental antiparasitic mechanisms of DOX and suggest the possibility of repurposing DOX as a faster-acting antimalarial at higher dosing, which remains well tolerated. Multiple mechanisms of DOX activity would be expected to limit parasite resistance.

Isopentenyl pyrophosphate (IPP) is an essential metabolic output of the apicoplast organelle in Plasmodium falciparum malaria parasites and is required for prenylation-dependent vesicular trafficking and other cellular processes. We have elucidated a critical and previously uncharacterized role for IPP in apicoplast biogenesis. Inhibiting IPP synthesis blocks apicoplast elongation and inheritance by daughter merozoites, and apicoplast biogenesis is rescued by exogenous IPP and polyprenols. Knockout of the only known isoprenoid-dependent apicoplast pathway, tRNA prenylation by MiaA, has no effect on blood-stage parasites and thus cannot explain apicoplast reliance on IPP. However, we have localized an annotated polyprenyl synthase (PPS) to the apicoplast lumen. PPS knockdown is lethal to parasites, rescued by IPP, and blocks apicoplast biogenesis, thus explaining apicoplast dependence on isoprenoid synthesis. We hypothesize that PPS synthesizes long-chain polyprenols critical for apicoplast membrane fluidity and biogenesis. This work critically expands the paradigm for isoprenoid utilization in malaria parasites and identifies a novel essential branch of apicoplast metabolism suitable for therapeutic targeting.

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