Malaria remains a serious global health problem, with hundreds of thousands of annual deaths due 69
to Plasmodium falciparum parasites. The absence of a potent, long-lasting vaccine and parasite 70 tolerance to frontline artemisinin combination therapies continue to challenge malaria elimination 71 efforts. Furthermore, there are strong concerns that the current COVID-19 pandemic will disrupt 72 malaria prevention and treatment efforts in Africa and cause a surge in malaria deaths that unravels 73 decades of progress (1) . Deeper understanding of basic parasite biology and the mechanisms of 74 current drugs will guide their optimal use for malaria prevention and treatment and facilitate 75 development of novel therapies to combat parasite drug resistance. 76
Tetracycline antibiotics like DOX are thought to kill eukaryotic P. falciparum parasites by 77 inhibiting prokaryotic-like 70S ribosomal translation inside the essential apicoplast organelle 78 ( Figure 1) (2). Although stable P. falciparum resistance to DOX has not been reported, clinical use 79 is largely limited to prophylaxis due to delayed activity against intraerythrocytic infection (3, 4) . 80
Parasites treated with 1-3 µM DOX, the drug concentration sustained in human serum with current 81 100-200 mg dosage (5), continue to grow for 72-96 hours and only die after the second 48-hour 82 intraerythrocytic growth cycle when they fail to expand into a third cycle (2). Slow antiparasitic 83 activity is believed to be a fundamental limitation of DOX and other antibiotics that block 84 apicoplast-maintenance pathways (4, 6). First-cycle anti-Plasmodium activity has been reported 85 for DOX and azithromycin concentrations >3 µM, but such activities have been ascribed to targets 86 outside the apicoplast (2, 7, 8). A more incisive understanding of the mechanisms and parameters 87 that govern first versus second-cycle DOX activity can inform and improve clinical use of this 88 valuable antibiotic for antimalarial treatment. We therefore set out to test and unravel the 89 mechanisms and apicoplast specificity of first-cycle DOX activity. 90
First-cycle activity by 10 µM DOX has an apicoplast-specific mechanism. Prior studies have 92
shown that 200 µM isopentenyl pyrophosphate (IPP), an essential apicoplast product, rescues 93 parasites from the delayed-death activity of 1-3 µM DOX, confirming an apicoplast-specific target 94 (7). To provide a baseline for comparison, we first used continuous-growth and 48-hour growth-95 inhibition assays to confirm that IPP rescued parasites from 1 µM DOX ( Figure 2A ) and that DOX 96 concentrations >5 µM killed parasites with first-cycle activity ( (2). To test the apicoplast specificity of first-cycle DOX 98 activity, we next asked whether 200 µM IPP could rescue parasites from DOX concentrations >5 99 µM. We observed that IPP shifted the 48-hour EC50 value of DOX from 5 ± 1 to 12 ± 2 µM 100 (average ± SD of 5 independent assays, P = 0.001 by unpaired t-test) ( Figure 2C and Figure 2 -101 figure supplement 1), suggesting that first-cycle growth defects from 5-10 µM DOX reflect an 102 apicoplast-specific mechanism but that DOX concentrations >10 µM cause off-target defects 103 outside this organelle. We further tested this conclusion using continuous growth assays performed 104 at constant DOX concentrations. We observed that IPP fully or nearly fully rescued parasites from 105 first-cycle growth inhibition by 10 µM but not 20 or 40 µM Dox ( with first-cycle activity by an apicoplast-specific mechanism. 108 109 10 µM DOX blocks apicoplast biogenesis in the first cycle: Inhibition of apicoplast biogenesis 110 in the second intraerythrocytic cycle is a hallmark of 1-3 µM DOX-treated P. falciparum, resulting 111 in unviable parasite progeny that fail to inherit the organelle (2). IPP rescues parasite viability after 112 the second cycle without rescuing apicoplast inheritance, such that third-cycle daughter parasites 113 lack the organelle and accumulate apicoplast-targeted proteins in cytoplasmic vesicles (7). We 114 treated synchronized ring-stage D10 (9) or NF54 (10) parasites expressing the acyl carrier protein 115 leader sequence fused to GFP (ACPL-GFP) with 10 µM DOX and assessed apicoplast morphology What is the molecular mechanism of faster apicoplast-specific activity by 10 µM DOX? We first 124 considered the model that both 1 and 10 µM DOX inhibit apicoplast translation but that 10 µM 125 DOX kills parasites faster due to more stringent translation inhibition at higher drug 126 concentrations. This model predicts that treating parasites simultaneously with multiple distinct 127 apicoplast-translation inhibitors, each added at a delayed death-inducing concentration, will 128 produce additive, accelerated activity that kills parasites in the first cycle. To test this model, we 129 treated synchronized D10 parasites with combinatorial doses of 2 µM DOX, 2 µM clindamycin, 130 and 500 nM azithromycin and monitored growth over 3 intraerythrocytic cycles. Treatment with 131 each antibiotic alone produced major growth defects at the end of the second cycle, as expected 132 for delayed-death activity at these concentrations (6). Two-and three-way drug combinations 133 caused growth defects that were indistinguishable from individual treatments and provided no 134 evidence for additive, first-cycle activity ( Figure 3A and results contradict a simple model that 1 and 10 µM DOX act via a common translation-blocking 136 mechanism and suggest that the first-cycle activity of 10 µM DOX is due to a distinct mechanism. 137 138 Exogenous iron rescues parasites from first-but not second-cycle effects of 10 µM DOX. 139
Tetracycline antibiotics like DOX tightly chelate a wide variety of di-and trivalent metal ions via 140 their siderophore-like arrangement of exocyclic hydroxyl and carbonyl groups (Figure 1) , with a 141 reported affinity series of Fe 3+ >Fe 2+ >Zn 2+ >Mg 2+ >Ca 2+ (11, 12). Indeed, tetracycline interactions 142 with Ca 2+ and Mg 2+ ions mediate cellular uptake and binding to biomolecular targets such as the 143 tetracycline repressor and 16S rRNA (12, 13). We next considered a model that first-cycle effects 144 of 10 µM DOX reflect a metal-dependent mechanism distinct from ribosomal inhibition causing 145 second-cycle death. To test this model, we investigated whether exogenous metals rescued 146 parasites from 10 µM DOX. We failed to observe growth rescue by 10 µM ZnCl2 (toxicity limit 147 (14)) or 500 µM CaCl2 in continuous-growth ( Figure 3B and We also observed that 500 µM FeCl3 but not CaCl2 rescued first-cycle apicoplast-155 branching in 10 µM DOX ( Figure 3E and Figure 3 -figure supplement 2). These observations 156 contrast with IPP, which rescued parasite viability in 10 µM DOX but did not restore apicoplast 157 branching ( Figure 2E ). We further noted that FeCl3 selectively rescued parasites from the 158 apicoplast-specific, first-cycle growth effects of 10 µM DOX but did not rescue parasites from the We first considered whether exogenous FeCl3 might selectively rescue 10 µM DOX 174 activity by blocking or reducing its uptake into the parasite apicoplast, since metal chelation has 175 been reported to influence the cellular uptake of tetracycline antibiotics in other organisms (12). 176
However, 500 µM FeCl3 or MgCl2 did not rescue second-cycle parasite death in continuous growth 177 assays with 10 µM (Figures 3D) or 1 µM DOX ( Figure 3F ). Furthermore, exogenous iron resulted 178
in only a small, 1.5-µM shift in EC50 value from 0.5 to 2 µM in a 96-hour growth inhibition assay, 179 in contrast to the 10.5-µM shift provided by IPP (Figure 3-figure supplement 1) . These results 180 strongly suggest that DOX uptake into the apicoplast is not substantially perturbed by exogenous 181 iron. The inability of 500 µM FeCl3 to rescue first-cycle activity by ≥20-µM DOX ( Figure 3G ) 182 further suggests that general uptake of DOX into parasites is not substantially affected by 183 exogenous iron. 184
We propose two distinct models to explain the metal-dependent effects of 10 µM DOX, 185
both of which could contribute to apicoplast-specific activity. First, DOX could directly bind and 186 sequester labile iron within the apicoplast, reducing its bioavailability for Fe-S cluster biogenesis 187 and other essential iron-dependent processes in this organelle. Indeed, prior work has shown that 188 apicoplast biogenesis requires Fe-S cluster synthesis apart from known essential roles in 189 isoprenoid biosynthesis (17). In this first model, rescue by exogenous FeCl3 would be due to 190 restoration of iron bioavailability, while modest rescue by 500 µM MgCl2 may reflect competitive 191 displacement of DOX-bound iron to restore iron bioavailability. RPMI growth medium already 192 contains ~400 µM Mg 2+ prior to supplementation with an addition 500 µM MgCl2, and thus Mg 2+ 193 availability is unlikely to be directly limited by 10 µM DOX. Consistent with a general mechanism 194 that labile-iron chelation can block apicoplast biogenesis, we observed in preliminary studies that 195 the anti-Plasmodium growth inhibition caused by the highly-specific iron chelator, deferoxamine In a second model, DOX could bind to additional macromolecular targets within the 205 apicoplast (e.g., a metalloenzyme) via metal-dependent interactions that inhibit essential functions 206 required for organelle biogenesis. Exogenous 500 µM Fe 3+ would then rescue parasites by 207 disrupting these inhibitory interactions via competitive binding to DOX. This second model would 208
be mechanistically akin to diketo acid inhibitors of HIV integrase like raltegravir that bind to active 209 site Mg 2+ ions to inhibit integrase activity but are displaced by exogenous metals (19, 20) . To test 210 this model, we are developing a DOX-affinity reagent to identify apicoplast targets that interact 211 with doxycycline and whose inhibition may contribute to first-cycle DOX activity. There has been a prevailing view in the literature that delayed-death activity is a 226 fundamental limitation of antibiotics like DOX that block apicoplast maintenance (21, 22). Our 227 results emphasize that DOX is not an intrinsically slow-acting antimalarial drug and support the 228 emerging paradigm (23-25) that inhibition of apicoplast biogenesis can defy the delayed-death 229 phenotype to kill parasites on a faster time-scale. The first-cycle, iron-dependent impacts of 10 230 µM DOX or 15 µM DFO on apicoplast biogenesis also suggest that this organelle may be 231 especially susceptible to therapeutic strategies that interfere with acquisition and utilization of iron, 232 perhaps due to limited uptake of exogenous iron and/or limited iron storage mechanisms in the 233
Finally, this work suggests the possibility of repurposing DOX as a faster-acting 235 antiparasitic treatment at higher dosing, whose multiple mechanisms would be expected to limit 236 parasite resistance. Prior studies indicate that 500-600 mg doses in humans achieve sustained 237 serum DOX concentrations ≥5 µM for 24-48 hours with little or no increase in adverse effects (26, 238 27). DOX is currently contraindicated for long-term prophylaxis in pregnant women and young 239 children, two of the major at-risk populations for malaria, due to concerns about impacts on fetal 240 development and infant tooth discoloration, respectively, based on observed toxicities for other 241 tetracyclines (28). Recent studies suggest that these effects are not associated with short-term DOX 242 use (28, 29), but more work is needed to define the safety parameters that would govern short-term Imaging experiments were independently repeated twice. Parasite nuclei were visualized by 295
incubating samples with 1-2 µg/ml Hoechst 33342 (Thermo Scientific Pierce 62249) for 10-20 296 minutes at room temperature. The parasite apicoplast was visualized in D10 (9) or NF54 297 mevalonate-bypass (10) cells using the ACPleader-GFP expressed by both lines. Images were taken 298 on DIC/brightfield, DAPI, and GFP channels using either a Zeiss Axio Imager or an EVOS M5000 299 imaging system. Fiji/ImageJ was used to process and analyze images. All image adjustments, Hoechst stain). 20-40 parasites were examined for each treatment condition on each given day for 501 duplicate experiments, and data were plotted as the average percentage of parasites in each 502
population that displayed an elongated, punctate, or dispersed apicoplast GFP signal. For clarity, 503 error bars are not displayed but standard deviations were <10% in all conditions. Cell-percentage 504 differences were analyzed by unpaired t-test (P values in parentheses, ns = not significant) .  505  506  507  508  509  510  511  512  513  514  515  516  517  518  519  520  521  522  523  524 later (green = ACPL-GFP, blue = nuclear Hoechst stain). 532