Restructured mitochondrial-nuclear interaction in P. falciparum dormancy and persister survival of artemisinin exposure

Artemisinin and its semi-synthetic derivatives (ART) are fast acting, potent antimalarials; however, their use in malaria treatment is frequently confounded by recrudescences from bloodstream Plasmodium parasites that enter into and later reactivate from a dormant persister state. Here we show that the mitochondria of dihydroartemisinin (DHA)-exposed persisters are dramatically altered and enlarged relative to the mitochondria of young, actively replicating ring forms. Persister forms exhibit restructured mitochondrial-nuclear associations and an altered metabolic state consistent with stress from reactive oxygen species. New contacts between the mitochondria and nuclei may support communication pathways of mitochondrial retrograde signaling, resulting in transcriptional changes in the nucleus as a survival response. Further characterization of the organellar interactions and metabolic dependencies of persisters may suggest strategies to combat recrudescences of malaria after treatment.


Introduction 39
Artemisinin, from the Chinese natural Qinghaosu remedy, provides the basis for 40 first line treatments of malaria worldwide, particularly for the deadliest form 41 caused by Plasmodium falciparum [1]. Artemisinin and its semi-synthetic 42 derivatives (collectively abbreviated here as ART) are among the best 43 antimalarials for rapid clearance of parasitemia and resolution of illness [2,3]. 44 Yet, despite these invaluable qualities, lasting cures from ART treatment are not 45 assured: since the time of its introduction, frequent recrudescences have been 46 reported after ART monotherapy, necessitating the utilization of a partner drug 47 for their prevention [4]. Various studies have identified dormant persister forms 48 from erythrocytic-stage populations that give rise to these recrudescences [5][6][7][8]. 49 Much remains to be understood about the nature of these persisters and how 50 they develop from only a small fraction (estimated less than ~1% [5,7]) of the 51 circulating ART treated parasite population. 52 53 Although dormant hypnozoites in the liver stage cycle of certain Plasmodium spp. 54 were well known, evidence was not reported until 1995 that dormant parasites 55 could be found in erythrocytes after drug and toxin exposure. Nakazawa et al. [9] 56 demonstrated the presence of a small number of the dormant parasites following 57 destruction of the great majority of actively replicating parasites by D-sorbitol 58 treatments or exposure to pyrimethamine. Importantly, the dormant parasites 59 were able to survive these exposures for several days, after which the dormant 60 parasites gave rise to recrudescent populations that were as susceptible to the 61 exposure. GB4 or 803 parasites at 0 -3 hours ring-stage (post-invasion) were 154 placed in cultures at timepoint t = 0 hours with control vehicle (0.1% DMSO) or 155 700 nM DHA/0.1% DMSO. Control or DHA-treated samples were washed twice 156 at t = 6 hours and placed back into culture. At t = 30 hours, the DHA-treated 157 samples were passed through a magnetically activated cell sorting (MACS) 158 column to deplete mature-stage, hemozoin-containing parasites that had 159 survived drug treatment, reducing but not completely removing the contribution of 160 these stages from reinvasion into the next cycle. To isolate and compare the 161 populations of control vehicle-and DHA-exposed GB4 and 803 parasite 162 populations at the beginning of the 2 nd erythrocytic-stage cycle, we performed 163 quadrant of the plot, the gate region labeled "PYK" counts pyknotic parasites that 170 lack mitochondrial potential whereas, in the upper right quadrant of the plot, the 171 gate region labeled MT+ counts parasites that exhibit mitochondrial activity. In 172 the MT+ gate region, increasing SG and MT signals moving upward and to the 173 right arise from the progression of young ring stages to mature stage parasites, 174 as has been described with fluorescent markers in previous studies [27]. Clusters 175 of ring and late stage schizonts are evident in the control populations of both 176 GB4 and 803. Referring to the timeline (Fig. 1C), the clusters of schizonts derive 177 from the end of the first erythrocytic-stage cycle while the more numerous ring 178 stages derive from the beginning of the second cycle. FACS results from the 179 DHA-treated parasites show large numbers of pyknotic parasites that lack 180 mitochondrial potential in the "PYK" region, as expected from drug killing. 181 Surviving parasites, however, are evident in both the GB4 and 803 populations, 182 with greater counts of 803, consistent with their higher RSA survival levels 183 compared to GB4 parasites (Fig. 1A). Thus, in the DHA-treated 803 MT+ 184 distribution, the presence of trophozoites from the delay in the first 185 intraerythrocytic cycle contributes to the appearance of a more continual 186 representation of developmental stages across the intensity spectrum. 187

188
While the SG+MT+ subsets of the GB4 and 803 control populations contained 189 predominantly ring-stage forms, the DHA-treated parasites after passage through 190 the magnetic column at t = 30 hrs were enriched for persister forms [11].Thus,191 FACS at t = 50 hours identified a distribution of persisters as well as rings and 192 mature stages that escaped removal by the single magnetic column. From this 193 distribution, we were able to use the criteria of size and fluorescence intensity to 194 identify and individually study these different forms.  After DHA treatment of the synchronized GB4 parasites followed by magnetic 218 column separation at t = 30 hrs, actively growing parasites were mostly 219 eliminated, and surviving persisters greatly predominated in the DHA-treated 220 GB4 population captured for analysis at t = 50 hours. Figure 3A (right panel) 221 presents the nuclear and mitochondrial morphologies of six parasites from this 222 population: one exhibited a smooth oblate mitochondrion separate from the 223 nucleus as is characteristic of an actively growing ring-stage form, whereas the 224 five others had distinctly different appearances, with rumpled and corrugated 225 mitochondria in close approximation to the nuclei. The mitochondria partially 226 enwrapped the nuclei and were often enlarged compared to the control ring-227 stage parasites. Contact sites appeared to be present between the nuclear DNA 228 and mitochondria as another potential feature of the persister phenotype. Mitochondrial volume comparisons of control and DHA-exposed GB4 or 803 265 parasites revealed an increase in the mitochondrial volume after DHA treatment. 266 In control GB4 parasites ( Figure 4A, right), the median mitochondrial volume and 267 IQR was 0.26 (0.20 -0.34) µm 3 , which was much smaller than the post DHA 268 treatment mitochondrial median volume of 1.30 (0.64 -1.9) µm 3 , representing a 269 5-fold increase (p < 0.0001, unpaired t-test). In control 803 parasites, the median 270 mitochondrial volume was 0.31 (0.20 -0.43) µm 3 while the DHA-exposed 271 median volume was 0.54 (0.23 -1.5) µm 3 ( Figure 4B, right) (p < 0.0001, 272 unpaired t-test). Compared to the results from GB4 parasites, the less dramatic 273 increase of median mitochondrial volume in DHA-exposed 803 parasites at t = 50 274

DHA Treatment 290
After examining the morphological characteristics of persister mitochondria and 291 quantifying their close apposition to nuclei at t = 50 hours, we looked for the 292 presence of these features in persisters 1 -2 weeks old, before their 293 recrudescence in culture. Accordingly, we followed the in vitro cultures of 294 synchronized 803 and GB4 parasites after treatment with 700 nM DHA for 6 295 hours and three successive 5% D-Sorbitol treatments at 24, 48 and 72 hours, as 296 described previously [32]. Figure Figure 4E for day 11. On day 5, persister morphology 305 was observed in the 803 line, as evidenced by the large and rumpled 306 mitochondrial volumes enwrapping the nucleus ( Figure 4D). On day 11, this 307 persister morphology continued to characterize the GB4 and 803 parasites and 308 was similar to the morphology observed earlier, at t = 50 hours and at 5 days. 309 However, 803 parasites of the day 11 population were also found with smaller 310 oblate mitochondria separate from the nucleus, i.e. with features that resembled 311 the actively growing ring-stage forms of the original 803 control group ( Figure 4E, 312 compared with Figure 3B). Such evidence for actively growing ring-stage forms 313 among persisters in the 11-day 803 population is consistent with waking from 314 dormancy at the beginning of the recrudescence curve shown in Figure 4C. (1.2 -2.2) µm 3 ; p = 0.09), unpaired t-test). However, on day 5, persister 803 332 parasites had a smaller nuclear volume of 1.2 (1.1 -1.5) µm 3 than on day 11 (p < 333 0.0001, unpaired t-test). These responses may relate to an innate growth bistability phenomenon whereby 480 a fraction of the drug-exposed population switches into the metabolic quiescence 481 of persister forms, as we have discussed elsewhere [53]. 482

483
Our findings from autofluorescence FLIM-phasor analysis indicate an increased 484 ratio amount of free NADH in persisters relative to actively replicating ring-stage 485 parasites. This measure relates to the redox state of the cells. In other systems, 486 human breast cell lines exposed to potassium cyanide (KCN) treatment or 487 exposed to hypoxic conditions, autofluorescence FLIM revealed a higher amount 488 of free NADH due to inhibition of cellular respiration [54]. Increased 489 concentrations of free NADH in the mitochondria have been shown to promote 490 increased ROS production [55]. When assaying ART activity in tumor cell lines, 491 ETC function in the mitochondria was found to be essential for apoptosis through 492 ROS production [43]. Taken together, these findings suggest that activation of 493 the endoperoxide bridge of artemisinin by mitochondrial heme leads to altered 494 ETC function, increased free NADH, and increased ROS species with 495 concomitant mitochondrial damage. Columbia, MD), at 5% hematocrit, 37˚C, and a gas mixture containing 90% N2, 522 5% CO2, and 5% O2. To monitor parasitemia, thin blood films were prepared on 523 glass slides, fixed with methanol and stained for 15 minutes with 20% Giemsa 524 staining (Sigma-Aldrich, St. Louis, MO). Using bright-field microscopy and an 525 100× oil objective, an estimated 1,000 erythrocytes were counted, and the 526 number of parasitized cells was used to estimate percentage parasitemia. In 527 recrudescence experiments, media was changed every other day and fresh 528 erythrocytes were added every four days. When media was changed, blood films 529 were made and used to monitor parasitemia. 530 531

Ring-stage Survival Assay (RSA) 532
803 and GB4 cultures were tightly synchronized to obtained 0 -3 hour ring 533 stages through two successive 5% D-sorbitol treatments 46 hours apart [24]. 534 Each sorbitol treatment lasted 10 minutes at room temperature. Immediately 535 following the second sorbitol treatment, cultures were adjusted to 2% parasitemia 536 and 5% hematocrit in a total volume of 10 mL in a T25 flask (Thermo Fisher 537 Scientific, Waltham, MA). Thin blood smears were prepared just before drug 538 exposure. Cultures were then treated at a final concentration of 0.1% DMSO or 539 700 nM DHA/0.1% DMSO by addition of 10 l of DMSO or 10 l of 700 µM DHA 540 for 6 hours. After 6 hours of incubation, cells were washed and returned to 541 culture in drug-free cRPMI. Sixty-six to ninety hours post DHA treatment, thin 542 blood smears were prepared, fixed with 100% methanol, and stained with 20% 543 were suspended in 2 mL of cold cRPMI and passed three times through the 555 column over a period of 3 -5 hours. Afterwards, the column was washed with 30 556 mL of cRPMI. Eluates were combined and pelleted at 2,500 rpm for 5 minutes, 557 resuspended in 10 mL of cRPMI, and returned to culture. As the parasitized 558 erythrocytes approached maturity, the cultures were placed on a rotator in the 559 37˚C incubator to minimize the occurrence of multiply-infected relative to singly-560 infected cells. a polystyrene tube with 1× HBSS/2% FBS at a final concentration of 1 mL. One 683 hundred µL was taken from these samples for a blood film, and the cells in the 684 samples were pelleted at 2,500 rpm for 5 minutes, resuspended in 180 µL of 685 cRPMI, and transferred to a 96 well plate. Twenty µL of a 50% erythrocyte stock 686 were added to each well to obtain a 5% hematocrit culture. Media were changed 687 on alternate days and 150 µL of 3.3% hematocrit solution were added every 4 or 688 5 days to replenish erythrocytes lost to sampling. The parasitemia in each well 689 was monitored through thick and thin smears, using 1 µL of parasitized 690 erythrocytes for each smear, and the time it took for the parasitemia to reach 691 0.5% was recorded. consistent with a more mixed population of cells, many with the rumpled, 967 corrugated mitochondria closely apposed to nuclei and others with the smooth, 968 oblate mitochondria separate from the nuclei. 969 970 971