Restructured Mitochondrial-Nuclear Interaction in Plasmodium falciparum Dormancy and Persister Survival after Artemisinin Exposure

Abstract

Artemisinin and its semisynthetic 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 provide evidence that the mitochondria of dihydroartemisinin (DHA)-exposed persisters are dramatically altered and enlarged relative to the mitochondria of young, actively replicating ring forms. Restructured mitochondrial-nuclear associations and an altered metabolic state are 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 organelle communication and metabolic dependencies of persisters may suggest strategies to combat recrudescences of malaria after treatment. IMPORTANCE The major first-line treatment for malaria, especially the deadliest form caused by Plasmodium falciparum, is combination therapy with an artemisinin-based drug (ART) plus a partner drug to assure complete cure. Without an effective partner drug, ART administration alone can fail because of the ability of small populations of blood-stage malaria parasites to enter into a dormant state and survive repeated treatments for a week or more. Understanding the nature of parasites in dormancy (persisters) and their ability to wake and reestablish actively propagating parasitemias (recrudesce) after ART exposure may suggest strategies to improve treatment outcomes and counter the threats posed by parasites that develop resistance to partner drugs. Here, we show that persisters have dramatically altered mitochondria and mitochondrial-nuclear interactions associated with features of metabolic quiescence. Restructured associations between the mitochondria and nuclei may support signaling pathways that enable the ART survival responses of dormancy.

Keywords: Airyscan microscopy; artemisinin-based combination therapy; drug resistance; fluorescence lifetime imaging; malaria; mitochondrial retrograde response.

FIG 1

Recrudescence of DHA-treated P. falciparum parasites in vitro and sorting of their dormant persister forms by FACS. (A) Recrudescence curves of GB4 and 803 parasites after exposure of early ring stages to 700 nM DHA for 6 h and three daily 5% d-sorbitol treatments. (B) Schematic of GB4 and 803 parasite preparations for study by ASM and FLIM-phasor analyses. The parasites were synchronized through two sorbitol treatments, 46 h apart, to obtain 0- to 3-h rings. At the start of the experiment (t = 0 h), the GB4 or 803 populations of 0- to 3-h rings were treated at 2% parasitemia with either 700 nM DHA or 0.1% DMSO vehicle for 6 h, washed, and returned to culture. In one arm of the study, DHA-treated parasites at t = 30 h were passed through a magnetic depletion column to remove mature-stage parasites that were present after DHA treatment. The recovered parasites were then returned to culture on a rocking incubator for 20 h at 37°C along with the vehicle control parasites. At t = 50 h, 1 ml of each culture was stained with either SG plus MT for FACS and ASM or with MT only for FACS and FLIM-phasor autofluorescence analyses. In the other arm of the study, parasite populations exposed to DHA or DMSO only were given three daily treatments with 5% sorbitol to select for persister forms. On days 5 and 11, 1 ml of each culture was stained and studied in the same way as the magnetically purified samples. Schematic created with BioRender (Toronto, Canada). (C) SG-positive parasites at t = 50 h were gated into two populations based on MT fluorescence intensity. Large numbers of pyknotic forms in the PYK gates of the DHA-treated populations represent parasites killed by the DHA. Parasites from the MT+ gate were used for ASM. SG, SYBR green I; MT, MitoTracker Deep Red FM. Uninfected erythrocytes that lack SG and MT signals are indicated in the lower left quadrant of the plots.

FIG 2

ASM images of control and DHA-treated GB4 and 803 parasites at t = 50 h. Individual panels show processed ASM images of the mitochondrial (red) and nuclear (green) fluorescence signals from six representative parasites in each treatment group separated by FACS. (A) GB4 parasites from control 0.1% DMSO vehicle-treated samples present smooth, oblate mitochondrial and nuclear volumes. Images in this control group are consistent with young ring-stage parasites in the culture at t = 50 h. DHA-treated parasites show the distinctly different morphologies of persisters, with rumpled and corrugated mitochondrial volumes that are in close approximation to the nuclei. (B) Images of the control 803 parasites at t = 50 h also have the smooth, oblate appearance of mitochondria separated from the nucleus as expected of actively replicating young parasite ring forms. Images of DHA-treated 803 parasites compared to those of the treated GB4 population are consistent with a mixed population of cells, many with the rumpled, corrugated mitochondria characteristic of persisters, while others have the smooth, oblate mitochondria apart from nuclei that is characteristic of ring-stage parasites.

FIG 3

Quantifications of mito-nuclear distances and mitochondrial volumes of GB4 and 803 parasites at t = 50 h, day 5, and day 11 after DHA treatment. (A) Scatterplots, medians, and interquartile ranges (IQR) of mito-nuclear distances in the populations of GB4 and 803 parasites exposed to 0.1% DMSO (control) or 700 nM DHA for 6 h, selected, sorted, and analyzed as described in the text. In the mito-nuclear distance scatterplots, there were five outliers beyond the bounds of the y axis in the GB4 control group (at 2.8 μm, 2.5 μm, 2.2 μm, 2.7 μm, and 6.8 μm), one outlier in the 803 control group (at 3.7 μm), and three outliers in the 803 DHA group (at 2.6 μm, 2.0 μm, 5.5 μm). (B) Mitochondrial volume scatterplots, medians, and IQR from the same populations used for mito-nuclear distance determinations. (C) Middle panel presents representative ASM images of DHA-treated/sorbitol-selected 803 parasites on day 5 posttreatment. The six examples of persisters show rumpled, corrugated mitochondria in close approximation to the nuclei. The right and left panels present the surface images of DHA-treated/sorbitol-selected GB4 and 803 parasites at day 11. Persister morphologies are evident in the images from each parasite line, including rumpled and corrugated mitochondria in close approximation to the nuclei.

FIG 4

Metabolic phenotyping of untreated control versus DHA-treated GB4 and 803 parasites. (A) Schematic flow of FLIM-phasor data collection and analysis. Parasites were stained only with MT, sorted by FACS, and seeded into a 10- by 10-μm polydimethylsiloxane (PDMS) stencil microwell covered with 0.01% poly-lysine. The region of interest (ROI) in a parasite is established by the boundary of MT fluorescence, and this ROI mask is applied to the lifetime datafile represented by its FLIM intensity image. At least 10 million photon events are counted while limiting the time of exposure of these parasites to 750 nm excitation. Next, Fourier transformation of the FLIM data is performed on the data from each pixel, and the frequency domain results are averaged to represent a data point on the 2D phasor plot. Each position has coordinates G (from 0 to 1) and S (from 0 to 0.5); single exponential decays fall upon the semicircle, whereas complex multiexponential decays, such as occur with fluorescent lifetimes of metabolic coenzymes, fall within the semicircle. A shift to the right (shorter fluorescence lifetime) indicates increased free NADH, whereas a shift to the left (longer lifetime) indicates increased enzyme-bound NADH. (B) Autofluorescence FLIM-phasor graphs from the GB4 and 803 parasite populations at t = 50 h. The phasor distribution of untreated control GB4 parasites has a mean coordinate of 0.7542, 0.2150, whereas the distribution of DHA-treated parasites has a mean coordinate of 0.7971, 0.1879 (P < 0.0001, Pillai trace). This shift after DHA treatment is toward increased free NADH, consistent with the quiescent metabolic state of persisters. The distribution of untreated 803 parasites at t = 50 h (right) has a mean coordinate of 0.6341, 0.1400, and the phasor distribution of DHA-treated parasites has a mean coordinate of 0.6270, 0.1570. The distribution of DHA-treated 803 parasites is slightly shifted upward relative to control (P < 0.05, Pillai trace) but not to the left or right, consistent with the presence of actively replicating 803 parasites that survived ring-stage exposure to DHA. Asterisks mark the mean phasor positions of the control and DHA-treated parasite distributions. Comparison of intensity images in the same field of view before and after FLIM acquisition confirmed little or no photobleaching. (C) Autofluorescence FLIM-phasor graphs for DHA-treated/sorbitol-selected GB4 and 803 on day 5 and day 11. In the phasor distributions from day 5 (left), the treated GB4 and 803 parasites have mean coordinates of 0.72, 0.21 and 0.77, 0.21, respectively. Consistent with depletion of the many actively replicating parasites from the 803 population by three daily sorbitol treatments, the 803 phasor distribution is shifted right with quiescence and increased levels of free NADH in persisters. The genetic backgrounds and baseline metabolic states of African GB4 and Southeast Asian 803 parasites may contribute to the different positions of the 803 and GB4 coordinates (P < 0.0001, Pillai trace).