Screening the MMV Pathogen Box reveals the mitochondrial bc1-complex as a drug target in mature Toxoplasma gondii bradyzoites
- P 6: Metabolism of Microbial Pathogens, Robert Koch Institute, Germany
- ZBS 4: Advanced Light and Electron Microscopy, Centre for Biological Threats and Special Pathogens 4, Robert Koch-Institute, Germany
- FG 16: Mycotic and Parasitic Agents and Mycobacteria, Robert Koch Institute, Germany
Figures
Figure 1 with 1 supplement
An in vitro screen reveals tachy- and bradyzocidal properties of the Medicines for Malaria Venture (MMV) Pathogen Box compounds.
(A) Experimental scheme of the MMV compound screen on T. gondii tachyzoites in human foreskin fibroblast cells. (B) Growth curves of tachyzoites treated with 10 µM of each MMV compound for 7 days (shades of gray), a solvent control (green), and an inhibition control (red). (C) Experimental scheme of the MMV compound screen on bradyzoites matured in myotubes. (D) Growth curves of bradyzoites treated with 10 µM of each compound (shades of gray) or a solvent control (green) for 7 days, followed by a tachyzoite regrowth phase. (E) Pie chart displaying the number of Pathogen Box compounds and their effects on our in vitro cultures.
Figure 1—figure supplement 1
Longitudinal screening procedure.
All 400 compounds of the Medicines for Malaria Venture (MMV) Pathogen Box were screened against (A) tachyzoites (n=4) and (B) in vitro tissue cysts (n=3). Plotted fluorescence values were background-subtracted and reflect the abundance of parasites per treatment over time (black line = solvent control, green line = inhibitory compound, red line = ineffective compound, dashed line = growth threshold).
Figure 2 with 1 supplement
Confirmed Pathogen Box compounds that target both tachyzoites and mature bradyzoites.
(A) shows the growth curve of MMV689480 (buparvaquone), one of the 16 dually active compounds, in a tachyzoite growth assay (n=8). The infected cultures were treated for 7 days, and regrowth in absence of the inhibitor was observed for another week. Depending on the development of the DMSO control, the half-maximal inhibitory concentration (IC50) was determined at either day 4 or 7 (arrow). (B) Shown is the respective data plotted as relative fluorescence units depending on the concentration at day 7. (C) With 4 weeks of age, mature bradyzoite cultures (n=6), the half-maximal lethal concentration (LC50) was determined at the time point which ideally reflected the dose-response curve (in the case of buparvaquone at day 31; arrow). (D) The LC50 of buparvaquone on encysted bradyzoites determined at day 31. All IC50 and LC50 of dually active compounds are summarized in (E) for tachyzoite and (F) for bradyzoite cultures. The gray dotted line indicates 1 µM in both charts. (G) Comparison of the minimal lethal concentration of each compound on tachyzoites and bradyzoites. Mann-Whitney test of eight replicative measurements on tachyzoites and six replicates of bradyzoites from two independent experiments each ***p<0.001.
Figure 2—figure supplement 1
Properties of dually active screening hits.
(A) Table showing the relative amount of tachyzocidal, bradyzocidal, and dually active compounds per originally indicated target species. For example, 11 compounds were indicated by Medicines for Malaria Venture (MMV) to be active against Cryptosporidium, and 36% and 27% of these are bradyzocidal and dually active, respectively. In contrast, 67% of the 15 compounds indicated as active against Toxoplasma possessed activity against tachyzoites, but none were active against bradyzoites. (B) Estimated lipophilicity approximated by predicted partition coefficient suggests that active compounds generally exhibit an increased lipophilicity compared to inactive compounds. (C) A Venn diagram showing intersections of tachyzocidal and bradyzocidal compounds as well as their predicted gastrointestinal absorption capability (GIA) and blood-brain barrier (bbb) permeability. (D) Tabulation of compound IC50 and LC50 data of confirmed active compounds. The calculated IC50s and LC50s of 16 dually active compounds from Figure 2 are summarized in this table. The tachyzoite IC50 values represent eight replicates, the LC50 values were generated from six replicate bradyzoite cultures. Lowest cytotoxicity values were recorded during the primary screen as detailed in the Materials and methods section.
Figure 3 with 1 supplement
Untargeted metabolomics of tachyzoites treated with six screening hits effective against both stages of T. gondii.
Volcano plots showing the parasitic, intracellular metabolic phenotype caused by Medicines for Malaria Venture (MMV) Pathogen Box compounds that inhibited tachyzoites and bradyzoites during the screen. The metabolome was measured with two columns of complementary chemistries to enhance metabolite coverage. Blue dots indicate metabolites that were significantly reduced compared to DMSO-treated parasites, and red dots indicate significantly accumulated metabolites (p<0.05, log2 fold change <-1 or >1, 3× biological replicates with 3× technical replicates each, n=9). Abbreviations: AcCoA, acetyl-CoA; AcCys, acetylcysteine; AcPhe, acetylphenylalanine; AMP, adenosine monophosphate; ATP, adenosine triphosphate; CarbAsp, carbamoyl aspartate; cGMP-AMP, cyclic guanosine monophosphate-adenosine monophosphate; CMP, cytosine monophosphate; DHO, dihydroorotate; GABA, gamma-aminobutyric acid; GluCys, glutamylcysteine; GluGln, glutamylglutamine; Gro1P, glyceraldehyde 1-phosphate; Gro3P, glyceraldehyde 3-phosphate; GSH, glutathione oxidized; GSSG, glutathione reduced; GTP, guanosine triphosphate; HP4, hexose phosphate No. 4 (fourth chromatographic peak of 260.02972); HydKyn, hydroxylkynurenine; MEP, 2-C-methyl-D-erythritol 4-phosphate; NAcAla, N-acetylalanine; NAcCys, N-acetylcysteine; NAcMet, N-acetylmethionine; NAcPhe, N-acetylphenylalanine; OAcSer, O-acetylserine; PalmCarn, palmitoylcarnitine; PEP, phosphoenolpyruvate; PseudoU, pseudouridine; Trp, tryptophan; UMP, uridine monophosphate; UTP, uridine triphosphate.
Figure 3—figure supplement 1
Principal component analysis of LCMS metabolomes of treated parasites using two chromatographic columns.
(A) Principal component analysis of metabolites from extracellular tachyzoites that were treated with indicated Medicines for Malaria Venture (MMV) compounds. Metabolites were separated using BEH-amide column-HILIC chromatography and detected by mass spectrometry. (B) The same metabolite extracts have been re-analyzed using pHILIC-column HILIC chromatography for separation instead. (C) Chemical structures of dually active and tested compounds as provided by MMV.
Figure 4 with 1 supplement
The metabolic response to electron transport chain inhibitors and MMV1028806.
(A) Scheme of the affected pathways upon bc1-complex inhibition. Intracellular tachyzoites were grown in the presence of U-13C-glucose (B, C) or 15N-amide-glutamine (D, E) instead of unlabeled carbon sources and treated with atovaquone (ATQ), buparvaquone (BPQ), and MMV1028806 (MMV) for 3 hr. (B, D) The treatment-induced changes of mitochondrial electron transport chain (mETC)-related metabolite abundances are shown as log2-fold changes in comparison to DMSO-treated cultures. Shown are three replicate measurements. (C, E) Shown is the average isotopologue distribution of mETC-related metabolites. The pie charts depict the number of heavy carbon or nitrogen atoms incorporated into the respective metabolites (M+0 in light gray represents the unlabeled metabolite, while M+1 in light blue indicates the integration of one heavy atom into the molecule). Shown are the means of three replicate measurements. Abbreviations: ATQ, atovaquone; BPQ, buparvaquone; CarbAsp, carbamoyl-aspartate; DHO, dihydroorotate; DHODH, dihydroorotate dehydrogenase; GABA, gamma-aminobutyrate; MMV, MMV1028806; PPP, pentose phosphate pathway; SSA, succinic semialdehyde.
Figure 4—figure supplement 1
Intracellular tachyzoites exhibit differential 15N incorporation after treatment with mitochondrial electron transport chain (mETC) inhibitors.
RH∆ku80 were treated with atovaquone, buparvaquone, and MMV1028806, labeled with 15N-amide-L-glutamine for 3 hr, followed by their isolation from the human foreskin fibroblast (HFF) cells. The heatmap shows the overall isotopic labeling (100-’M+0’) of each replicate.
Figure 5 with 3 supplements
Atovaquone (ATQ), buparvaquone (BPQ), and MMV1028806 reduce mitochondrial potential and respiration in T. gondii.
(A) Human foreskin fibroblast (HFF) cells were infected with tachyzoites and treated with DMSO, ATQ, BPQ, and MMV1028806 for 24 hr, and mitochondria were stained with MitoTracker (MT, red), and DNA was stained with DAPI (blue). Scale bar = 5 µm. (B) The ratios of fluorescence intensities between MT and DAPI dyes were calculated per vacuole (DMSO n=56, ATQ n=45, BPQ n=49, MMV1028806 n=40; blue lines represent the median, two-sided Mann-Whitney test). (C) 4 weeks of age, myotube-derived ME49 in vitro cysts were treated with DMSO, ATQ, BPQ, and MMV1028806 for 24 hr, and mitochondria were stained with MT (red), the cyst wall was stained with Dolichos biflorus agglutinin and Streptavidin-Cy2 (green), and DNA was stained with DAPI (blue). Scale bar = 20 µm. (D) The ratios of fluorescence intensities between MT and DAPI dyes were calculated per vacuole (DMSO n=56, ATQ n=42, BPQ n=45, MMV1028806 n=38; blue lines represent the median, two-sided Mann-Whitney test). (E) 10 million freshly egressed tachyzoites were incubated with known bc1-complex inhibitors ATQ and BPQ, specific control compounds antimycin and 4-(trifluoromethoxy)phenylhydrazone (FCCP), and MMV1028806. Every treatment significantly impacts the oxygen consumption rate (OCR) (blue line represents the median, n=3) (Welch’s test; **p<0.005).
Figure 5—figure supplement 1
Direct mitochondrial inhibitors reduce MitoTracker (MT) signal intensity and MT/DAPI ratio in T. gondii tachyzoites.
Human foreskin fibroblast (HFF) cells were infected with RH-S9 tachyzoites and treated for 24 hr with DMSO (n=61), atovaquone (ATQ, n=32), buparvaquone (BPQ, n=35), MMV1028806 (MMV, n=36), pyrimethamine (Pyri, n=58), clindamycin (Clin, n=53), and 6-diazo-5-oxonorleucine (DON, n=68). * and **** denote p<0.05 and p<0.000 in Mann-Whitney tests. MT signal appears red, DAPI stain is blue, and the mitochondrial signal originates from GFP. Scale bars represent 10 µm.
Figure 5—figure supplement 2
bc1-complex inhibitors partially reduce mitochondrial profiles of bradyzoites.
Thin section electron microscopy of cysts that were treated with atovaquone (ATQ), buparvaquone (BPQ), or MMV1028806 (MMV) for 24 hr. Scale bar 0.5 µm. (A). (B) Measured areas of mitochondrial profiles from 21, 12, 15, and 26 images showing DMSO-, ATQ-, BPQ-, and MMV1028806-treated parasites (* denotes p<0.05 in Mann-Whitney tests).
Figure 5—video 1
Fluorescence microscopy of T. gondii tissue cyst with mitochondria.
Fluorescence microscopic reconstruction of a 4-week-old tissue cyst of the ME49 strain. Dolichos biflorus agglutinin (DBA) staining shows the cyst wall in green, DAPI stains DNA shown in blue, and MitoTracker was used to stain mitochondria in red.
Figure 6 with 1 supplement
Untargeted metabolomic analysis of bradyzoites treated with bc1-complex inhibitors shows an energy imbalance.
(A) Four-week-old in vitro cysts were treated for 3 hr. The cell monolayer was scraped off and the cysts were syringe-released. With Dolichos biflorus agglutinin (DBA) coated magnetic beads, the isolated cysts were captured and washed with PBS. The metabolites were extracted with 80% acetonitrile (AcN) in water and sonication, and analyzed via hydrophilic-interaction ultra-high pressure liquid chromatography coupled mass spectrometry (HILIC-UHPLC-MS). The results are shown in three separate volcano plots: Metabolic phenotypes of bradyzoites treated with atovaquone (B), buparvaquone (C), and MMV1028806 (D). (n=3, significance analyzed with a two-sided Mann-Whitney U test). Abbreviations: ATQ, atovaquone; BPQ, buparvaquone; DMSO, dimethyl sulfoxide; LA-carnitine, linoleoylcarnitine; MMV, MMV1028806; NADH, reduced nicotinamide adenine dinucleotide; PA-carnitine, palmitoylcarnitine; SA-carnitine, stearoylcarnitine.
Figure 6—figure supplement 1
Principal component analysis of bradyzoites treated with atovaquone (ATQ), buparvaquone (BPQ), and MMV1028806.
Four-week-old intracellular cysts were exposed for 3 hr, purified from their host cells using magnetic beads, and analyzed by LCMS.
Figure 7 with 1 supplement
Differential efficacy and ATP depletion by atovaquone and 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ) in tachyzoites and bradyzoites.
Atovaquone and HDQ were tested against T. gondii Pru parasites. (A) Showing the normalized half-maximal inhibitory concentration (IC50) against tachyzoites in fibroblasts at day 4, n=8. (B) 4-week-old bradyzoites were treated for a week with atovaquone or HDQ. Cultures were observed for tachyzoite regrowth for 3 weeks. The half-maximal lethal concentration (LC50) was determined at the time point which ideally reflected the dose-response curve, in this case day 14, n=6. 105 tachyzoites and bradyzoites were lysed and the ATP concentration was determined with an enzymatic luciferase assay. (C) Showing the calculated concentration of ATP within an average parasite. Lines reflect the median, error bars represent SEM, significance test Mann-Whitney, n=12. (D) Parasites were treated with 1 μM atovaquone or HDQ. RFUs were normalized to the untreated control of either the tachyzoite or bradyzoite dataset. Shown are means, error bars represent SD, significance test is pairwise Mann-Whitney, n=6.
Figure 7—figure supplement 1
Luminescence-based ATP assay.
(A) The linear response of the BacTiter-Glo assay has a certain range, depending on the ATP concentration of the sample. The assay is reliable between 100 fM and 1 nM ATP. (B) Increasing numbers of freshly isolated parasites were analyzed to determine a minimal parasite density necessary for steady readouts. 105 parasites per sample were both manageable and accurate.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Toxoplasma gondii) | PruTom Prugniaud ∆hxgprt tdTomato | John et al., 2009 PMID:19578440 | RRID:NCBITaxon_748168 | |
| Strain, strain background (Toxoplasma gondii) | ME49 | Suzuki et al., 1989 PMID:2926171 | RRID:NCBITaxon_748168 | |
| Strain, strain background (Toxoplasma gondii) | RH-Rep1/2-S9(33–159)-GFP (RH-S9) | DeRocher et al., 2000; Thomsen-Zieger et al., 2003; PMID:11058084; PMID:12860390 | RRID:NCBITaxon_748168 | |
| Biological sample (Homo sapiens) | BJ-5ta human foreskin fibroblasts (HFF) | ATCC | Cat#: CRL-4001 RRID:CVCL_6573 | Synonymous with BJ1-hTERT |
| Biological sample (Homo sapiens) | Human myoblast cells (KD3) | Shiomi et al., 2011; PMID:21490680 | Immortalized primary cells. Availability statement here: PMID:38213326 | |
| Chemical compound, drug | MMV Pathogen Box | Medicines for Malaria Venture | Provided by MMV free of charge | |
| Software | ImageJ v1.54 | Schindelin et al., 2012; PMID:22743772 | RRID:SCR_003070 | Version 2.16.0 |
| Software | GraphPad Prism | RRID:SCR_002798 | Version 8.4.1 | |
| Software | Compound Discoverer | Thermo Fisher | RRID:SCR_028693 | Version 3.1 |
| Other | DAPI stain | Sigma-Aldrich | Cat#: D9542 | More information in the Fluorescence stain of mitochondrial activity section |
| Other | Streptavidin-Cy2 | Jackson Antibodies | Not available | More information in the Fluorescence stain of mitochondrial activity section |
| Other | Dolichos biflorus agglutinin | Sigma-Aldrich | Cat#: L6533 | More information in the Fluorescence stain of mitochondrial activity section |
| Other | MitoXpress Xtra Oxygen Consumption Assay | Agilent Technologies | Cat#: MX-200-4 | Following the manufacturer’s high-sensitivity (HS) method with modifications. More information in the Fluorescence stain of mitochondrial activity section |
Additional files
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Supplementary file 1
Screening data.
- https://cdn.elifesciences.org/articles/102511/elife-102511-supp1-v1.xlsx
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Supplementary file 2
Tachyzoite mass spectrometry profiling data and its curation.
- https://cdn.elifesciences.org/articles/102511/elife-102511-supp2-v1.xlsx
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Supplementary file 3
Tachyzoite 13C-labeling mass spectrometry data and its curation.
- https://cdn.elifesciences.org/articles/102511/elife-102511-supp3-v1.xlsx
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Supplementary file 4
Tachyzoite 15N-labeling mass spectrometry data and its curation.
- https://cdn.elifesciences.org/articles/102511/elife-102511-supp4-v1.xlsx
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Supplementary file 5
Bradyzoite mass spectrometry profiling data and its curation.
- https://cdn.elifesciences.org/articles/102511/elife-102511-supp5-v1.xlsx
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Supplementary file 6
Supplementary file containing abbreviations.
- https://cdn.elifesciences.org/articles/102511/elife-102511-supp6-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/102511/elife-102511-mdarchecklist1-v1.docx
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Screening the MMV Pathogen Box reveals the mitochondrial bc1-complex as a drug target in mature Toxoplasma gondii bradyzoites
eLife 13:RP102511.
https://doi.org/10.7554/eLife.102511.4
