Doxycycline has distinct apicoplast-specific mechanisms of antimalarial activity
- Department of Biochemistry, University of Utah School of Medicine, United States
Figures
Figure 1
Scheme of intraerythrocytic P. falciparum parasite depicting doxycycline, its canonical delayed-death mechanism at 1 µM inhibiting apicoplast genome translation, the novel metal-dependent mechanism(s) in the apicoplast explored herein at 10 µM, and off-target activity outside the apicoplast at >20 µM.
Figure 2 with 2 supplements
10 µM doxycycline kills P. falciparum with first-cycle, apicoplast-specific activity.
(A) Continuous growth assay of synchronized Dd2 parasites treated with 1 or 10 µM DOX ±200 µM IPP with (B) Giemsa-stained blood smears for days 1–3. (C) 48 hr growth-inhibition curve for DOX-treated Dd2 parasites ± 200 µM IPP. (D) Continuous growth assay of synchronized Dd2 parasites treated with 10–40 µM DOX and 200 µM IPP. (E) Epifluorescence images of synchronized parasites treated as rings with 10 µM DOX ±200 µM IPP and imaged 36 or 65 hr later (green = ACPL GFP, blue = nuclear Hoechst stain). Data points in growth assays are the average ± SD of two to four biological replicates. All growth assays were independently repeated two to four times using different batches of blood (shown in Figure 2—figure supplement 1).
Figure 2—figure supplement 1
Additional, independent growth assays with DOX and IPP.
(A) Additional continuous growth assays of synchronized Dd2 parasites treated with 1 or 10 µM DOX ±200 µM IPP. (B) Independent 48 hr growth-inhibition assays for DOX-treated Dd2, D10, NF54 parasites in the absence or presence of 200 µM IPP or 50 µM mevalonate. Dd2 and NF54 parasites were synchronized by single treatment with 5% D-sorbitol. D10 parasites were synchronized by magnet purification of schizonts followed by 5 hr incubation with uninfected erythrocytes and treatment with 5% D-sorbitol. Data points in individual plots are the ave ± SD of two to four biological replicates. Each plot reflects an independent assay performed with a different batch of blood. (C) Scatter plot of doxycycline EC50 values from independent assays ± IPP with calculated ave ± SD and analysis by two-tailed unpaired t-test. (D) Independent continuous growth assay of synchronized Dd2 parasites treated with 10–40 µM DOX and 200 µM IPP.
Figure 2—figure supplement 2
Additional epifluorescence images of DOX-treated D10 parasites.
Magnet plus sorbitol-synchronized parasites were treated as rings with 10 µM DOX ±200 µM IPP and imaged 1 or 3 days later (green = ACPL GFP, blue = nuclear Hoechst stain). Twenty to 40 total parasites were examined for each treatment condition on each given day for duplicate experiments, and data were plotted as the average percentage of parasites in each population that displayed an elongated, punctate, or dispersed apicoplast GFP signal. For clarity, error bars are not displayed but standard errors of the mean were ≤15% in all conditions. Cell-percentage differences were analyzed by two-tailed unpaired t-test (p values in parentheses, ns = not significant).
Figure 3 with 3 supplements
10 µM DOX kills P. falciparum with a first-cycle, metal-dependent mechanism.
Continuous growth assays of synchronized Dd2 parasites treated with (A) DOX, clindamycin (CLI), and/or azithromycin (AZM) and (B) 10 µM DOX and 10 µM ZnCl2 or 500 µM CaCl2. (C) 48 hr growth inhibition assay of D10 parasites treated with DOX without or with 500 µM FeCl3 or CaCl2. (D) Continuous growth assay of synchronized Dd2 parasites treated with 10 µM DOX and 500 µM FeCl3 or MgCl2. (E) Epifluorescence images of synchronized parasites treated as rings with 10 µM DOX ±500 µM FeCl3 or CaCl2 and imaged 36 hr later (green = ACPL GFP, blue = nuclear Hoechst stain). (F) Continuous growth assay of synchronized Dd2 parasites treated with 1 µM DOX and 500 µM FeCl3 or MgCl2. (G) Continuous-growth assay of synchronized Dd2 parasites treated with 20 or 40 µM DOX and 500 µM FeCl3. Data points in growth assays are the average ± SD of two to four biological replicates. All growth assays were independently repeated using different batches of blood (shown in Figure 3—figure supplement 1).
Figure 3—figure supplement 1
Additional, independent growth assays with DOX, other antibiotics, and metals.
Additional continuous growth assays of synchronized Dd2 parasites treated with (A) DOX, clindamycin (CLI), and/or azithromycin (AZM); (B) 500 µM of CaCl2, MgCl2, or FeCl3 alone; (C) 10 µM DOX and 5 µM ZnCl2 or 250 µM CaCl2; (D) 10 µM DOX treated with 50–500 µM FeCl3 or 500 µM MgCl2. Individual data points are the ave ± SD from two to four biological replicates. (E) 96 hr growth-inhibition assays for DOX-treated D10 parasites in the absence or presence of 200 µM IPP, 500 µM FeCl3, or 500 µM CaCl2. Parasites were synchronized by magnet purification of schizonts followed by 5 hr incubation with uninfected erythrocytes and treatment with 5% D-sorbitol. Data points are the ave ± SD of 3 biological replicates.
Figure 3—figure supplement 2
Additional epifluorescence images of D10 parasites treated with DOX and metals.
Magnet plus sorbitol-synchronized parasites were treated as rings with 10 µM DOX ±500 µM FeCl3 or CaCl2 and imaged 36 hr later (green = ACPL GFP, blue = nuclear Hoechst stain). Twenty to 40 total parasites were examined for each treatment condition on each given day for duplicate experiments, and data were plotted as the average percentage of parasites in each population that displayed an elongated, punctate, or dispersed apicoplast GFP signal. For clarity, error bars are not displayed but standard errors of the mean were ≤15% in all conditions. Cell-percentage differences were analyzed by two-tailed unpaired t-test (p values in parentheses, ns = not significant).
Figure 3—figure supplement 3
Effect of deferoxamine on parasite growth and apicoplast biogenesis.
(Left) Continuous growth assay of sorbitol-synchronized Dd2 parasites cultured without or with 15 µM deferoxamine (DFO), 15 µM FeCl3, or 200 µM IPP. (Right) Fluorescence microscopy of live, synchronized D10 parasites untreated or treated with 15 µM DFO and imaged 36 hr later (green = ACPL GFP, blue = nuclear Hoechst stain). Twenty to 30 total parasites were examined for each treatment condition on each given day for duplicate experiments, and data were plotted as the average percentage of parasites in each population that displayed an elongated, punctate, or dispersed apicoplast GFP signal. For clarity, error bars are not displayed but standard errors of the mean were <15% in all conditions. Cell-percentage differences were analyzed by two-tailed unpaired t-test (p value in parenthesis).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Plasmodium falciparum) | Dd2 | PMID:1970614 | ||
| Cell line (Plasmodium falciparum) | D10 ACPL-GFP | PMID:10775264 | ||
| Cell line (Plasmodium falciparum) | NF54-PfMev ACPL-GFP | PMID:32059044 | ||
| Software, algorithm | Prism 8 | GraphPad | RRID:SCR_002798 | |
| Chemical compound, drug | Doxycycline | Sigma-Aldrich | Cat. No. D3447 | |
| Chemical compound, drug | Isopentenyl pyrophosphate | Isoprenoids | Cat. No. IPP001 | |
| Chemical compound, drug | Ferric chloride | Sigma-Aldrich | Cat. No. 236489 | |
| Chemical compound, drug | Clindamycin | Sigma-Aldrich | Cat. No. C6427 | |
| Chemical compound, drug | Azithromycin | Sigma-Aldrich | Cat. No. 75199 | |
| Chemical compound, drug | Deferoxamine | Sigma-Aldrich | Cat. No. D9533 |
Additional files
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Source data 1
Source Data for Microscopy Analyses.
- https://cdn.elifesciences.org/articles/60246/elife-60246-data1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/60246/elife-60246-transrepform-v2.pdf
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Doxycycline has distinct apicoplast-specific mechanisms of antimalarial activity
eLife 9:e60246.
https://doi.org/10.7554/eLife.60246
