Article Figures & Data
Figures
- FIG 1
Screening of LOPAC1280 for the ability to decrease viability of Blastocystis. The cutoff was set at less than or equal to 20% viability relative fluorescence units.
- FIG 2
Relative fluorescence units (RFU) of Blastocystis cultures treated with compounds from LOPAC1280. Each compound was compared to Mz of the same isolate, and we determined whether the RFU was significantly lower. **, P > 0.01; ***, P > 0.001. DPI, diphenyleneiodonium chloride; AUR, auranofin; BIX, BIX 01294 trihydrochloride hydrate; Mz, metronidazole.
- FIG 3
Blastocystis cultures show different apoptotic profiles when treated with drugs from LOPAC1280 for 6 h. In general, ST4-WR1 showed the least proportion of viable cells compared to the other isolates after treatment. Diphenyleneiodonium chloride caused the lowest viability on ST4-WR1 cultures. ST7-B cultures are relatively resistant to the drugs. ST1-NUH9 and ST4-WR1 cultures had lower viability even without treatment, suggesting their fragility when exposed to aerobic conditions. DPI, diphenyleneiodonium chloride; AUR, auranofin; BIX, BIX 01294 trihydrochloride hydrate; Mz, metronidazole.
- FIG 4
Flow cytometry histograms of Hoechst-stained Blastocystis cells showed no changes in nucleic acid content when treated with selected compounds from LOPAC1280 except with BIX. The latter compound increased nucleic acid content in three Blastocystis isolates. DPI, diphenyleneiodonium chloride; AUR, auranofin; BIX, BIX 01294 trihydrochloride hydrate; Mz, metronidazole.
- FIG 5
Image gallery showing various forms of Blastocystis obtained and identified using an imaging flow cytometer. (A) Round and vacuolar cells are typically found in cultures. (B) Granular cells were identified as having high intensity values in the side-scatter channel. (C) The irregularly shaped cells have lower aspect-ratio values in the brightfield channel. (D and E) The EDF setting made it possible to perform spot counting in Hoechst-stained cells. These spots could indicate nuclear and other nucleic acid-containing structures in the cells.
- FIG 6
Changes in phenotypic features of Blastocystis cultures treated with drug hits from LOPAC1280 for 24 h as determined using imaging flow cytometry. (A) The proportion of irregular-shaped cells increased after treatment. (B) There was an increase in the proportion of granular cells of Blastocystis upon treatment compared to untreated controls. (C) Most drugs caused Blastocystis to decrease in size. (D) Multinucleated cells determined by Hoechst staining increased in Blastocystis ST7-B isolates when treated with the drugs. Each isolate treated with the compounds was compared to untreated cultures of the same isolate. *, P < 0.05; **, P < 0.01; ***, P < 0.001. DPI, diphenyleneiodonium chloride; AUR, auranofin; BIX, BIX 01294 trihydrochloride hydrate; Mz, metronidazole.
- FIG 7
N-Acetylcysteine (NAC) reduced the effect of AU in Blastocystis. A resazurin-based assay showed lower metabolic activities in the three Blastocystis isolates when treated with AU, as indicated by the lower RFU emitted by Blastocystis cultures. There was a dose-dependent rescue when NAC was added to the cultures, as shown by significantly higher RFU values. ***, P < 0.001 (comparing the RFU values of each culture conditions with the culture treated with 10 μM auranofin [AU] of the same isolate).
Tables
- TABLE 1
Drugs that inhibit growth of Blastocystis isolates and their target class
Drug Target class Decreased viability of: ST1-NUH9 ST4-WR1 ST7-B Emetine dihydrochloride hydrate Apoptosis + NSC348884 hydrate Apoptosis + + 1,10-Phenanthroline monohydrate Biochemistry + 2,3-Dimethoxy-1,4-naphthoquinone Cell stress + (–)-Scopolamine hydrobromide Cholinergic + + Idarubicin DNA metabolism + Pregnenolone sulfate sodium GABA + BIX 01294 trihydrochloride hydrate Gene regulation + + + Diphenhydramine hydrochloride Histamine + + Sanguinarine chloride Ion pump + Tetrabenazine Neurotransmission + Diphenyleneiodonium chloride Nitric oxide + + + JS-K Nitric oxide + Ammonium pyrrolidinedithiocarbamate Nitric oxide + BNTX maleate salt hydrate Opioids + Azathioprine P2 receptor + BTO-1 Phosphorylation + Auranofin Phosphorylation + + + SR 57227A Serotonin + + (±)-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride Sphingolipid + - TABLE 2
IC50s of drugs that inhibit growth of Blastocystis isolates
Druga Mean IC50 (μg/ml) ± SDb ST1-NUH9 ST4-WR1 ST7-B Metronidazole 5.26 ± 1.32 (30.7) 5.74 ± 1.65 (33.5) 31.5 ± 12 (184.3) DPI 0.0972 ± 0.113 (0.309) 0.105 ± 0.00226 (0.334) 0.0759 ± 0.0397 (0.241) AU 0.0622 ± 0.00314 (0.0917) 0.122 ± 0.00735 (0.180) 0.113 ± 0.0424 (0.166) BIX 0.467 ± 0.251 (0.778) 0.648 ± 0.158 (1.08) 1.39 ± 0.150 (2.32)
Supplemental material
- Supplemental file 1 -
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