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. 2022 Sep 26;13(1):5653.
doi: 10.1038/s41467-022-33400-y.

High-throughput small molecule screen identifies inhibitors of microsporidia invasion and proliferation in C. elegans

Brandon M Murareanu  1 , Noelle V Antao  2 , Winnie Zhao  1 , Aurore Dubuffet  3 , Hicham El Alaoui  3 , Jessica Knox  1   4 , Damian C Ekiert  2   5 , Gira Bhabha  2 , Peter J Roy  1   4 , Aaron W Reinke  6
Affiliations

High-throughput small molecule screen identifies inhibitors of microsporidia invasion and proliferation in C. elegans

Brandon M Murareanu et al. Nat Commun. .

Abstract

Microsporidia are a diverse group of fungal-related obligate intracellular parasites that infect most animal phyla. Despite the emerging threat that microsporidia represent to humans and agricultural animals, few reliable treatment options exist. Here, we develop a high-throughput screening method for the identification of chemical inhibitors of microsporidia infection, using liquid cultures of Caenorhabditis elegans infected with the microsporidia species Nematocida parisii. We screen a collection of 2560 FDA-approved compounds and natural products, and identify 11 candidate microsporidia inhibitors. Five compounds prevent microsporidia infection by inhibiting spore firing, whereas one compound, dexrazoxane, slows infection progression. The compounds have in vitro activity against several other microsporidia species, including those known to infect humans. Together, our results highlight the effectiveness of C. elegans as a model host for drug discovery against intracellular pathogens, and provide a scalable high-throughput system for the identification and characterization of microsporidia inhibitors.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. High-throughput drug screen of the Spectrum Collection identifies compounds that restore progeny production in C. elegans infected with N. parisii.
A Schematic of small molecule inhibitor screen (see methods). Bleach-synchronized L1 animals were incubated with 60 μM of each compound from the Spectrum Collection for 6 days at 21 °C in the presence of N. parisii spores. 2,560 total compounds were screened once, yielding 25 initial hits. The initial hits were retested once, yielding 11 confirmed hits. The effectiveness of these 11 compounds was then quantified across multiple replicates of the screening assay using WorMachine. Figure made using Biorender.com. B Representative images of wells containing worms. (B Far Left) Normal worm growth in the absence of spores. (B Middle Left) Microsporidia infection leads to inhibition of progeny production. (B Middle & Far Right) Treatment with anti-microsporidial compounds restores progeny production. C, D Effect of compounds on progeny production in (C) infected and (D) uninfected animals (n = 6). Progeny levels expressed as a percentage of the DMSO uninfected control. Statistical significance evaluated in relation to DMSO controls using two-sided t-tests: ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant (p > 0.05). Bars represent the sample mean, error bars represent + /− 1 standard error from the mean. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. Identified compounds inhibit microsporidia infection.
A Schematic of continuous infection assay (see methods). Bleach-synchronized L1 animals were incubated with compounds for 4 days at 21 °C in the presence of N. parisii spores in liquid. Animals were subsequently fixed with acetone, and stained with DAPI (blue) and DY96 (green). Figure made using Biorender.com. B Representative images taken at 5x magnification; scale bars are 500 μm. (B Far Left) Normal worm growth in the absence of spores. (B Middle Left) Microsporidia infection results in production of new spores highlighted in bright green by DY96, and slows growth thereby preventing development of gravid adults. (B Middle Right & Far Right) Treatment with anti-microsporidial compounds reduces formation of new spores, and restores the development of gravid adults. C Percentage of animals that contain embryos (n = 3, N = ≥ 200 animals counted per biological replicate). D Percentage of animals that contain newly formed spores (n = 3, N = ≥ 200 animals counted per biological replicate). Significance evaluated in relation to DMSO infected controls using one-way ANOVA with Dunnett’s post-hoc test: ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant (p > 0.05). Bars represent the sample mean, error bars represent + /−1 standard error from the mean. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Dexrazoxane prevents microsporidia proliferation.
A Schematic of pulse-chase infection assay (see methods). Bleach-synchronized L1 animals were pulse infected with N. parisii spores for 3 h at 21 °C on NGM plates. Excess spores were washed away, and infected animals were incubated with compounds for 2 or 4 days at 21 °C in liquid. Animals were fixed in acetone or PFA, FISH stained, and then stained with DY96 (green) and DAPI (blue). Figure made using Biorender.com. B Representative images of acetone-fixed animals 4 days post infection taken at 5x magnification; scale bars are 500 μm. (B Far Left) Normal growth in uninfected worms. (B Middle Left) Pulse infection results in sporoplasms and meronts highlighted in red by microB FISH probes and new spores highlighted in bright green by DY96. Pulse infection also slows growth thereby preventing development of gravid adults. (B Middle Right & Far Right) Treatment of pulse-infected animals with fumagillin or dexrazoxane reduces sporoplasms and meronts, new spores, and restores development of gravid adults. C Representative images (z-stack maximum intensity projections; 7 slices, 0.25 μm spacing) of PFA-fixed animals 2 days post infection taken at 63x magnification; scale bars are 20 μm. (C Left) In the absence of drug treatment, pulse-infected animals develop large meronts with many nuclei. (C Middle) Fumagillin treatment restricts proliferation; both large and small meronts are observed. (C Right) Dexrazoxane treatment restricts proliferation; only small meronts with one or two nuclei are observed. D Percentage of animals with embryos (n = 3, N = ≥ 170 animals counted per biological replicate). E Percentage of animals with newly formed spores (n = 3, N = ≥ 170 animals counted per biological replicate). F Percentage of animals with FISH signal (n = 3, N = ≥ 170 animals counted per biological replicate). ANOVA not significant (p = 0.111). G Quantitation of FISH fluorescence per worm (n = 3, N = 15 animals quantified per biological replicate). H Effects of iron chelation with BP on infection, and effects of iron supplementation with FAC on dexrazoxane activity. Percentage of animals with newly formed spores (n = 3, N = ≥ 120 animals counted per biological replicate) after 4 days of continuous infection is shown. Significance evaluated in relation to DMSO infected controls using one-way ANOVA with Dunnett’s post-hoc test: ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant (p > 0.05). Bars represent the sample mean, error bars represent  + /−1 standard error from the mean. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Several compounds prevent microsporidia infection by inhibiting spore firing.
A Schematic of spore firing assay (see methods). Bleach-synchronized L1 animals were incubated with compounds for 3 h at 21 °C in the presence of N. parisii spores in liquid. Spore prep control was generated by incubating spores in liquid in the absence of C. elegans. Animals and spores were fixed in acetone and stained with microB FISH probe (red) and DY96 (green). Figure made using Biorender.com. B Schematic of modified spore firing assay (see methods). Spores were incubated with compounds for 24 h at 21 °C, and then used to infect beach-synchronized L1 animals either with or without prior washing to remove excess compounds. Figure made using Biorender.com. C Representative images of unfired and fired spores at 63x magnification; scale bars are 5 μm. Unfired spore is indicated by white arrowhead. D, E Effects of microsporidia inhibitors on (D) the percentage of fired spores in the C. elegans intestinal lumen (n = 3, N = ≥ 60 spores counted per biological replicate) and (E) the average number of sporoplasms per animal (n = 3, N = ≥ 40 animals counted per biological replicate) in a spore firing assay. F, G Effects of serine protease inhibitors on (F) spore firing and (G) sporoplasm invasion in a modified spore firing assay without washing away compounds prior to infection (n = 3, N = ≥ 39 animals and ≥60 spores counted per biological replicate, except for one chymostatin replicate where only 12 animals and 19 spores were counted). H, I Effects of spore firing inhibitors on (H) spore firing and (I) sporoplasm invasion in a modified spore firing assay with compounds washed away prior to infection (n = 3, N = ≥ 40 animals and ≥ 70 spores counted per biological replicate). Significance evaluated in relation to DMSO controls using one-way ANOVA with Dunnett’s post-hoc test: ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant (p > 0.05). Bars represent the sample mean, error bars represent + /−1 standard error from the mean. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Identified inhibitors display activity against multiple diverse microsporidia species.
A, B Bleach-synchronized L1 animals were pulse infected with P. epiphaga spores for 3 h on NGM plates. Excess spores were washed away, and infected animals were incubated with 350 μM fumagillin or 60 μM dexrazoxane for 4 days at 21 °C in liquid. Animals were fixed and stained with DAPI (blue) and a FISH probe (red). A Representative images of animals 4 days post infection; scale bars are 25 μm. B Quantitation of FISH fluorescence per worm (n = 3, N = 15 animals quantified in each condition per biological replicate). C, D Cells infected with A. algerae spores for 3 h with 60 dexrazoxane for 4 days. C Representative images of an infected cell stained with dapi (blue), FISH (red), and DY96 (green); scale bars are 5 μm. Meronts undergoing divisions are indicated by arrowheads and meront not currently dividing is indicated by arrow. Spore attached to the cells after initial infection indicated with star. D Average number of A. algerae divisions per cell (n = 2, N = 30–243 cells analyzed per biological replicate). E Host cell viability (n = 3). ANOVA for control and dexrazoxane treatment conditions was not significant (p = 0.325) (F–H) E. intestinalis spores were treated with 60 μM inhibitors for 24 h. Spores were either induced to fire (F) or used to infect cells for 24 h and then stained with a FISH probe and DAPI (G, H). F Percentage of spores that have undergone complete germination (n = 5, N = ≥ 100 spores counted per biological replicate). G Representative images of cells infected with E. intestinalis that were either untreated or treated with ZPCK; scale bars are 10 μm. DIC, Differential interference contrast microscopy. H Percentage of cells infected (n = 4, N = ≥ 100 cells counted per biological replicate). Significance evaluated in relation to DMSO infected controls using one-way ANOVA with Dunnett’s post-hoc test: ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant (p > 0.05). Bars represent the sample mean, error bars represent + /− 1 standard error from the mean. Source data are provided as a Source Data file.
Fig. 6
Fig. 6. Compound structures and mechanisms.
A Structures of compounds containing a quinone moiety (circled in red) that were identified in the initial screen as inhibitors of microsporidia infection. B Microsporidia infection model depicting the stages at which various microsporidia inhibitors act. Two protease inhibitors and four quinone-derivatives were shown to act directly on N. parisii spores to prevent spore firing and subsequent invasion of sporoplasms. Fumagillin and dexrazoxane act after invasion has occurred, inhibiting proliferation of sporoplasms and meronts, ultimately reducing parasite burden and preventing the production of microsporidia spores. Figure made using Biorender.com.
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