Boromycin Has Potent Anti- Toxoplasma and Anti- Cryptosporidium Activity

Abstract

Toxoplasma gondii and Cryptosporidium parvum, members of the phylum Apicomplexa, are significant pathogens of both humans and animals worldwide for which new and effective therapeutics are needed. Here, we describe the activity of the antibiotic boromycin against Toxoplasma and Cryptosporidium Boromycin potently inhibited intracellular proliferation of both T. gondii and C. parvum at half-maximal effective concentrations (EC₅₀) of 2.27 nM and 4.99 nM, respectively. Treatment of extracellular T. gondii tachyzoites with 25 nM boromycin for 30 min suppressed 84% of parasite growth, but T. gondii tachyzoite invasion into host cells was not affected by boromycin. Immunofluorescence of boromycin-treated T. gondii showed loss of morphologically intact parasites with randomly distributed surface antigens inside the parasitophorous vacuoles. Boromycin exhibited a high selectivity for the parasites over their host cells. These results suggest that boromycin is a promising new drug candidate for treating toxoplasmosis and cryptosporidiosis.

Keywords: Cryptosporidium parvum; Toxoplasma gondii; antiparasitic; boromycin; drug discovery.

FIG 1

Boromycin displays a dose-dependent inhibitory effect against intracellular and extracellular T. gondii. (A) Parasites were allowed to infect host cells for 24 h, and then dilutions of compound were added and the incubation was continued for 24 h. Boromycin exhibits EC₅₀s of 2.27 nM (95% CI, 1.92 to 2.63 nM) and 5.31 nM (95% CI, 4.2 to 6.8 nM) for TgRH GFP::Luc and TgME49ΔHPT::Luc, respectively. (B) Extracellular TgRH GFP::Luc tachyzoites were exposed to boromycin for 2 h, washed to remove the compound, and then allowed to infect the HFF monolayer for 24 h. Boromycin inhibited the establishment of infection in HFF after 2 h of drug exposure with an EC₅₀ of 9.60 nM (95% CI, 8.52 to 10.86 nM). EC₅₀s were calculated using the log(inhibitor) versus response-variable slope (four-parameter) regression equation in GraphPad Prism 8 (GraphPad, La Jolla, CA). Means ± standard deviations (SD) of results from three independent experiments consisting of three replicates per condition are shown.

FIG 2

Boromycin rapidly kills T. gondii. Proliferating TgME49ΔHPT::Luc parasites were treated with 25 nM boromycin or DMSO for 0.5, 1, 2, 4 and 8 h. (A and B) Relative luminescence (RLUs) of the parasites after 72 h and 96 h of incubation posttreatment, respectively. (C) Percent growth inhibition calculated using the results shown in panel A. Eighty-four percent of parasite growth was inhibited after 30 min of boromycin treatment. Samples were run in triplicate, and the means ± SD of results from three independent experiments (A) or two independent experiments (B) are shown.

FIG 3

Morphology of intracellular and extracellular T. gondii RH strain treated with DMSO or boromycin. (A to C) Parasites were visualized with antibody against T. gondii SAG-1 (red), and nuclei are visualized with DAPI (blue). Images were captured using an SP8 confocal microscope (Leica Microsystems, Buffalo Grove, IL, USA). Differential interference contrast (DIC) images are to the right of the immunofluorescence image. (A) DMSO-treated parasites. (B) Intracellular T. gondii exposed to 2 nM boromycin. Tachyzoite swelling was observed in the majority of treated parasites (arrows). (C) Intracellular T. gondii exposed to 11 nM boromycin. Parasitophorous vacuoles containing randomly distributed surface antigens with complete loss of morphologically intact parasites (arrowheads) were commonly observed after treatment with 11 nM boromycin. (D and E) Images of GFP-expressing tachyzoites were captured using a wide-field fluorescence microscope (Leica Microsystems, Buffalo Grove, IL, USA). (D) DMSO-treated tachyzoites exhibit their normal crescent shape. (E) Cell swelling was observed in extracellular tachyzoites exposed to 25 nM boromycin for 2 h. Scale bar, 10 μm.

FIG 4

Boromycin did not significantly affect T. gondii invasion into host cells. Invasion of T. gondii tachyzoites was first synchronized by exposing the parasites to high-K⁺ buffer in the HFF monolayer for 20 min. High-K⁺ buffer was then aspirated, and parasites on the host cell surface were exposed to 25 nM, 50 nM boromycin, or DMSO in CM for 5 min before fixation. (A) Number of parasites counted in 10 randomly selected fields. (B) Percent invasion was calculated as (number of intracellular parasites/total number of parasites) × 100. A significant reduction in tachyzoite invasion of HFF was not observed with 25 nM boromycin. Increasing the concentration to 50 nM resulted in a slight, but not statistically significant, reduction in invasion compared to the DMSO control (P = 0.0650). Means ± SD of results from two independent experiments are shown.

FIG 5

Boromycin potently inhibits in vitro growth of C. parvum. NLuc C. parvum Iowa oocysts were used to infect HCT-8 cells for 24 h. Infected cells were treated with 2-fold serial dilutions of boromycin, and parasite growth was evaluated 48 h posttreatment. C. parvum growth was monitored by nanoluciferase activity. Boromycin exhibited an EC₅₀ of 4.99 nM (95% CI, 3.77 to 6.49 nM) against C. parvum. Samples were run in triplicate, and the means ± SD of results from three independent experiments are shown.

FIG 6

Boromycin is not toxic to host cells at therapeutic concentrations. Host cells used in the in vitro parasite inhibition assays (HFF, HCT-8) were grown to 40% confluence and then treated with boromycin from 11 μM to 0.089 μM. The viability of the cells was determined by quantification of ATP with a CellTiter Glo luminescent assay after 24 h of drug exposure. Boromycin’s half-maximal cytotoxic concentration (CC₅₀) was calculated to be 20.0 μM (95% CI, 13.32 to 39.30 μM) for HFF and 27.46 μM (95% CI, 13.80 to 88.44 μM) for HCT-8 cells. Samples were run in triplicate, and the means ± SD of results from three independent experiments are shown.