The antifungal Aureobasidin A and an analogue are active against the protozoan parasite Toxoplasma gondii but do not inhibit sphingolipid biosynthesis

Abstract

Toxoplasma gondii is an obligate intracellular protozoan parasite of the phylum Apicomplexa, and toxoplasmosis is an important disease of both humans and economically important animals. With a limited array of drugs available there is a need to identify new therapeutic compounds. Aureobasidin A (AbA) is an antifungal that targets the essential inositol phosphorylceramide (IPC, sphingolipid) synthase in pathogenic fungi. This natural cyclic depsipeptide also inhibits Toxoplasma proliforation, with the protozoan IPC synthase orthologue proposed as the target. The data presented here show that neither AbA nor an analogue (Compound 20), target the protozoan IPC synthase orthologue or total parasite sphingolipid synthesis. However, further analyses confirm that AbA exhibits significant activity against the proliferative tachyzoite form of Toxoplasma, and Compound 20, whilst effective, has reduced efficacy. This difference was more evident on analyses of the direct effect of these compounds against isolated Toxoplasma, indicating that AbA is rapidly microbicidal. Importantly, the possibility of targeting the encysted, bradyzoite, form of the parasite with AbA and Compound 20 was demonstrated, indicating that this class of compounds may provide the basis for the first effective treatment for chronic toxoplasmosis.

Keywords: Toxoplasma; Aureobasidin A; bradyzoite; sphingolipid biosynthesis.

Fig. 1.

The structures of the cyclic depsipeptide compounds Aureobasidin A and its analogue Compound 20 (Wuts et al. 2015).

Fig. 2.

ED₅₀ of Aureobasidin A (AbA, A-D) or Compound 20 (Cpmd 20, E-H) – μg mL⁻¹; (95% Confidence Interval) – against the Toxoplasma RH tachyzoite form in HFF cells. 6 days post addition of the compounds. In agreement with Sonda et al. (2005), both compounds were non-toxic to HHF cells under the conditions employed. A and B: no wash out post-compound addition; C and D: wash out 2 h post-compound addition; E and F: wash out 8 h post-compound addition; G and H: 2 h pre-treatment of isolated parasites pre-infection. Calculated using GraphPad Prism 7, log(inhibitor) vs normalized response – Variable slope. >10 µg mL⁻¹ – ED₅₀ could not be determined. Representative in triplicate dataset.

Fig. 3.

Yeast dependent on the expression of the Toxoplasma AUR1p orthologue TgSLS (YPH499-HIS-GAL-AUR1 pRS426 TgSLS) are resistant to Aureobasidin A (AbA) and Compound 20 (Cmpd 20) at 5 and 10 µg mL⁻¹. This contrasts to the sensitivity of yeast dependent on AUR1 expression (YPH499-HIS-GAL-AUR1 pRS426 AUR1).

Fig. 4.

Vero cells (Host), isolated Toxoplasma tachyzoites (Toxo) and Saccharomyces cerevisiae (Yeast), labelled for 1 h with NBD-C6-ceramide and complex sphingolipids then fractionated by HPTLC. Like the host cells, Toxoplasma parasites synthesize sphingomyelin (SM) and ethanolamine phosphorylceramide (EPC), two unique sphingolipids are also produced (X and Y). However, unlike in S. cerevisiae, no labelled inositol phosphorylceramide (IPC) is evident from either host or Toxoplasma cells. Representative dataset.

Fig. 5.

Isolated Toxoplasma tachyzoites treated with Aureobasidin A (AbA) and Compound 20 (Cmpd 20) at 10 µg mL⁻¹ for 1 (A), 4 (B) and 7 (C) hours before labelling with NBD-C6-ceramide for 1 h. Neither compound affected the complex sphingolipid profile synthesized at any time point when compared with the vehicle control (DMSO). SM – Sphingomyelin (SM); EPC – Ethanolamine PhosphorylCeramide; X and Y – Unclassified sphingolipids. Representative dataset.

Fig. 6.

ED₅₀ of Aureobasidin A (A, AbA) or Compound 20 (B, Cpmd 20) – μg mL⁻¹ (95% Confidence Interval) – against the Toxoplasma Pru bradyzoite form in Human Foreskin Fibroblast (HFF) cells. Three days post addition of the compounds. In agreement with Sonda et al. (2005), both compounds were non-toxic to HHF cells under the conditions employed. Calculated using GraphPad Prism 7, log(inhibitor) vs normalized response – Variable slope. Representative in triplicate dataset.