Characterization of the inositol phosphorylceramide synthase activity from Trypanosoma cruzi

Abstract

IPC (inositol phosphorylceramide) synthase is an enzyme essential for fungal viability, and it is the target of potent antifungal compounds such as rustmicin and aureobasidin A. Similar to fungi and some other lower eukaryotes, the protozoan parasite Trypanosoma cruzi is capable of synthesizing free or protein-linked glycoinositolphospholipids containing IPC. As a first step towards understanding the importance and mechanism of IPC synthesis in T. cruzi, we investigated the effects of rustmicin and aureobasidin A on the proliferation of different life-cycle stages of the parasite. The compounds did not interfere with the axenic growth of epimastigotes, but aureobasidin A decreased the release of trypomastigotes from infected murine peritoneal macrophages and the number of intracellular amastigotes in a dose-dependent manner. We have demonstrated for the first time that all forms of T. cruzi express an IPC synthase activity that is capable of transferring inositol phosphate from phosphatidylinositol to the C-1 hydroxy group of C6-NBD-cer {6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]hexanoylceramide} to form inositol phosphoryl-C6-NBD-cer, which was purified and characterized by its chromatographic behaviour on TLC and HPLC, sensitivity to phosphatidylinositol-specific phospholipase C and resistance to mild alkaline hydrolysis. Unlike the Saccharomyces cerevisiae IPC synthase, the T. cruzi enzyme is stimulated by Triton X-100 but not by bivalent cations, CHAPS or fatty-acid-free BSA, and it is not inhibited by rustmicin or aureobasidin A, or the two in combination. Further studies showed that aureobasidin A has effects on macrophages independent of the infecting T. cruzi cells. These results suggest that T. cruzi synthesizes its own IPC, but by a mechanism that is not affected by rustmicin and aureobasidin A.

A plate-based assay system for analyses and screening of the Leishmania major inositol phosphorylceramide synthase

“…A similar problem has previously been identified with the use of Trypanosoma cruzi derived microsomes in an assay of TcIPCS turnover (Figueiredo, et al 2005 ). However, it has been demonstrated that washing microsomal membranes derived from the pathogenic yeast, Candida albicans with a zwitterionic detergent (CHAPS) removed endogenous substrate lipids without affecting IPCS activity (Aeed, et al 2004).…”

“…These data provide the first direct indication of the mechanism of action of the sphingolipid synthase family. Such analyses would not have been possible using a conventional assay system such as that previously employed for the T. cruzi orthologue (Figueiredo, et al 2005). However, by employing a complemented yeast system, large quantities of active LmjIPCS could be isolated in a cost effective manner avoiding the handling of pathogenic material.…”

“…Both unwashed and CHAPS-washed (1 µg) microsomal membranes were used in a 50 µl assay mix containing 1 mM PI, 100 mM Tris HCl (pH7.4) and 200 µM NBD-C 6 -ceramide (Figueiredo et al, 2005). The reaction was pre-incubated before the addition of the NBD-C 6 -ceramide at 30 °C (the established optimum temperature in the assay) for 30 minutes and subsequently for a further hour before quenching with 150 µl of chloroform:methanol:water (10:10:3).…”

Characterization of inositol phosphorylceramides from Leishmania major by tandem mass spectrometry with electrospray ionization

“…This transfer reaction is catalyzed by the phosphatidylinositol: ceramide phosphoinositol transferase (IPC synthase), a membrane-bound enzyme [11,12]. In S. cerevisiae and C. albicans, phytoceramide is the preferred receptor for phosphoinositol.…”

The Trypanosoma brucei sphingolipid synthase, an essential enzyme and drug target

“…The assay mix contained 100 µM donor substrate (bovine liver PI, PC or PE, Avanti Polar Lipids), 10 µg of microsomes, 100 mM Tris HCl, 10 mM EDTA, 6 mg/ml BSA and 5 µM NBD C 6 -ceramide [19]. Following incubation at 30 °C for 60 minutes the reaction was quenched by the addition of 150 µl of chloroform:methanol:water (10:10:3).…”