Molecular and functional characterization of the ceramide synthase from Trypanosoma cruzi

In this study, we characterized ceramide synthase (CerS) of the protozoan parasite Trypanosoma cruzi at the molecular and functional levels. TcCerS activity was detected initially in a cell-free system using the microsomal fraction of epimastigote forms of T. cruzi, [3H]dihydrosphingosine or [3H]sphingosine, and fatty acids or acyl-CoA derivatives as acceptor or donor substrates, respectively. TcCerS utilizes both sphingoid long-chain bases, and its activity is exclusively dependent on acyl-CoAs, with palmitoyl-CoA being preferred. In addition, Fumonisin B1, a broad and well-known acyl-CoA-dependent CerS inhibitor, blocked the parasite's CerS activity. However, unlike observations in fungi, the CerS inhibitors Australifungin and Fumonisin B1 did not affect the proliferation of epimastigotes in culture, even after exposure to high concentrations or after extended periods of treatment. A search of the parasite genome with the conserved Lag1 motif from Lag1p, the yeast acyl-CoA-dependent CerS, identified a T. cruzi candidate gene (TcCERS1) that putatively encodes the parasite's CerS activity. The TcCERS1 gene was able to functionally complement the lethality of a lag1Δ lac1Δ double deletion yeast mutant in which the acyl-CoA-dependent CerS is not detectable. The complemented strain was capable of synthesizing normal inositol-containing sphingolipids and is 10 times more sensitive to Fumonisin B1 than the parental strain.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (108 K)Download as PowerPoint slideHighlights► We characterized the Trypanosoma cruzi ceramide synthase (TcCerS) at the biochemical level. ► TcCerS is fully dependent on palmitoyl-CoA donor and is inhibited by Fumonisin B1. ► TcCERS1 gene was able to rescue the lethality of a CerSΔ deletion yeast mutant. ► At the molecular level, TcCERS1 encodes a ‘bona-fide’ acyl-CoA dependent CerS.