Molecular and functional characterization of the ceramide synthase from Trypanosoma cruzi

Abstract

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 lag1Δlac1Δ 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.

The Sphingolipid Biosynthetic Pathway Is a Potential Target for Chemotherapy against Chagas Disease

“…CerS activity has been identified [54] and characterized at the biochemical and molecular levels only in T. cruzi ([63], submitted). The TcCerS was initially identified by the incorporation of [ 3 H]palmitic acid into ceramides, which were chemically degraded to radiolabeled dihydrosphingosine and fatty acid [54].…”

Leishmania (Viannia) braziliensis Inositol Phosphorylceramide: Distinctive Sphingoid Base Composition

“…However, enzyme activities for Leishmania are not yet characterized, and so far are based on or inferred from the final SL biosynthetic product: IPC ceramide structure. In regard to other trypanosomatids, Figueiredo et al ( 2012 ) reported that TcCerS in T. cruzi preferentially uses dihydrosphingosine base as sphingosine base and palmitoylCoA as substrate, and Sevova et al ( 2010 ) showed that specific SL synthases in T. brucei, T. cruzi , and L. major transfer polar head groups from phosphoglycerolipid donors to generate SM, ethanolamine phosphorylceramide, and IPC using 6-(( N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine C 6 -ceramide.…”

Everybody needs sphingolipids, right! Mining for new drug targets in protozoan sphingolipid biosynthesis

“…However, clear divergence is observed in the second and the third steps, both of which represent a cell-fate modulator process. CerSs exhibit differential preferences for the chain length of the acyl-CoA substrate (Park et al 2014 ) and its hydroxylation pattern (Layre & Moody, 2013 ), with 6 isoforms present in humans suggesting a different role for each CER species produced (Levy & Futerman, 2010 ; Figueiredo et al 2012 ). To-date, one or, maximum, two genes encoding CerS function have been identified in protozoan parasite species (Koeller & Heise, 2011 ).…”

“…Experimental evidence (from our laboratory and others) has previously indicated the presence of CerS activity in Leishmania spp (Zhang et al , 2003 ; Denny et al , 2004 , 2006 ) and in T. cruzi (De Lederkremer et al 2011 ). More recently LAG1 orthologues have been identified and functionally and molecularly characterized in the latter parasite (Figueiredo et al 2012 ). Other results indirectly suggest the presence of such activity in T. brucei (Patnaik et al 1993 ; Richmond et al 2010 ; Smith & Bütikofer, 2010 ).…”

“…However, to-date all available chemotherapeutic agents suffer from being toxic (Chappuis et al 2007 ) or inaccessible, both geographically and financially, in endemic areas where public health is under-resourced, poor and underdeveloped. Additionally, the lack of effective vaccines (de Oliveira et al 2009 ) and the alarming emergence of resistance to these drugs (Croft et al 2006 ), combined with the short-lived prevention resulting from applying measures such as vector and reservoir host control (WHO, 2004 ; Figueiredo et al 2012 ), demand an intensive search for alternative anti-leishmanials to enable effective treatment and control.…”