Leishmania amazonensis Glycosphingolipids, Glycosylinositolphospholipids and Inositol Phosphorylceramide Expression during Amastigote-Promastigote Differentiation

This study was focused in the analysis of the expression of glycosphingolipids (GSLs), glycosylinositolphospholipids (GIPLs) and inositol phosphorylceramide (IPC) in promastigote and amastigote forms of Leishmania (Leishmania) amazonensis, the main causative agent of cutaneous leishmaniasis in South America. Parasite (glyco)(sphingo)lipids expression were analyzed by high performance thin layer chromatography, ESI/MS, indirect immunofluorescence and flow cytometry. Levels of IPC synthase (ipcs) gene and inositol phosphosphingolipid phospholipase C-Like (iscl) gene were analyzed by real-time PCR. Two mAbs, LST-1 and LST-2 were used to identify promastigote IPC and GIPLs. LST-1 detected IPC preferentially located at the inner leaf of plasma membrane, whereas LST-2 detected GIPLs at promastigote surface. Both LST-1 and LST-2 reacted only with promastigotes, indicating that IPC and GIPLs are promastigote-stage-specific antigens. On the other hand, mAb ST-4, directed to L. amazonensis glycosphingolipids only recognized amastigote forms. When amastigotes were cultured at 23°C, differentiation to promastigotes is observed by morphological changes, decrease of parasite reactivity with mAb ST-4, and a progressive increase of parasite reactivity with mAbs LST-1 and LST-2. In amastigotes the expression of ipcs gene was similar to that observed in promastigotes. Thus the low expression level or absence of IPC in amastigotes may result from the high levels of iscl expression. As already described for L. amazonensis GSLs (Straus et al, 1993), and now for GIPLs and IPC, these lipids are expressed differentially in promastigotes and amastigotes, i.e. IPC is expressed preferentially in promastigotes and it is modulated mainly by the iscl expression whereas, GIPLs and neutral glycosphingolipids are expressed mainly in amastigotes and promastigotes, respectively. The steps involved in the L. amazonensis genetic control responsible for switching GSLs to GIPLs on amastigote–promastigote differentiation are under investigation, and may contribute for the development of new therapeutical strategies in Leishmaniasis.

Supported by FAPESP, CNPq and CAPES.