Ceramide glycosylation and fatty acid hydroxylation influence serological reactivity in Trypanosoma cruzi glycosphingolipids.

Ceramide mono (CMH) or dihexoside (CDH) fractions from Trypanosoma cruzi (Dm28c clone) were identified as glucosyl and lactosylceramides containing non-hydroxylated fatty acids. The di-glycosylated form was much more efficiently recognized by sera from T. cruzi-immunized rabbits, indicating that glycosylation influences antigenicity. Fatty acid hydroxylation was also a determinant of serological reactivity, since an alpha-hydroxylated CMH, only present at the Y clone, was recognized by the hyperimmune sera. In summary, these data indicate that T. cruzi CMHs with non-hydroxylated fatty acids are unable to induce antibody responses in animal hosts, which is reverted by the addition of a sugar residue or an alpha-hydroxyl group.

Glycoinositolphospholipids from Trypanosoma cruzi interfere with macrophages and dendritic cell responses.

To investigate the possible effects of glycoinositolphospholipid (GIPL) from Trypanosoma cruzi on human antigen presenting cells, we tested their effects on lipopolysaccharide (LPS)-stimulated human macrophages and dendritic cells (DC). Human macrophages or DC were incubated with GIPL (50 microg/ml) and LPS (500 pg/ml) and tumor necrosis factor alpha (TNF-alpha), interleukin 8 (IL-8), IL-10, and IL-12p40 levels in supernatants were analyzed by enzyme-linked immunosorbent assay. TNF-alpha, IL-10, and IL-12 secretion were significantly decreased by GIPL both in macrophages and DC. In contrast, GIPL did not alter IL-8 production. We also analyzed the expression of CD80, CD86, HLA-DR, CD40, and CD57 on the macrophage surface after stimulation with LPS in the presence or absence of T. cruzi GIPL. GIPL led to a down-regulation in the expression of all tested molecules. We additionally examined the influence of T. cruzi GIPL on the response of human DC to LPS. LPS-induced HLA-DR, CD83, and CD86 up-regulation was significantly inhibited by GIPL. A slight down-regulation in CD80 and CD40 expression on DC surfaces in the presence of GIPL was also noticed. Similarly, GIPL led to down-modulation of CD83, CD80, CD86, and HLA-DR surface expression and TNF-alpha and IL-10 production when DC were stimulated by CD40L. The ceramide portion of GIPL was responsible for most of the activity exhibited by the whole molecule. Considering the important role of the immune response in determining the fate of the host-parasite relationship, the immunoregulatory activities of T. cruzi GIPL are potentially important for parasite evasion and then pathogenesis of infection with protozoan parasites.

Structure elucidation of sphingolipids from the mycopathogen Paracoccidioides brasiliensis: an immunodominant beta-galactofuranose residue is carried by a novel glycosylinositol phosphorylceramide antigen.

Two major acidic glycolipid components (Pb-1 and Pb-2) have been extracted from the mycopathogen Paracoccidioides brasiliensis, a thermally dimorphic fungus endemic to rural areas of South and Central America. Sera of all patients exhibiting paracoccidioidomycosis were found to be reactive with Pb-1, but not with Pb-2; no reactivity was observed with sera of healthy patients or those with histoplasmosis [Toledo, M. S., Suzuki, S., Straus, A. H., and Takahashi, H. K. (1995) J. Med. Vet. Mycol. 33, 247-251]. We report here the complete structure elucidation of both P. brasiliensis glycolipids using monosaccharide, fatty acid, sphingosine, and inositol component analysis by GC-MS; 1H- and 31P NMR spectroscopy; ESI-MS and -MS/CID-MS, linkage analysis, and exoglycosidase digestion. The compounds were found to be glycosylinositol phosphorylceramides (GIPCs) with the following structures: Pb-2, Manpalpha1-->3Manpalpha1-->2Ins1-P-1Cer; and Pb-1, Manpalpha1-->3[Galfbeta1-->6]Manpalpha1-->2Ins1- P-1Cer. The serologically nonreactive Pb-2 appears to be a biosynthetic intermediate between mannosylinositol phosphorylceramide (MIPC), which is widely distributed among fungi, and the antigenic Pb-1. Pb-1 is a novel glycosphingolipid, similar to a triglycosyl IPC (Hc-VI) reported from Histoplasma capsulatum [Barr, K., Laine, R.A, and Lester, R. L. (1984) Biochemistry 23, 5589-5596], but differing in the anomeric configuration of the terminal Galf1-->6 residue, which is immunodominant. The significance of these structures as serological and taxonomic markers, as well as their potential utility as targets for immunodiagnostic agents, is discussed.