Survival and infectivity of trypanosomatids rely on cell-surface and secreted glycoconjugates, many of which contain a variable number of galactose residues. Incorporation of galactose to proteins and lipids occurs along the secretory pathway from UDP-galactose (UDP-Gal). Before being used in glycosylation reactions, however, this activated sugar donor must first be transported across the endoplasmic reticulum and Golgi membranes by a specific nucleotide sugar transporter (NST). In this study, we identified an UDP-Gal transporter (named TcNST2 and encoded by the TcCLB.504085.60 gene) from Trypanosoma cruzi, the etiological agent of Chagas disease. TcNST2 was identified by heterologous expression of selected putative nucleotide sugar transporters in a mutant Chinese Hamster Ovary cell line. TcNST2 mRNA levels were detected in all T. cruzi life-cycle forms, with an increase in expression in axenic amastigotes. Confocal microscope analysis indicated that the transporter is specifically localized to the Golgi apparatus. A three-dimensional model of TcNST2 suggested an overall structural conservation as compared with members of the metabolite transporter superfamily and also suggested specific features that could be related to its activity. The identification of this transporter is an important step toward a better understanding of glycoconjugate biosynthesis and the role NSTs play in this process in trypanosomatids.

Vaccination remains the best hope for control of all forms of leishmaniasis, and the development of a safe and effective vaccine is a critical global public-health priority. Our previous work showed that immunization with non-persistent phosphomannomutase-deficient (ΔPMM) Leishmania major parasites confers significant protection in susceptible BALB/c mice due to increased T-cell numbers and suppression of IL-10 and IL-13 early during infection. Here, we complemented the ΔPMM L. major parasites with human PMM2 to determine whether we could further improve the protection. Complemented ΔPMM parasites have restored glycoconjugate biosynthesis, while retaining avirulence of the parental knockout strain. Immunization with hPMM2 add-back parasites showed similar Th1/Th2 cytokine profiles to that observed in ΔPMM-vaccinated mice. However, the numbers of the activated CD4+CD44hi and CD8+CD44hi T cells recruited to the draining lymph nodes early after Leishmania infection were reduced, leading to decreased protection following hPMM2-immunization. Thus, the magnitude of T-cell responses early in the infection and the absence of mannose-rich glycoconjugates determine the protective outcome of anti-leishmanial immunity.

Glycans at the surface of adult Schistosoma mansoni were investigated with gold-labelled lectins. The fragile complex of the glycans with the outer membranes could be preserved for electron microscopy by avoiding extensive pre-fixation with aldehydes and by introducing osmium-ferrocyanide as a membrane fixative. Male and female worms were entirely covered with glycans that intensely bound lectins from Erythrina cristagalli and Datura stramonium, suggesting that galactose(β1–4)N-acetylglucosamine residues occur in high numbers in the surface glycans. Similar staining was obtained with lectins from Triticum vulgaris, Glycine max and Ricinus communis agglutinin I, which react with N-acetylglucosamine or terminal galactose residues and bind non-selectively with high affinity to N-acetyllactosamine. Fucose, N-acetylgalactose and sialic acid were not detected with lectins and sialidase treatment. The tegument contained an abundance of glycans with the same lectin reactivities as the surface-expressed molecules, indicating that the worms synthesize and replenish their surface glycans and do not merely adsorb host substances. Glycomimesis is discussed as a mechanism of immune evasion in view of N-acetyllactosamine being a common and weakly immunogenic component in glycans of vertebrate hosts. S. mansoni might disguise themselves with the glycans against attack by immune effectors.

Invasive amoebiasis is the result of infection of Entamoeba histolytica. The closely related Entamoeba dispar can colonize the human gut but does not cause invasive disease. In this study, E. dispar was analysed for the presence of the lipophosphoglycan-like (LPG) glycoconjugate known to be present on the cell surface of E. histolytica. E. dispar cells were radio-isotope labelled with [3H]galactose or [3H]inositol. The acidic glycoconjugates were extracted and analysed by hydrophobic chromatography over phenyl–Sepharose and by sodium dodecyl sulphate polyacrylamide gel electrophoresis. No LPG-like molecules could be identified in E. dispar in contrast to E. histolytica, suggesting that these molecules may be absent in the non-pathogenic species.