Inhibition of Leishmania donovani promastigote internalization into murine macrophages by chemically defined parasite glycoconjugate ligands

C B Palatnik, R Borojevic, J O Previato, L Mendonça-Previato
1989 Infection and Immunity  
Leishmania donovani, the agent of human visceral leishmaniasis, is an intracellular parasite that must be recognized and internalized by host macrophages to complete its biological cycle. In a search for possible ligands for macrophage surface receptors, glycoconjugates were obtained from Leishmania promastigotes by aqueous, phenol-aqueous, and alkaline extraction. A fucose-mannose glycoproteic ligand, a lipopeptidephosphoglycan, and a phosphate mannogalactan ligand were purified from
more » ... fied from promastigotes and analyzed for their chemical contents, with special attention to their glycidic moieties. Sugars that were identified as components of these glycoconjugates were tested for their capacity to inhibit promastigote internalization by BALB/c peritoneal macrophages in vitro. Neutral hexoses showed little inhibitory activity; fucose, charged monosaccharides, and a mannose polymer showed the highest activity. Two of the glycoconjugates (fucose-mannose glycoproteic ligand and phosphate mannogalactan ligand) purified from promastigotes were potent inhibitors of internalization, 75% inhibition being obtained at concentrations of 6 to 10 ,ug/ml. The simultaneous presence of both ligands in low concentrations yielded an increase in inhibitory activity above that found for each ligand alone, indicating that promastigotes may use at least two receptor sites for penetration into macrophages. These ligands are specific inhibitors of L. donovani promastigote phagocytosis, since 10 ,ug of each ligand per ml interfered neither with internalization of yeast cells nor with phagocytosis of Leishmania adleri promastigotes. Leishmania donovani, the etiological agent of human kala-azar (human visceral leishmaniasis), is an obligatory intracellular parasite. Leishmania promastigotes are introduced into the host blood circulation by the insect vector. They need to be recognized and internalized by host macrophages to complete their biological cycle. Once they reach the intraphagolysosomal environment, they become amastigotes and multiply until they disrupt the macrophage and propagate the infection to other cells. Recognition and penetration into macrophages is therefore a very critical point in the Leishmania cell cycle. It involves several complex phenomena that are still incompletely understood. In recent research, evidence has been gathered which indicates that both recognition and penetration of leishmanias into macrophages are mediated by carbohydrates. Dwyer et al. (13) suggested that L. donovani glycidic coat participates in adhesion of parasites to cells and to other substrates. Chang (9) indicated that mannose and glucosamine are the principal sugars involved in adhesion of promastigotes to hamster macrophages. Wilson and Pearson (41) have shown that mannose-fucoseand mannose-phosphate-containing polymers might be the ligands on the parasite surface that are recognized by receptors responsible for parasite internalization by human macrophages. These observations are consistent with studies of macrophage receptors that interact with Leishmania species and that have been shown to have a lectinlike activity. A mannose-fucosyl receptor, associated with the C3b complement receptor complex, is responsible in mouse macro-* Corresponding author. phages for the general mechanism of internalization of parasites (7, 42) and for the associated respiratory burst (10). Most of the evidence indicating the participation of sugars in interaction of Leishmania species with macrophage receptors and in control of their internalization has been obtained from indirect studies on inhibition of parasite penetration into macrophages in vitro by purified monosaccharides or neoglycoproteins. Knowledge of the composition and structure of the L. donovani glycidic ligand is still rudimentary. Recent observations of other Leishmania species have demonstrated the relevance of glycidic moieties of surface glycoconjugates in parasite interaction with host cells. An integral membrane glycoprotein containing glucosamine, mannose, and galactosamine was characterized in Leishmania mexicana (36), and a lipopolysaccharide containing galactose was identified in Leishmania major (19) . In the present work, we report our findings on the chemical and biological characterization of three different carbohydrate-containing fractions obtained from L. donovani (LD1S-Sudan): a glycoproteic fraction containing fucose and mannose that was isolated from the aqueous extract, an acidic phosphate-mannogalactan glycoconjugate that was released by alkaline extraction, and a lipopeptidephosphoglycan (LPPD) from the phenolic extract. Monosaccharides identified in these fractions were tested in vitro for their capacity to inhibit parasite penetration into macrophages. Subsequently, the fractions purified from Leishmania promastigotes were used in the same in vitro test. Two of them (fucose-mannose glycoproteic ligand [FML] and phosphate mannogalactan ligand [PMGL]) were shown to behave as separate specific ligands for parasite internalization by mouse peritoneal macrophages. 754 on May 5, 2020 by guest http://iai.asm.org/ Downloaded from
doi:10.1128/iai.57.3.754-763.1989 fatcat:45i52xsrzjdxhiqcrhsychxrey