Antibodies directed against O-acetylated sialoglycoconjugates accelerate complement activation in Leishmania donovani promastigotes

Abstract

Background: An enhanced presence of 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) triggers the alternate pathway (AP) in Indian visceral leishmaniasis (VL). Antibodies directed against these ePI⁺topes are present in high titers. The biological relevance of these antibodies, with regard to activation of the classical pathway (CP), was investigated. Methods: Complement activators were affinity purified, complement activation via the CP, AP, and lectinmediated complement pathway was measured by use of an Anti-C3 radiO-binding assay, and the number of C3 molecules was quantitated by Scatchard analysis. Cell death induced via the complement pathways was measured by use of MTT (tetrazolium salt 3- [4, 5-dimethylthiazol-2-yl] -2, 5-diphenyltetrazolium bromide) assay, and uptake of proPI+dium iodide (PI⁺) was measured by flow cytometry. Results: Anti-O-AcSGs from both healthy donors and patients with VL elicited C3 deposition as early as 3 min, which triggered parasite lysis, as demonstrated by use of MTT assay and corroborated by the high rate of uptake of PI⁺. Analysis of complement activation by mannan-binding lectin and C-reactive protein demonstrated their negligible contribution during the 3-min time frame. Conclusions: Anti-O-AcSGs were identified as an important source of CP activation under normal physiological conditions, suggesting that they play a role in conferring host protection against parasite infection.