Characterization of ligand of heparin in trypanosoma cruzi and determination of the domain of heparan sulfate involved in the process of invasion T. cruzi-cardiomyocyte in vitro.
The ability of Trypanosoma cruzi to recognize molecules at the surface of both the phagocytic and non-phagocytic cells is essential to its survival in the vertebrate host. Therole of the sulfated proteoglycans in the cell recognition process has been reported in several human pathogens, including T. cruzi. Data from our group have demonstrated the participation of heparan sulfated proteoglycan (HSPG) of cardiomyocytes in the invasion for forms trypomastigotes. However, the structure of the HSPG molecule involved in the receptor-ligand interaction and the role of other sulfated glycosaminoglycans (GAGs) in theinvasion process have not been elucidated yet. To evaluate the participation of GAGs in T. cruzi invasion, trypomastigotes, clone Dm28c, were pre-treated with 20amp;#956;g/ml of heparin, keratan sulfate (KS) or three distinctfragments of heparan sulfate (HS) obtained by enzymatic (heparitinase I and II) and nitrous acid treatments. For competition assays, the untreated or soluble GAGs pre-treated parasites were incubated for 2h at 37°C with the ca rdiomyocyte cultures and the percentageof infection was determined after Giemsa staining. Our results revealed a significant inhibition of the infection index of 84.8% and 45% after treatment of the parasites withheparin and the N-acetylated/ N-sulfated fragment, respectively, suggesting the important role of the ([IdoUA-GlcNAc]-[GlcUA-GlcNS]3-[GlcUA-GlcNAc]4[GlcNAc]) domain of the HS chain in the T. cruzi-cardiomyocyte recognition. In contrast, the treatment of the parasites with KS, Nacetylated or N-sulfated fragments did not display any effect in the invasion process. The role of sulfation in the recognition and invasion process was evaluated by treating the cardiomyocytes cultures for 16h at 37°C with differ ent concentrations of sodium chlorate, followed by trypomastigotes infection (2h). The decline of sulfation resulted in the reduction (dose dependent) of the infection index, achieving inhibition levels of 26%, 48.5% and 73.6%after treatment of cardiomyocyte cultures with 25mM, 50mM and 75mM of sodium chlorate, respectively, suggesting the participation of negative charge as modulator of the specific recognition with the NA/NS domain of HS chain.Additionately, biochemical assays were performed to characterize the heparin binding protein present at the surface of T. cruzi. Two major protein bands of 65.8 kDa and 59 kDa were identified in the total protein extract of all evolutive forms of T. cruzi by Western blotting,using biotin conjugated-heparin, -chondroitin sulfate (CS) and -HS. The T. cruzi heparin ligand was isolated by association of Triton X-114 extraction method and heparin-Sepharose affinity chromatography. After metabolic labeling (35S-Methionine), the hydrophobic proteinswere isolated by affinity column and separated by SDS-PAGE, revealing a protein profile, similar to total protein extract, with two major bands (65.8 kDa and 59 kDa) eluted with 0.5M and 1M NaCl in trypomastigotes and epimastigotes, respectively. The isotopic analysis also revealed a higher expression of heparin ligand (1.3-2X) in trypomastigotes when compared to epimastigotes. The heparin binding proteins were detected at membrane fraction ofepimastigotes obtained by cell fractionation methodology associated to the affinity column purification. The detection of proteins eluted from heparin affinity column with biotinilated heparin, CS and HS by Western blotting revealed intense labeling mainly at the 59 kDaprotein. In addition, the analysis of the proteins by denaturant electrophoresis revealed the presence of two protein band in both trypomastigotes and epimastigotes forms of T. cruzi. Complementary biochemical assays will be carried out to get more detailed information aboutthe T. cruzi heparin binding protein.
Advisor:Maria de Nazareth Silveira Leal de Meirelles; Mirian Claudia de Souza Pereira
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Trypanosoma cruzi Glicosaminoglicanas Hidrofobicidade Chagas Disease Myocytes Cardiac Glycosaminoglycans Heparin Hydrophobicity
Date of Publication:04/09/2007