Involvement of host cell heparan sulfate proteoglycan in Trypanosoma cruzi amastigote attachment and invasion

Cell surface glycosaminoglycans (GAGs) play an important role in the attachment and invasion process of a variety of intracellular pathogens. We have previously demonstrated that heparan sulfate proteoglycans (HSPG) mediate the invasion of trypomastigote forms of Trypanosoma cruzi in cardiomyocytes. Herein, we analysed whether GAGs are also implicated in amastigote invasion. Competition assays with soluble GAGs revealed that treatment of T. cruzi amastigotes with heparin and heparan sulfate leads to a reduction in the infection ratio, achieving 82% and 65% inhibition of invasion, respectively. Other sulfated GAGs, such as chondroitin sulfate, dermatan sulfate and keratan sulfate, had no effect on the invasion process. in addition, a significant decrease in infection occurred after interaction of amastigotes with GAG-deficient Chinese Hamster Ovary (CHO) cells, decreasing from 20% and 28% in wild-type CHO cells to 5% and 9% in the mutant cells after 2 h and 4 h of infection, respectively. These findings suggest that amastigote invasion also involves host cell surface heparan sulfate proteoglycans. the knowledge of the mechanism triggered by heparan sulfate-binding T. cruzi proteins may provide new potential candidates for Chagas disease therapy.Fundacao Oswaldo Cruz (FIOCRUZ)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Programa Estrategico de Apoio a Pesquisa em SaudeFundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fiocruz MS, Lab Ultraestrutura Celular, Inst Oswaldo Cruz, BR-21045900 Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Dept Bioquim, UNIFESP, SP, BR-04044020 São Paulo, BrazilInst Biol Mol Parana FIOCRUZ, Inst Carlos Chagas, BR-81350010 Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Bioquim, UNIFESP, SP, BR-04044020 São Paulo, BrazilCNPq: 473087/2007-3Programa Estrategico de Apoio a Pesquisa em Saude: 403504/2008-2FAPERJ: E-26/111.624/2008FAPESP: 07/59801-1Web of Scienc

Effects of chlorate on the sulfation process of Trypanosoma cruzi glycoconjugates. Implication of parasite sulfates in cellular invasion

Sulfation, a post-translational modification which plays a key role in various biological processes, is inhibited by competition with chlorate. In Trypanosoma cruzi, the agent of Chagas’ disease, sulfated structures have been described as part of glycolipids and we have reported sulfated high-mannose type oligosaccharides in the C-T domain of the cruzipain (Cz) glycoprotein. However, sulfation pathways have not been described yet in this parasite. Herein, we studied the effect of chlorate treatment on T. cruzi with the aim to gain some knowledge about sulfation metabolism and the role of sulfated molecules in this parasite. In chlorate-treated epimastigotes, immunoblotting with anti-sulfates enriched Cz IgGs (AS-enriched IgGs) showed Cz undersulfation. Accordingly, a Cz mobility shift toward higher isoelectric points was observed in 2D-PAGE probed with anti-Cz antibodies. Ultrastructural membrane abnormalities and a significant decrease of dark lipid reservosomes were shown by electron microscopy and a significant decrease in sulfatide levels was confirmed by TLC/UV-MALDI-TOF-MS analysis. Altogether, these results suggest T. cruzi sulfation occurs via PAPS. Sulfated epitopes in trypomastigote and amastigote forms were evidenced using AS-enriched IgGs by immunoblotting. Their presence on trypomastigotes surface was demonstrated by flow cytometry and IF with Cz/dCz specific antibodies. Interestingly, the percentage of infected cardiac HL-1 cells decreased 40% when using chlorate-treated trypomastigotes, suggesting sulfates are involved in the invasion process. The same effect was observed when cells were pre-incubated with dCz, dC-T or an anti-high mannose receptor (HMR) antibody, suggesting Cz sulfates and HMR are also involved in the infection process by T. cruzi.Fil: Ferrero, Maximiliano Ruben. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Soprano, Luciana Lía. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Acosta, Diana Maria. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Garcia, Gabriela Andrea. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Esteva, Monica I.. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Couto, Alicia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Duschak, Vilma Gladys. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; Argentin