Participation of heparin binding proteins from the surface of Leishmania (Viannia) braziliensis promastigotes in the adhesion of parasites to Lutzomyia longipalpis cells (Lulo) in vitro

Particle and Fibre Toxicology

Carlos Roberto Alves⁶  Felio J Bello⁴  Maria de Fátima Madeira⁵  Helena Bonciani Nader²  Carolina Meloni Vicente²  Leny Toma²  Reginaldo Peçanha Brazil¹  Renata Oliveira de Araújo Soares⁶  Francisco Odêncio de Oliveira Junior³  Bernardo Acácio Santini Pereira⁶  Franklin Souza da Silva⁶  Mirian Claudia de Souza Pereira³  Luzia Monteiro de Castro Côrtes⁶ 

[1] Laboratório de Bioquímica e Fisiologia de Insetos - IOC - Fiocruz, Av. Brasil 4365, Rio de Janeiro - CEP, 21040-360, Brazil;Departamento de Bioquímica Universidade Federal de São Paulo, UNIFESP, SP, Brazil;Laboratório de Ultraestrutura Celular, Av. Brasil 4365, Rio de Janeiro - CEP, 21040-360, Brazil;Universidad Del Rosario, Escuela de Medicina, Carrera 24 no 63 C-69, Bogotá, D.C, Colombia;Laboratório de Vigilância em Leishmanioses - IPEC - Fiocruz, Av. Brasil 4365, Rio de Janeiro - CEP, 21040-360, Brazil;Laboratório de Biologia Molecular e Doenças Endêmicas, Av. Brasil 4365, Rio de Janeiro - CEP, 21040-360, Brazil

关键词: Surface plasmon resonance;    Glycosaminoglycans;    Lulo cells;    Promastigotes;    L. (V.) braziliensis;   

Others  :  1231755
DOI  :  10.1186/1756-3305-5-142

 received in 2012-02-23, accepted in 2012-07-02,  发布年份 2012

【 摘 要 】

Background

Leishmania (V.) braziliensis is a causative agent of cutaneous leishmaniasis in Brazil. During the parasite life cycle, the promastigotes adhere to the gut of sandflies, to avoid being eliminated with the dejection. The Lulo cell line, derived from Lutzomyia longipalpis (Diptera: Psychodidae), is a suitable in vitro study model to understand the features of parasite adhesion. Here, we analyze the role of glycosaminoglycans (GAGs) from Lulo cells and proteins from the parasites in this event.

Methods

Flagellar (F_f) and membrane (M_f) fractions from promastigotes were obtained by differential centrifugation and the purity of fractions confirmed by western blot assays, using specific antibodies for cellular compartments. Heparin-binding proteins (HBP) were isolated from both fractions using a HiTrap-Heparin column. In addition, binding of promastigotes to Lulo cells or to a heparin-coated surface was assessed by inhibition assays or surface plasmon resonance (SPR) analysis.

Results

The success of promastigotes subcellular fractionation led to the obtainment of F_f and M_f proteins, both of which presented two main protein bands (65.0 and 55.0kDa) with affinity to heparin. The contribution of HBPs in the adherence of promastigotes to Lulo cells was assessed through competition assays, using HS or the purified HBPs fractions. All tested samples presented a measurable inhibition rate when compared to control adhesion rate (17 ± 2.0% of culture cells with adhered parasites): 30% (for HS 20μg/ml) and 16% (for HS 10μg/ml); HBP M_f (35.2% for 10μg/ml and 25.4% for 20μg/ml) and HBP F_f (10.0% for 10μg/ml and 31.4% for 20μg/ml). Additionally, to verify the presence of sulfated GAGs in Lulo cells surface and intracellular compartment, metabolic labeling with radioactive sulfate was performed, indicating the presence of an HS and chondroitin sulfate in both cell sections. The SPR analysis performed further confirmed the presence of GAGs ligands on L. (V.) braziliensis promastigote surfaces.

Conclusions

The data presented here point to evidences that HBPs present on the surface of L. (V.) braziliensis promastigotes participate in adhesion of these parasites to Lulo cells through HS participation.

【 授权许可】

   
2012 de Castro Côrtes et al.; licensee BioMed Central Ltd.

【 图 表 】

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