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.
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 (Ff) and membrane (Mf) 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 Ff and Mf proteins, both of which presented two main protein bands (65.0 and 55.0 kDa) 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 20 μg/ml) and 16% (for HS 10 μg/ml); HBP Mf (35.2% for 10 μg/ml and 25.4% for 20 μg/ml) and HBP Ff (10.0% for 10 μg/ml and 31.4% for 20 μg/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.
Laboratório de Biologia Molecular e Doenças Endêmicas, Av, Brasil 4365, Rio de Janeiro - CEP, 21040-360, Brazil.
Luzia Monteiro
LM
de Castro Côrtes
Mirian Claudia
MC
de Souza Pereira
Franklin Souza
FS
da Silva
Bernardo Acácio Santini
BA
Pereira
Francisco Odêncio
FO
de Oliveira Junior
Renata Oliveira
RO
de Araújo Soares
Reginaldo Peçanha
RP
Brazil
Leny
L
Toma
Carolina Meloni
CM
Vicente
Helena Bonciani
HB
Nader
Maria
M
de Fátima Madeira
Felio J
FJ
Bello
Carlos Roberto
CR
Alves
D000818:Animals; D002448:Cell Adhesion / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000502:physiology; D007892:Leishmania braziliensis / Q000378:metabolism*; D011576:Psychodidae / Q000166:cytology*
D015815:Cell Adhesion Molecules; C074285:heparin-binding protein (Leishmania)