Blood-engorged females were separated and maintained at 26?C

Blood-engorged females were separated and maintained at 26?C. the native protein with anti-rLuloG during sand fly infections, we demonstrated strong binding of rLuloG to whole surface of promastigotes. Conclusions We characterized a novel O-glycoprotein from sand fly parasites in vitro. We propose a role of this molecule in attachment to sand fly midgut. protozoans, the causative agents of leishmaniases, are transmitted by bites of female phlebotomine sand flies. In the sand fly vector, parasites must overcome various barriers to generate transmissible infections and ensure continuation of the life-cycle (reviewed by [1]). A crucial step in the vector phase of the life-cycle is the binding of promastigotes to the sand fly midgut. Promastigotes insert their flagella into the microvillar border of the Dacarbazine midgut epithelium and anchor themselves to the midgut surface [2, 3]. Colec11 This attachment enables them to persist in the sand fly gut when the blood meal remnants are defecated. A series of studies on the parasite-vector combination established an important paradigm; galactose residues on mono-galactosylated phosphodisaccharide repeats of the major surface glycoconjugate lipophosphoglycan (LPG) have been shown to bind to a galectin located on the surface of the sand fly midgut [4C7]. However, a more recent study reported partial involvement of the flagellar protein FLAG1/SMP1 in attachment of to the midgut epithelium of [8], and earlier experiments with LPG-deficient mutants revealed that LPG is not required for the attachment in many other sand fly species [9C11]. These studies indicate that alternative attachment molecules can be involved in midgut binding in addition to LPG-galectin. Laboratory studies on vector competence to parasites suggest that sand flies fall into two broad groups. Three species, Dacarbazine i.e. and are termed specific or restricted vectors that support the development of one species only (and and spp. In laboratory conditions parasites are capable of developing in Dacarbazine any permissive vector, if given the opportunity (reviewed by [1, 12]). In a study exploring the molecular basis of vector competence My?kov and colleagues [9] revealed a remarkable correlation between specificity permissivity of the vector and the glycosylation of its midgut proteins. The agglutinin (HPA), a lectin specific for terminal N-acetyl-galactosamine (GalNAc) present Dacarbazine on O-linked glycoconjugates, bound to midgut proteins from permissive but not from specific vectors. All five permissive species tested possessed HPA positive Dacarbazine bands, whereas none were detected in the three specific vectors examined [9, 10, 13]. These findings suggested a hypothesis for the role of O-linked glycoconjugates in binding, which was supported by two further observations. Fluorescent-labelled HPA (FITC-HPA) showed specific localisation of O-glycosylated epitopes in the microvillar border of sand fly midgut, the location required for attachment, and the O-linked glycoconjugates recognised by FITC-HPA bound to the surface of promastigotes [9]. In the present work we describe an O-linked glycoconjugate of the permissive vector and show that it has mucin-like properties. Functional testing was performed using the recombinant form of the glycoconjugate (rLuloG) and specific anti-rLuloG antibodies using in vivo and in vitro methods. For in vivo studies, experimental infections of with were performed as described previously for and galectin [6]: parasites were mixed with blood reconstituted with specific anti- rLuloG serum to test for blocking of parasite development in the sand fly midgut. In vitro experiments were performed to investigate the binding of rLuloG to parasites. Methods Sand fly colonies and parasites Laboratory colonies of two sand fly species were used: (origin from Jacobina, Brazil) and (origin from Turkey). Colonies were maintained in conditions described previously [14] and fed on 50?% sucrose..