Leishmania enriettii: caracterização preliminar dos lipofosfoglicanos e glicoinositolfosfolípides e infectividade para Cavia porcellus

Abstract

Leishmania enriettii is a non-infectious species to man, whose reservoir is Cavia porcellus. Some aspects of the parasite-host interaction in this model are still unknown, especially aspects involving parasite surface molecules. Many Lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) of species from the Old and New World have already been described. However, some glicobiology aspects of L. enriettii are still unknown. In this study, we have preliminarly characterized the LPGs and GIPLs from two strains of L. enriettii (L88 and Cobaia) isolated from their vertebrate hosts in 1945 and 1985, respectively. Moreover, the role of those molecules was evaluated during in vitro interaction with mice peritoneal macrophages and Chinese Hamsters ovary cells (CHO). Additionally, their infectivity was evaluated in vivo in Cavia porcellus. LPGs were extracted, purified and analysed by western-blot, showing that LPG from type L88 was longer than that of Cobaia strain. LPGs and GIPLs were depolymerized and their sugar was analysed through electrophorese of carbohydrates. The region of repetitive units of the two types of LPGs did not present lateral chains, being represented by disaccharide Gal(1,4)Man(1)-PO4. The GIPL of the strain L88 presented galactose in its structure, similar to GIPL of type II. On the other hand, the GIPL of Cobaia strain presented an abundance of glucose, a characteristic not yet observed. In order to evaluate the type of receptor recognized by LPGs and GIPLs, mice peritoneal macrophages of C57BL/6 and Knock-out (TLR2 -/- and TLR4 -/-) were primed with IFN-y and stimulated with glycoconjugates and their respective live parasites (MOI 10:1). No activation of NO or cytokines was observed with live parasites. On the other hand, LPGs and GIPLs were able to activate the production of NO and cytokines (IL-6, IL-12 and TNF). Furthermore, LPGs were more powerful agonists than GIPLs, preferably via TRL2 and secondarily via TRL4. To evaluate separately the role of each TRL, we used CHO cells. In this model, it was observed that LPGs and GIPLs were not able to activate neither TLR2 nor TRL4, suggesting a co-participation of both receptors. In order to evaluate the infectivity the two strains, male guinea pigs (cobaia) of the species Cavia porcellus were used. These animals were inoculated with parasites of the two strains in the presence or absence of the salivar gland extract (GSF) from te sandfly Lutzomyia longipalpis and evaluated weekly for 91 days. The infected animals started to develop protuberance and/or lesion in the 4th and 5th week after the infection. But only L88 strain developed ulcerated lesions. Size, development and scaring time were greater when the parasites were inoculated in the presence of GSF (p< 0.01). In conclusion, the L88 strain of L. enriettii exhibited a longer LPG and GIPL type II. Those features may be related to a more pro-inflammatory profile than the Cobaia strain. Those in vitro data were confirmed in vivo, where L88 species was able to develop ulcerated lesions.