Vector Biological Studies in Leishmaniasis

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Abstract

The analysis of Leishmania mutants deficient in lipophosphoglycan expression have revealed dual roles for phosphoglycans as virulence molecules in the sand fly vector: the sereted molecules protect the parasite from proteolytic digestion in the bloodfed midgut, and the surface LPG mediates attachment of the parasite to the gut wall so as to prevent loss of infection during bloodmeal excretion. The role of LPG in mediating attachment to the midgut suggests that gut-associated lectins or lectin-like molecules serve as parasite attachment sites, and that these molecules can vary between phlebotomine species. We established cDNA libraries of sand fly midguts of different species, including P. papatasi, P. argentipes, P. sergenti, and Lutzomyia Longipaplpis. A full length cDNA, PpGal, encoding a galectin with two recognition sites for ?-galactoside sugars was identified as an abundantly expressed gene from a midgut library of Phlebotomus papatasi. This gene is not expressed in the midgut of other sand fly species. The recombinant galectin protein from an E. coli cell-free system bound specifically to L. major parasites but not to LPG-deficient mutants, providing strong evidence that the galectin is the midgut receptor for attachment of L. major parasites. The use of RNA interference in silencing the expression of PpGal is being developed. Other genes targeted for silencing by RNAi include those encoding chymoptrypsin (PpChym2), involved in bloodmeal digestion, and chitinase, responsible for the breakdown of the peritrophic memebrane. Several groups of one hundred P. papatasi sand flies were microinjected intrathoracically with 20nl (1-5mg/ml) dsRNA of PpChym2 and PpGal. In each group, the surviving flies and non-injected controls were blood fed 24hrs post injection. RNA was isolated from individual midguts 24hrs PBM and analyzed for the expression of both PpChym2 and PpGal by RT-PCR. Some 30% of the injected flies showed complete or partial specific silencing of target mRNA. For PpChym2, the effect on the mRNA expression was supported by a reduction in protein levels and enzyme activity.