Developmental Biology Of Leishmania Promastigotes

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Plasmid DNA encoding the Phlebotomus paptasi salivary protein SP15 was able to protect vaccinated mice challenged with parasites plus salivary gland homogenate (SGH). Vaccinated mice developed a strong DTH reaction to sand fly bites, and were protected against challenge using L. major plus SGH. The homologue of SP15 has been cloned from a salivary gland cDNA library of Phlebotomus perniciosus, the main vector of canine leishmaniasis in Mediterranean regions. Dogs immunized with SP15DNA develop a DTH response at the site of P. perniciosus bite. Naive dogs tranferred to endemic zone in southern France have been immunized with SP15 or control DNA and will be followed up over 12 months for seroconversion and/or development of visceral leishmaniasis. The analysis of Leishmania mutants deficient in LPG 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, which have been described for sand flies, serve as parasite attachment sites, and that these molecules can vary between phlebotomine species. We have established cDNA libraries of sand fly midguts from newly emergent sand flies of different species, including P. papatasi, P. argentipes, P. sergenti, and Lutzomyia Longipaplpis. About 500 randomly picked genes were sequenced from the 5' end of the gene followed by sequence similarity searches using the BLAST program. This was followed by blasting each sequence within the library with the remaining genes, thus grouping them in clusters. An important finding was the discovery of the gene encoding for galectin, a galactose-binding lectin. This gene was only found in the midgut of P. papatasi when compared to first round libraries of P. sergenti, P. argentipes and Lu. Longipalpis. Small amounts of properly folded recombinant midgut galectin has been purified from a cell free expression system and used in binding assays to LPG or to cultured promastigotes of different species. Specific binding to L. major LPG was observed that did not occur with L. major mutants lacking the side-chain poly galactose epitope.