The Vector Molecular Biology Unit focuses on the molecular aspects of salivary and midgut genes in vector/host and vector/parasite interactions. We are interested in the use of salivary genes as vaccines to block parasite or pathogen transmission. We have previously identified and isolated a salivary gene from the sand fly Phlebotomus papatasi that confers protection against Leishmania major infection in mice. The approach to use vector salivary proteins or genes to block parasite infection may be applicable to other species of Leishmania and also to other vector borne diseases. By using high-throughput approaches we have isolated and sequenced a large number of cDNAs from various species of sand flies, mosquitoes, and from the tick Ixodes scapularis, the vector of Lyme disease. We have selected a number of these salivary genes and prepared DNA vaccines using a high-throughput cloning approach. We tested these salivary DNA constructs in animals and this resulted in the identification of a set of salivary proteins which produced a strong immune response in the skin. Future work is directed to test these molecules for protection against pathogen transmission and to understand the mechanism of this protection. Another interest of the unit is the identification of the Leishmania major attachment site on the Phlebotomus papatasi sand fly midgut. Classical biochemical attempts have failed to identify and isolate this attachment site in the vector midgut. We have isolated and sequenced a large set of full-length genes expressed on the midgut of different phlebotomines and identified a candidate gene homologous to a galactose binding protein. This protein has been produced as recombinant form in different expression systems including, insect cells, mammalian cells and cell-free expression system. Antibodies were produced against this recombinant protein and these antibodies recognized a sand fly midgut protein of 34 kiloDaltons, the predicted molecular mass of the recombinant protein. Future work is directed to demonstrate the binding specificity of Leishmania to this midgut sand fly protein. The midgut of sand flies is a completely unexplored area of research, digestive enzymes, receptors for parasites, anti-bacterial peptides and many other activities have not been examined in detail. Massive sequencing of midgut cDNA libraries revealed a variety of cDNAs never been reported from a Phlebotomine, including chitinases, digestive enzymes, anti-bacterial peptides and many other novel genes which may have an impact in parasite survival and development inside the sand fly vector. We are in the process of categorizing these cDNAs and fully sequence many of them to study in detail their characteristics and activities in the sand fly midgut.
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