Trapping cDNAs encoding secreted and membrane proteins from the Anopheles gambiae salivary glands.

The research project proposed here aims at the isolation and molecular characterization of cDNAs encoding membrane and secreted proteins from the salivary glands of the most efficient malaria vector in the subsaharan Africa, the mosquito Anopheles gambiae. Salivary glands of vector mosquitoes are interesting both because they play an important role in malaria transmission and because they produce substances that facilitate blood-feeding. The current knowledge on the A. gambiae salivary glands is limited to the identification of biochemical activities in their saliva, while the only salivary gland-specific genes so far isolated belong to the dengue and yellow fever vector Aedes aegypti. The Signal Sequence Trap (SST) method allows the trapping, through screening in COS-7 cells, of molecules containing signal peptides such as receptors and secreted proteins and it has been successfully applied to mammalian cDNAs. In this system a 5'end-enriched cDNA expression library is directionally cloned in an appropriate plasmid vector between a promoter that confers strong expression in COS-7 cells and a reporter gene, the α-chain of the interleukin-2 receptor (Tac). cDNAs containing a signal sequence and cloned in frame with the Tac molecule can be expressed as fusion proteins on the surface of COS-7 cells and can be easily detected by a fluoruescent anti-Tac monoclonal antibody. The vectors necessary for the SST methods have been kindly provided from Prof. T. Honjo (Kyoto Univ., Japan) and a 5'end-enriched salivary gland cDNA expression library has already been constructed at the Institute of Parasitology (Univ. di Roma "La Sapienza") where well equiped mosquito insectaries are available. Preliminary screening of the salivary gland cDNA library has shown that insect signal peptides are recognized in COS-7 cells and it has led to the identification of immunofluoruescet positive pools. The objective of the project is to identify potential receptors expressed on the surface of salivary gland cells mediating invasion of Plasmodium sporozoites. The SST system proposed should also result in the isolation of molecules secreted in the saliva and involved in adaptation to blood-feeding; this will allow a deeper understanding of the functions of this specialized organ. The isolation of the receptor for Plasmodium sporozoites and of salivary gland specific promoters will be of great help in the designing of new strategies for vector control as soon as a mosquito transformation system becomes available