Identification of novel salivary gland genes from the malaria mosquito Anopheles gambiae by the Signal Sequence Trap.

The salivary glands of mosquito vectors are an interesting target for molecular entomology and for parasitology studies both because they are the site of production of tissue-specific factors involved in the feeding process and because they represent the final destination of the malaria parasites prior to their inoculation into the vertebrate host. The identification of genes specifically expressed in the glands and/or playing a role in the host-parasite interaction is of special interest not only for a better understanding of adaptation to haematophagy and of gland recognition/invasion by Plasmodium but also in view of the development of vector control strategies based on genetically engineered Plasmodium-resistant mosquitoes. The objective of this work is the identification of novel genes encoding secreted proteins and/or potential sporozoite receptors expressed in the salivary glands of the major african malaria vector Anopheles gambiae. We have previously employed the Signal Sequence Trap to identify the first genes specifically expressed in the salivary glands of An. gambiae (Arcà B et al., 1999, Proc Natl Acad Sci USA, 96: 1516-1521). Briefly, a salivary gland cDNA expression library is screened by transfection of COS7 cells. cDNAs coding for proteins containing a signal peptide, such as receptors and secreted factors, can be expressed as recombinant fusion proteins on the surface of transfected COS7 cells and can be revealed by immunostaining with a monoclonal antibody. We present here the results of an additional round of SST. Clones identified during the initial screen were subtracted by colony hybridization before proceeding to a new screening of the salivary gland cDNA library in COS7 cells. A total of approximately 1100 clones have been screened until now leading to the isolation of twenty-three individual clones. Thirteen clones have been sequenced and they represent eight different cDNA fragments whose size ranges from approximately 320 to 550 base pairs. According to prediction analysis all these cDNA fragments have the potential to encode putative proteins with a signal peptide at their amino-terminus suggesting that they are likely to represent secreted or membrane anchored molecules. Sequence comparison revealed that one cDNA fragment represented the already known An. gambiae lysozyme whereas the other seven represented novel genes. Five of these cDNAs did not show similarity to known proteins, indicating that they encode novel functions, and the other two, 55F and 11B, showed respectively 35% and 43% identity (40% and 47% similarity) to gSG1 and gSG2, two An. gambiae salivary gland-specific genes previously isolated (Arcà B et al., 1999, Proc Natl Acad Sci USA, 96: 1516-1521). The tissue-specific expression profile was analysed by reverse transcription-PCR using as template total RNA from adult female salivary glands, female carcasses (adult females deprived of salivary glands) and adult males. Four classes of genes can be distinguished: (i) two expressed only in female salivary glands, whose function is presumably related to blood feeding; (ii) one expressed both in female glands and in males and whose role may be related to sugar-feeding or to more general physiological functions of the glands; (iii) two highly enriched in female glands; (iv) three expressed at approximately the same level in the three tissue analyzed. It is obviously anachronistic at this stage to draw any conclusion, however, the isolation of full-length cDNAs and the characterization of the additional clones that is in progress will certainly provide a more comprehensive picture of the function and the physiology of this organ of crucial importance in parasite transmission.