The salivary glands of mosquito vectors are interesting organs for at least two reasons: (i) they play a crucial role in pathogen transmission and several evidences suggest that recognition and entry of malaria parasite into salivary glands may be mediated by interactions of the ligand-receptor type (Rosenberg R., 1985, Am.J.Trop.Med.Hyg., 34: 687-691; Barreau C. et al., 1995, Exp. Parasitol., 81: 332-343); (ii) they specifically express and secrete substances with antihaemostatic activities (such as anti-coagulants, vasodilators and inhibitors of platelet aggregation) that increase the ability of mosquitoes to efficiently feed on blood (James A.A., 1994, Bull. Inst. Pasteur 92: 133-150). The salivary glands of Anopheles gambiae are still poorly characterized, specially at the molecular level; moreover, the identification of the sporozoite receptor and the isolation of salivary gland-specific promoters would represent important steps toward the design of new vector control strategies based on the development of transgenic mosquitoes incapable of transmitting malaria. In order to identify receptors and secreted proteins expressed in the salivary glands of An. gambiae we used the Signal Sequence Trap (SST) method (Tashiro K. et al., 1993, Science, 261: 600-603) which allows the trapping, through screening in COS-7 cells, of molecules containing signal peptides. 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 human interleukin-2 receptor (Tac) lacking the endogenous signal sequence. cDNAs containing a signal sequence and cloned in frame with Tac can be expressed as fusion proteins on the surface of COS-7 cells and can be easily detected by immunostaining with an anti-Tac monoclonal antibody. The cDNA library is screened by transfecting pools of clones in COS-7 cells. Positive pools are divided in sub-pools and re-screened by immunostaining leading to the identification and isolation of single positive clones. We used poly-A(+) RNA from salivary glands of An. gambiae females to construct a 5'end-enriched cDNA expression library and we did our screening in COS-7 cells using the SST method (plasmid vectors were kindly provided from Prof. T. Honjo, Kyoto Univ., Japan). In a first round of screening we were able to isolate eighteen immunofluoruescent positive clones; these cDNA fragments range in size from ~200 to 550 bp and fifteen of them were unique as shown by sequence analysis. All of them, with only one exception, seem to have a signal peptide fused to the reporter gene so they are likely to code for secreted or type I membrane proteins. RT-PCR analysis revealed that five cDNAs are specifically expressed in female salivary glands while other two are expressed both in male and in female glands. As shown by database similarity searches one of these salivary gland-specific clones is the An. gambiae apyrase (see Lombardo F. et al., this meeting) while other three seem to represent different members of a family of proteins that show similarity to the D7 protein of Aedes aegypti (James A.A. et al., 1991, Mol. Biochem. Parasitol., 44:245-254). The remaining three salivary gland-specific cDNA fragments did not show significant similarity to known proteins in the databases. Preliminary results, obtained by RNA in situ hybridization to whole glands, suggest that also in An. gambiae, as in the yellow fever mosquito A. aegypti, female-specific genes are expressed in the distal-lateral and medial lobes while transcripts expressed both in male and in female glands can be localized only in the proximal-lateral lobes. In conclusion using the SST method we have identified the first genes specifically expressed in the salivary glands of the malaria vector An. gambiae. The method worked very efficiently allowing for the trapping of seven salivary gland-specific cDNAs out of 300 clones screened. Several other cDNAs of interest are expected to be trapped by a more exhaustive screening.

Trapping cDNAs coding for receptors and secreted proteins from the salivary glands of the malaria vector Anopheles gambiae.

ARCA', Bruno;LOMBARDO, Fabrizio;DELLA TORRE, Alessandra;COLUZZI BARTOCCIONI, Caio Mario
1998

Abstract

The salivary glands of mosquito vectors are interesting organs for at least two reasons: (i) they play a crucial role in pathogen transmission and several evidences suggest that recognition and entry of malaria parasite into salivary glands may be mediated by interactions of the ligand-receptor type (Rosenberg R., 1985, Am.J.Trop.Med.Hyg., 34: 687-691; Barreau C. et al., 1995, Exp. Parasitol., 81: 332-343); (ii) they specifically express and secrete substances with antihaemostatic activities (such as anti-coagulants, vasodilators and inhibitors of platelet aggregation) that increase the ability of mosquitoes to efficiently feed on blood (James A.A., 1994, Bull. Inst. Pasteur 92: 133-150). The salivary glands of Anopheles gambiae are still poorly characterized, specially at the molecular level; moreover, the identification of the sporozoite receptor and the isolation of salivary gland-specific promoters would represent important steps toward the design of new vector control strategies based on the development of transgenic mosquitoes incapable of transmitting malaria. In order to identify receptors and secreted proteins expressed in the salivary glands of An. gambiae we used the Signal Sequence Trap (SST) method (Tashiro K. et al., 1993, Science, 261: 600-603) which allows the trapping, through screening in COS-7 cells, of molecules containing signal peptides. 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 human interleukin-2 receptor (Tac) lacking the endogenous signal sequence. cDNAs containing a signal sequence and cloned in frame with Tac can be expressed as fusion proteins on the surface of COS-7 cells and can be easily detected by immunostaining with an anti-Tac monoclonal antibody. The cDNA library is screened by transfecting pools of clones in COS-7 cells. Positive pools are divided in sub-pools and re-screened by immunostaining leading to the identification and isolation of single positive clones. We used poly-A(+) RNA from salivary glands of An. gambiae females to construct a 5'end-enriched cDNA expression library and we did our screening in COS-7 cells using the SST method (plasmid vectors were kindly provided from Prof. T. Honjo, Kyoto Univ., Japan). In a first round of screening we were able to isolate eighteen immunofluoruescent positive clones; these cDNA fragments range in size from ~200 to 550 bp and fifteen of them were unique as shown by sequence analysis. All of them, with only one exception, seem to have a signal peptide fused to the reporter gene so they are likely to code for secreted or type I membrane proteins. RT-PCR analysis revealed that five cDNAs are specifically expressed in female salivary glands while other two are expressed both in male and in female glands. As shown by database similarity searches one of these salivary gland-specific clones is the An. gambiae apyrase (see Lombardo F. et al., this meeting) while other three seem to represent different members of a family of proteins that show similarity to the D7 protein of Aedes aegypti (James A.A. et al., 1991, Mol. Biochem. Parasitol., 44:245-254). The remaining three salivary gland-specific cDNA fragments did not show significant similarity to known proteins in the databases. Preliminary results, obtained by RNA in situ hybridization to whole glands, suggest that also in An. gambiae, as in the yellow fever mosquito A. aegypti, female-specific genes are expressed in the distal-lateral and medial lobes while transcripts expressed both in male and in female glands can be localized only in the proximal-lateral lobes. In conclusion using the SST method we have identified the first genes specifically expressed in the salivary glands of the malaria vector An. gambiae. The method worked very efficiently allowing for the trapping of seven salivary gland-specific cDNAs out of 300 clones screened. Several other cDNAs of interest are expected to be trapped by a more exhaustive screening.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11573/472818
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