Several parasitic and viral diseases that represent a severe threat to human health are transmitted through the bites of arthropod vectors. The mosquito Anopheles gambiae is the most important vector of human malaria, a disease that is a global public health problem and one of the leading causes of mortality in Sub-saharan Africa. The Plasmodium parasite undergoes through complex developmental transitions in the mosquito vector and, as a final step, invades the salivary glands and can be transmitted to the vertebrate host during the next blood meals. The salivary glands of arthropod disease vectors are an interesting object of study not only in virtue of their role in pathogen transmission but also in view of the large variety of pharmacological activities that they secrete. Indeed, hematophagous arthropods saliva contains anti-hemostatic factors, that are essential for an efficient blood-feeding, as well as immuno-modulators, that interfere with the host immune response and that may enhance the transmission of pathogens (as shown for Leishmania by sandflies or for different viruses by mosquitoes and ticks). Moreover, salivary antigens may be strongly immunogenic inducing intense allergic reactions or evoking delayed-type hypersensitivity responses. Because of their immunogenic potential the use of salivary antigens as possible vaccine components is presently being evaluated (Valenzuela et al, 2001 J Exp Med, 194: 331-342). We started a few years ago a molecular study on the An. gambiae salivary glands with a special emphasis on secreted factors and potential sporozoite receptors. For this reason we used the Signal Sequence Trap (SST), a method that would allow for the isolation of cDNAs encoding secreted and transmembrane proteins independently from their functions. In two different rounds of SST screening (Arcà et al, 1999 Proc Natl Acad Sci USA, 96: 1516-1521; Lanfrancotti et al, 2002 FEBS Letters, in press) we identified 22 novel genes which are either specifically expressed in the salivary glands (10 female gland-specific, 6 expressed both in male and female glands) or whose expression is highly enriched in female glands. We studied to a certain extent the platelet inhibitor apyrase (Lombardo F et al, 2000 J Biol Chem, 275: 23861-23868) and a family of D7-related (D7r) genes (Arcà B et al, 2002 Insect Mol Biol, 11:47-55), however, several other proteins such as an Antigen 5 family member (gVAG), putative anticoagulants (cE5, gSG6, gSG7) and a novel family of proteins that we named glandins were identified. A striking result is that we could not assign a possible function to most of the genes identified; this observation underlines the complexity of mosquito saliva and points out that we have identified several novel activities. The accompanying abstracts by Lanfrancotti A et al. and by Lombardo F et al. report in detail some of the main outcome of our study. We summarize below the main properties of the An. gambiae D7r protein family. The D7r represent a cluster of four genes located in a region of approximately 6 kb on chromosome arm 3R. Tissue and developmental RT-PCR expression analysis showed that they are specifically and abundantly expressed in the An. gambiae adult female salivary glands, suggesting that they may play some essential role in blood-feeding. They are similar in sequence to D7, a salivary gene of unknown function previously isolated from the mosquito Aedes aegypti. Sequence analysis shows that the D7r deduced proteins are significantly shorter in comparison to D7 suggesting that the D7 family may include two type of proteins, long and short forms. A high degree of divergence within this protein family is also confirmed by Southern analysis on a few representative mosquito species of the culicine and anopheline subfamilies. The D7r proteins can be aligned, in virtue of four highly conserved cysteine residues, to an heterogeneous group of insect proteins that includes odorant- and pheromone-binding proteins, as well as several other proteins secreted in acqueous media as different as hemolymph, saliva or seminal fluid. The structure of two of these proteins, the Tenebrio molitor THP12 and the Bombyx mori pheromone-binding protein have been determined. They contain six alfa-helix folded to delimit a pocket where small hydrophobic ligands can bind. Secondary structure prediction analysis suggests that the D7r proteins may have a very similar tridimensional structure and that, therefore, they may function as carriers or binders of small hydrophobic molecules.

Toward a better understanding of composition and functions of the salivary secretions of the African malaria mosquito Anopheles gambiae.

ARCA', Bruno;LOMBARDO, Fabrizio;LANFRANCOTTI, Alessandra;COLUZZI BARTOCCIONI, Caio Mario
2002

Abstract

Several parasitic and viral diseases that represent a severe threat to human health are transmitted through the bites of arthropod vectors. The mosquito Anopheles gambiae is the most important vector of human malaria, a disease that is a global public health problem and one of the leading causes of mortality in Sub-saharan Africa. The Plasmodium parasite undergoes through complex developmental transitions in the mosquito vector and, as a final step, invades the salivary glands and can be transmitted to the vertebrate host during the next blood meals. The salivary glands of arthropod disease vectors are an interesting object of study not only in virtue of their role in pathogen transmission but also in view of the large variety of pharmacological activities that they secrete. Indeed, hematophagous arthropods saliva contains anti-hemostatic factors, that are essential for an efficient blood-feeding, as well as immuno-modulators, that interfere with the host immune response and that may enhance the transmission of pathogens (as shown for Leishmania by sandflies or for different viruses by mosquitoes and ticks). Moreover, salivary antigens may be strongly immunogenic inducing intense allergic reactions or evoking delayed-type hypersensitivity responses. Because of their immunogenic potential the use of salivary antigens as possible vaccine components is presently being evaluated (Valenzuela et al, 2001 J Exp Med, 194: 331-342). We started a few years ago a molecular study on the An. gambiae salivary glands with a special emphasis on secreted factors and potential sporozoite receptors. For this reason we used the Signal Sequence Trap (SST), a method that would allow for the isolation of cDNAs encoding secreted and transmembrane proteins independently from their functions. In two different rounds of SST screening (Arcà et al, 1999 Proc Natl Acad Sci USA, 96: 1516-1521; Lanfrancotti et al, 2002 FEBS Letters, in press) we identified 22 novel genes which are either specifically expressed in the salivary glands (10 female gland-specific, 6 expressed both in male and female glands) or whose expression is highly enriched in female glands. We studied to a certain extent the platelet inhibitor apyrase (Lombardo F et al, 2000 J Biol Chem, 275: 23861-23868) and a family of D7-related (D7r) genes (Arcà B et al, 2002 Insect Mol Biol, 11:47-55), however, several other proteins such as an Antigen 5 family member (gVAG), putative anticoagulants (cE5, gSG6, gSG7) and a novel family of proteins that we named glandins were identified. A striking result is that we could not assign a possible function to most of the genes identified; this observation underlines the complexity of mosquito saliva and points out that we have identified several novel activities. The accompanying abstracts by Lanfrancotti A et al. and by Lombardo F et al. report in detail some of the main outcome of our study. We summarize below the main properties of the An. gambiae D7r protein family. The D7r represent a cluster of four genes located in a region of approximately 6 kb on chromosome arm 3R. Tissue and developmental RT-PCR expression analysis showed that they are specifically and abundantly expressed in the An. gambiae adult female salivary glands, suggesting that they may play some essential role in blood-feeding. They are similar in sequence to D7, a salivary gene of unknown function previously isolated from the mosquito Aedes aegypti. Sequence analysis shows that the D7r deduced proteins are significantly shorter in comparison to D7 suggesting that the D7 family may include two type of proteins, long and short forms. A high degree of divergence within this protein family is also confirmed by Southern analysis on a few representative mosquito species of the culicine and anopheline subfamilies. The D7r proteins can be aligned, in virtue of four highly conserved cysteine residues, to an heterogeneous group of insect proteins that includes odorant- and pheromone-binding proteins, as well as several other proteins secreted in acqueous media as different as hemolymph, saliva or seminal fluid. The structure of two of these proteins, the Tenebrio molitor THP12 and the Bombyx mori pheromone-binding protein have been determined. They contain six alfa-helix folded to delimit a pocket where small hydrophobic ligands can bind. Secondary structure prediction analysis suggests that the D7r proteins may have a very similar tridimensional structure and that, therefore, they may function as carriers or binders of small hydrophobic molecules.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11573/472833
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