gSG6, an Anopheles protein with a role in blood feeding.

The salivary glands of Anopheles gambiae get into multiple interactions with both Plasmodium parasite and human host. The cocktail of factors carried by mosquito saliva are involved in these interactions, not only playing a crucial role in the parasite life cycle but also affecting complex physiological responses such as hemostasis, inflammation and immunity. A large amount of genomic, transcriptomic and expression data obtained in the last few years allowed to shed light on the mosquito salivary protein repertoires, especially on those of An. gambiae and Aedes aegypti (1, 2). This analysis not only led to the identification of several mosquito-specific genes but also showed that anopheline-specific and culicine-specific protein families do exist (3). Overall an interesting outcome is that a quite large number of cDNAs with no similarity to known proteins in databases was identified, which testifies the complexity of mosquito salivary secretions and how much we still need to learn on the saliva of blood-feeding arthropods. To continue in our effort of understanding the evolution of blood-feeding in mosquito vectors and its implications for parasite-vector-host interactions we are proceeding toward functional analysis by expression of recombinant salivary proteins and silencing of salivary genes by RNAi. One of the polypeptides that we expressed and characterized to a certain extent is gSG6, a small protein found so far only in Anopheles species. gSG6 is a relatively abundant secreted protein that is injected into the host with the saliva while the mosquito feeds and it is specifically expressed in the distal portion of lateral-lobes, a region known to express female-specific salivary genes. Database searches showed some weak, but potentially relevant similarity to an anticoagulant from the distantly related Ancylostoma caninum AcAP6 (24% identity, 65% similarity) and to the N-terminal portion of a member of the tumor necrosis factor receptor superfamily (24% identity, 65% similarity). We expressed gSG6 in a Pichia pastoris-based expression system and used the Ni-NTA affinity purified protein fraction to produce a polyclonal antibody as well as for a few functional assays. The recombinant gSG6 did not affect coagulation cascade or show any antiplatelet activity and did not bind TNFα in vitro (R. De Cristofaro, Catholic University, Rome; I. Francischetti and J. Ribeiro, NIH-NIAID); although the precise function of gSG6 is still unknown, most likely it plays some important role in blood-feeding. This is also suggested by the analysis of the probing time and feeding behaviour of gSG6 dsRNA-injected mosquitoes. Actually, six days after dsRNA-injection, when the reduction of gSG6 protein level is >50%, blood-feeding ability and probing time appeared significantly affected. Transcriptome and proteome analysis of the An. gambiae salivary glands have some additional interesting implications. Indeed, it is known that under natural conditions the exposure to mosquito bites is able to elicit a humoral immune response in humans (4). Moreover, a possible correlation between IgG antibody levels against Anopheles saliva and malaria risk has been recently suggested (5, 6). Therefore, immunogenic salivary proteins may be very helpful as epidemiological markers, especially considering that the identification of anopheline- and culicine-specific salivary gene families could allow to overcome cross-reactivity problems and could be exploited to specifically address epidemiological studies targeting anopheline- or culicine-transmitted diseases. (1) Arcà B. et al. 2005 J Exp Biol 208: 3971-86. (2) Ribeiro J:M:C: et al. 2007 BMC Genomics 8: 6. (3) Arcà B. et al. 2006 Parassitologia 48: 573-80. (4) Peng Z. & Simons F.E. 2004 Int Arch Allergy Immunol 133: 198-209. (5) Remoue F. et al. 2006 Trans R Soc Trop Med Hyg 100: 363-70. (6) Waitayakul A. et al. 2006 Acta Trop 98: 66-73.