Lambda display and RNAi as tools in the study of the Anopheles gambiae salivary glands.

With the aim of investigating invasion of Anopheles salivary glands by Plasmodium parasites, we displayed on the surface of lambda phages selected sporozoite protein domains putatively involved in recognition/invasion of mosquito salivary glands. Adhesive domains from Plasmodium falciparum and Plasmodium berghei TRAP (Trombospondin Related Anonimous Protein), CS (CircumSporozoite), MAEBL (Membrane Antigen and Erythrocyte Binding Like) and CRMPs (Cysteine Rich Modular Proteins) were fused to the C-terminus of the lambda phage capsid protein gpD in D4. Therefore, two mini-collections were produced, independent recombinant lambda clones were isolated and expression of the expected fusion proteins was verified by western analysis. To test the capability of the displayed domains to recognize and bind mosquito salivary glands, we used an in vivo biopanning assay. The phage collections were injected into the haemocoel of An. gambiae adult females and, after a few hours, mosquitoes were dissected and phages bound to glands or other control tissues (i.e. ovaries) were recovered. Initial analysis indicates a slightly stronger binding of the recombinant phage collections as compared to wild type D4. Further in vivo and in vitro binding assays will be performed to optimize experimental conditions suitable for the selection of higher affinity binding domains. In order to develop additional tools to study salivary gland invasion and proceed toward functional analysis, we are also setting up in the lab salivary gene silencing by RNAi. As recently suggested (Boisson B et al, 2006) down-regulation of salivary gland genes in An. gambiae requires the injection of large amounts of dsRNA (approx 5-15 times higher as compared to knock down of midgut- or haemocyte-expressed genes). As a prototype we decided to use gSG6, a female salivary gland-specific gene encoding a short protein with similarity to an anticoagulant from a distantly related species. The function of this salivary protein is still uncharacterized but it appeared especially suitable since a mouse polyclonal anti-serum was available. Preliminary analysis by western blot suggests a significant reduction of gSG6 protein level in salivary extracts from mosquitoes injected with gSG6-dsRNA as compared to buffer-injected controls. Optimization of injection conditions, validation of silencing specificity and mRNA level determination by real-time quantitative RT-PCR are in progress.