The Anopheles gambiae odorant binding protein 1 (AgamOBP1) mediates indole recognition in the antennae of female mosquitoes

Abstract

Haematophagous insects are frequently carriers of parasitic diseases, including malaria. The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa and is thus responsible for thousands of deaths daily. Although the role of olfaction in A. gambiae host detection has been demonstrated, little is known about the combinations of ligands and odorant binding proteins (OBPs) that can produce specific odor-related responses in vivo. We identified a ligand, indole, for an A. gambiae odorant binding protein, AgamOBP1, modeled the interaction in silico and confirmed the interaction using biochemical assays. RNAi-mediated gene silencing coupled with electrophysiological analyses confirmed that AgamOBP1 binds indole in A. gambiae and that the antennal receptor cells do not respond to indole in the absence of AgamOBP1. This case represents the first documented instance of a specific A. gambiae OBP-ligand pairing combination, demonstrates the significance of OBPs in odor recognition, and can be expanded to the identification of other ligands for OBPs of Anopheles and other medically important insects.

Figure 1. Fluorescence screening system for the detection of native ligands for OBPs.

Three concentrations of each ligand (16 µM, 8 µM and 4 µM) were tested using 4 µM of r-OBP1 (left panel). Indole was found to bind r-OBP1 as increasing concentrations of indole displaced more dye from the AgamOBP1 binding pocket. (right panel) Binding curve of ³H indole to r-OBP1 in Ni-NTA FlashPlates. Specific binding was determined after background subtraction from uncoated wells. Curve fitting was performed with GraphPad Prism and the calculated K_D is 2.3 µM±0.3 (95 % CI).

Figure 2. In silico ligand binding studies on Anopheles gambiae OBP1.

(A) AgamOBP1 with indole fitted in the binding cavity. Cyan is the indole ring. Residues in red line the binding site. (B) Surface representation of the AgamOBP1 binding cavity (wired mesh) and indole (semi transparent continuous surface) showing the calculated binding position of indole fitted in the upper part of the binding site. (C) Predicted oleic acid (cyan) binding position indicating the L shaped binding cavity. Diagrams created with PYMOL (DeLano Scientific LLC).

Figure 3. qRT-PCR determination of mRNA levels in mosquitoes.

Pools of 5 female mosquitoes 4 days after injection with AgamOBP1-dsRNA or AgamOBP7-dsRNA were analysed. Values were normalized to RpS7(S7). While OBP mRNA levels of a control OBP (AgamOBP7-left, AgamOBP4-right) remained unchanged, mRNA levels of the corresponding injected dsRNA (AgamOBP1-left, AgamOBP7-right) were reduced about 10-fold; C control, I injected.

Figure 4. Western blot analyses for the detection of OBP1.

Individual head extracts of AgamOBP1-dsRNA-injected female mosquitoes (OBP1dsRNA), as well as AgamOBP7-dsRNA-injected (OBP7dsRNA) or uninjected females (F) (top panel) were subjected to SDS PAGE and Western blot. The membranes were subsequently incubated, without stripping, with an anti-AgamOBP48 antibody (middle panel) and, finally, after stripping, with an anti-tubulin antibody (lower panel).

Figure 5. Electroantennogram responses of Anopheles gambiae females.

Electroantennogram responses of Anopheles gambiae females to p-cresol (1), indole (2), 3-methyl indole (3) and geranylacetone (4) eluting from an apolar gas chromatographic column. For the top 3 traces, 20 ng quantities of p-cresol, indole, 3-methyl indole and geranylacetone were injected and for the next two traces down 2 ng of these ligands were injected. The column effluent was split (50:50) between the flame ionization detector (FID, bottom trace) of the chromatograph and the antennal preparations (control and treatments above). In recordings from the antennae of three females injected with AgamOBP1-dsRNA (treatments) the response to indole and 3-methyl indole was silenced whereas in two recordings from the antennae of a females injected with AgamOBP7-dsRNA (controls) the responses to indole and 3-methyl indole were no different to that of an uninjected female (small insert on right); mV scale common to treatment and control recordings.