The OR-mediated olfactory cascade in mosquitoes also involves another protein called the olfactory receptor co-receptor (Orco). This protein plays a key role in signal transduction cascade that originates with the ORs and ends with their cognate G-protein complexes. It is a homomeric protein that comprises four subunits that are symmetrically arranged around a central channel [28]. Interestingly, it has been suggested that Orco itself can function as a cationic channel upon activation by some OBP-odorant complexes and odorants when it is expressed heterologously [29]. This property of Orco makes it a promising target for repellant design [18] and the central piece of the odor biosensing described herein (Figure 1 ). Consequently, we genetically engineered P. pastoris to express the Orco protein of female A. gambiae mosquitoes. The use of this Pichiabiosensor for evaluation of the stimulatory or repellatory commences with incubation of the cells in a buffered solution that contains the calcium-sequestering dye, fluo-4-acetoxymethyl ester (abbreviated as fluo-4-AM), in a standard multi-well plate. The calcium-bound form of the dye is highly fluorescent whereas the fluorescence emitted by its free form is barely detectable. Calcium-bound fluo-4-AM has an excitation and emission wavelength of 485 and 520 nm, respectively.
Figure 1: Design of the odor biosensor. (A) The complete odor transduction pathway in mosquitoes involves odor binding proteins (OBPs), olfactory receptors (ORs) and the olfactory receptor co-receptor (Orco). (B) Previous studies have shown that Orco can function as a cationic channel and individually transduce some olfactory signals. As a consequence, the refactored olfactory transduction pathway was constructed in P. pastoris by solely employing Orco.
After allowing sufficient time for the dye to infuse the cells, we transfer the cells to a multi-well plate in a calcium-containing medium and later inject odorants into the solution. Stimulatory odorants activate Orco and trigger the influx of calcium ions from the solution into the cells, and the increase in intracellular calcium concentration generates a fluorescent signal that can be titrated by adjusting either the concentration of calcium ions or odorant in the solution. If repellatory or inert odorants are added to the solution instead of a stimulatory molecule, they will not change the intracellular concentration of calcium ions. However, a repellant can be differentiated from an inert odorant by preceding its addition to the medium with the introduction of a known stimulatory odorant. The repellant will dampen or plateau the fluorescent signal whereas an inert molecule that does not interact with Orco will have no effect on the fluorescence emitted by the cells.
We assessed the performance of the Pichia odor biosensor to quantify the stimulatory or repellatory effect of odorants that have been studied previously in exceptionally complicated investigative models such as empty neurons of Drosophila melanogaster [30]. Specifically, we exposed the biosensor to oct-1-en-3-ol, a known mosquito attractant that is present in human sweat but does not activate Orco [31]; 2-(4-ethyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-ylthio)-N-(4-ethylphenyl)acetamide or VUAA1, one of the strongest activators of Orco reported in literature [32], and citronella oil, whose effect on Orco remains poorly understood [12]. Not only does the sensitivity (based on EC50 values) and specificity of the Pichia biosensor compare favorably to previously reported systems, but it is manifold faster to construct and deploy. Moreover, its modular architecture allows easy and efficient expansion of its detection range by co-expression of Orco with any of the 79 ORs expressed by female A. gambiae mosquitoes. This simple biosensor can form the basis of a high-throughput, high-resolution platform for detecting chemical modulators of the mosquito’s sense of smell that, perhaps most significantly, is cheap, modular and compatible with robotic screening infrastructure in place in the pharmaceutical industry. Moreover, it could also be easily adapted to study pollination and insect aromachology.