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.