Female A. gambiae mosquitoes are primarily attracted by
oct-1-en-3-ol in human sweat and a previous study by another group
revealed that the molecule does not interact with Orco but instead
activates other ORs [48]. It has even been suggested that DEET
repels mosquitoes by reducing the volatility of oct-1-en-3-ol rather
than inhibiting any interactions with ORs [49,50]. We investigated
the interaction between oct-1-en-3-ol and Orco by injecting 1 µL of the
undiluted molecule and 1:1, 1:3 and 1:7 mixtures (volume basis) of
oct-1-en-3-ol and Hank’s buffer into the assay buffer. We performed the
study in Hank’s buffer containing 5 mM of Ca2+ but
noticed insignificant differences in the fluorescence emissions from
each sample (Figure 9 ). Subsequent addition of VUAA1 to a final
concentration of 1 mM in the solutions activated Orco expression by the
PP-Orco cultures equally (data not shown). These observations
confirm
that oct-1-en-3-ol does not interact with Orco of A. gambiae .
Figure 9: The Pichia biosensor does not respond
to oct-1-en-3-ol , which is corroborates previous reports that Orco is
not activated by the odorant. The dilutions correspond to volumetric
ratios of oct-1-en-3-ol and Hank’s buffer containing 5 mM
Ca2+ in an injection of 1 µL into the assay buffer.
‘None’ refers to no dilution.
4. Discussion
Most mosquito and insect repellants that are presently on the market
were discovered using an apparatus known as an olfactometer. It is
quintessentially a Y-shaped chamber that studies the behavior of
mosquitoes that are housed at the base of Y when they are exposed to
odorant samples in both or either one of its two arms [51]. Not only
are these experiments extremely cumbersome and slow to perform, but
olfactometers are error-prone and offer limited control over critical
experimental parameters [16,17]. The Pichia odor biosensor
described in this study directly addresses these limitations. We have
conclusively demonstrated that P. pastoris can functionally
express Orco and that the level of expression can be modulated by
varying the concentration of the transcriptional inducer. We have also
identified culturing conditions that facilitate optimal expression of
the protein. We then tested the biosensor by exposing it to VUAA1, one
of the strongest agonists of Orco reported in literature. Although it is
known that Orco is a cationic channel, we observed that it functions
like a TRP cationic channel after activation, even when expressed
heterologously. Moreover, the fluorescent signal from the assay can be
titrated by adjusting the concentration of either the stimulatory
odorant or concentration of extracellular Ca2+ in the
assay buffer. The EC50s of VUAA1 for the Pichia expressed Orco
were determined to be 0.83 mM and 0.41 mM when the
Ca2+ concentrations in the assay buffer are 1 mM and 5
mM, respectively.
We also exposed the biosensor to citronella oil and oct-1-en-3-ol.
Citronella oil is a widely used insect repellant, and oct-1-en-3-ol is a
metabolite present in human sweat that is the primary attractant of
mosquitoes. Previous studies have revealed that oct-1-en-3-ol does not
interact with Orco [52]. However, the effect of citronella oil on
the protein is poorly understood. In D. melanogaster ,
citronellal, the primary constituent of citronella oil, has been shown
to be an agonist of Orco as well as TRPA1 receptors [46]. ThePichia biosensor corroborates that oct-1-en-3-ol does not
interact with Orco of A. gambiae , but we determined that either
citronellal or a minor constituent of citronella oil interferes with
Orco. The ligand is either a negative allosteric modulator of Orco or an
antagonist that is weakly competitive with VUAA1. We believe it is
possible to further increase the sensitivity and signal-to-noise ratio
of the biosensor by maintaining a higher concentration of
Ca2+ in the assay buffer. P. pastoris has been
shown to grow normally at extracellular Ca2+concentrations as high as 100 mM [53]. We are also confident that
the fluorescence emitted by the cells is definitely a product of channel
activation and not any other phenomena since the physiological
concentration of Ca2+ ions in the cytoplasm ofP. pastoris ranges between 50 and 200 nM [53,54].
Similar biosensors have been constructed previously using HEK293 cells
[32], Sf9 cells [55,56] and Xenopus oocytes [57]. Not
only are these cell lines cumbersome and expensive to maintain, but they
also require the use of patch clamping to assess receptor activation or
deactivation, which is incompatible with high-throughput screening.
Among these competing platforms, only the Xenopus system has been
adapted to a relatively high-throughput microfluidics screening
platform. However, the transformation of Xenopus oocytes is slow
and has a low efficiency. In contrast, not only is the Pichiabiosensor comparably sensitive as these systems [28,58], but it is
also easier to maintain and deploy and simpler to modify and optimize.P. pastoris also has a faster doubling time [59] and does not
require the use of high doses of antibiotics, which is a significant
advantage over other screening platforms [29,30,32]. Moreover, since
all 79 ORs and Orco of female A. gambiae mosquitoes exhibit a
high degree of structural and topological similarity within the
membrane, the system is highly modular and can be used to investigate
any of these proteins by co-expressing them with Orco.
In closing, the Pichia biosensor developed in this study is
sensitive and could form the basis of miniaturized, high-throughput and
highly precise assays for identifying chemical modulators of the
mosquito’s sense of smell. The biosensor opens opportunities for
medicinal chemistry to be performed, which could then facilitate
systematic elucidation of structure-activity relationships and the
subsequent identification of effective repellants through lead
optimization [29]. Beyond repellant screening, the P.
pastoris odorant sensor could also be used as an investigative tool in
other fields such as entomology, agriculture and aromachology.
Funding
This work was supported by the Discovery Grants Program of the Natural
Sciences & Engineering Research Council of Canada (NSERC). JNV was
supported by a doctoral fellowship from NSERC’s Collaborative Research
& Training Experience (CREATE) Program.
Declaration of competing interests
The authors declare no conflicts of interest.
Acknowledgements
We would like to acknowledge Dr. Sandip Pawar for his technical
assistance. We would also like to thank Profs. Abby Collier and Philip
Hieter from the University of British Columbia, who allowed us to use
the ultracentrifuge and Western blotting equipment in their
laboratories, respectively, and Prof. Steven Hallam, also from the
University of British Columbia, for donating the P. pastorisGS115 strain and pPICZA plasmid used in this study.
Author contributions
VGY conceived the study. JNV performed the experiments and collected the
data. JNV and VGY designed the experiments, analyzed the data and wrote
the manuscript.