MicrobiologyOpen (Feb 2021)
A Pichia biosensor for high‐throughput analyses of compounds that can influence mosquito behavior
Abstract
Abstract Mosquitoes utilize their sense of smell to locate prey and feed on their blood. Repellents interfere with the biochemical cascades that detect odors. Consequently, repellants are highly effective and resource‐efficient alternatives for controlling the spread of mosquito‐borne illnesses. Unfortunately, the discovery of repellents is slow, laborious, and error‐prone. To this end, we have taken a giant stride toward improving the speed and accuracy of repellant discovery by constructing a prototypical whole‐cell biosensor for accurate detection of mosquito behavior‐modifying compounds such as repellants. As a proof‐of‐concept, we genetically engineered Pichia pastoris to express the olfactory receptor co‐receptor (Orco) of Anopheles gambiae mosquitoes. This transmembrane protein behaves like a cationic channel upon activation by stimulatory odorants. When the engineered Pichia cells are cultured in calcium‐containing Hank's buffer, induction of the medium with a stimulatory odorant results in an influx of calcium ions into the cells, and the stimulatory effect is quantifiable using the calcium‐sequestering fluorescent dye, fluo‐4‐acetoxymethyl ester. Moreover, the stimulatory effect can be titrated by adjusting either the concentration of calcium ions in the medium or the level of induction of the stimulatory odorant. Subsequent exposure of the activated Pichia cells to a repellant molecule inhibits the stimulatory effect and quenches the fluorescent signal, also in a titratable manner. Significantly, the modular architecture of the biosensor allows easy and efficient expansion of its detection range by co‐expressing Orco with other olfactory receptors. The high‐throughput assay is also compatible with robotic screening infrastructure, and our development represents a paradigm change for the discovery of mosquito repellants.
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