Development of an intein-based QF2 system for controllable protein expression in the nervous system of Aedes aegypti

SUMMARY Aedes mosquito-transmitted viruses such as dengue, Zika, Yellow fever and chikungunya affect millions of people each year, and more than half of the world’s population is at risk of infection. As such, there is an urgent need to reduce the spread of these viruses, which would have a significant positive impact on global public health. The overarching objective of this application is to harness powerful genetic and synthetic biology tools developed in classic genetic model systems such as N. crassa, S. cerevisiae and D. melanogaster for the rational design of novel, controllable protein expression modules in the mosquito Ae. aegypti, with the goal to disrupt effective propagation of this insect species. This application leverages the bimodal QF/QUAS expression system (referred to as Q-system) from N. crassa (referred herein as Q-system) to modulate key olfactory and neurosecretory circuits that control virtually all aspects of insect behavior and physiology. Specifically, it is proposed to re-engineer the Q-system and incorporate temperature-sensitive self-splicing intein modules (INTts) from the yeast S. cerevisiae into the transcription factor QF2 to generate temperature sensitive QF2 drivers and conditional alleles for Glucose-6- Phosphatase (G6P) and Olfactory receptor co-receptor (Orco). G6P plays a key role in Drosophila neuropeptide signaling, while Orco is essential in both D. melanogaster and Aa. aegypti for odor perception in general through its key role in odorant receptor trafficking to dendritic processes of olfactory neurons. The experimental strategy takes advantage of the evolutionary relationship between the diptera D. melanogaster and Ae. aegypti and harnesses the ease by which Drosophila can be molecular-genetically manipulated and functionally assayed. Specifically, synthetic knock-in and conditional transgenes creating novel, intein- containing proteins will be functionally validated in Drosophila before transferred to mosquitoes. Together these new synthetic biology tools will be highly valuable to both the Drosophila research and the vector biology communities and will allow new investigations into understanding and disrupting peptidergic and olfactory signaling in mosquitoes. The studies proposed in this application will significantly expand overall understanding of key genetic components that drive mosquito mating, feeding, and host-seeking behaviors and enable new, neural-based strategies for mosquito population control.