The role of chemosensory proteins in pyrethroid resistance

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We have recently shown that elevated expression of chemosensory proteins (CSP) is a potent new pyrethroid resistance mechanism in Anopheles gambiae, the primary vector of malaria in Africa. Resistant populations have elevated CSP levels in their legs and antennae which is further induced by insecticide exposure; partial silencing of one CSP, SAP2, in resistant strains dramatically increases pyrethroid sensitivity whilst over-expressing SAP2 in a susceptible strain confers increased resistance to this insecticide class. Insecticide exposure also induces CSP expression in several agricultural pests suggesting that this family of small proteins, normally associated with chemical communication, may have a wider role in insecticide resistance. In this proposal we will elucidate the mechanism by which SAP2 confers resistance to pyrethroids by testing four, non-mutually exclusive hypotheses. Firstly, as we have established that SAP2 has a high affinity for pyrethroid insecticides in vitro, we will establish whether SAP2 acts to sequester pyrethroids in the legs by using a tagged transgenic line to localise SAP2 expression. Secondly, we will determine whether elevated SAP2 expression accelerates the excretion of pyrethroids by using HPLC to quantify pyrethroid levels in excreta in mosquito populations differing in their SAP2 levels. Next we will determine whether SAP2 acts as a chaperone, transporting pyrethroids to the tissues primarily involved in insecticide detoxification and thus accelerating their metabolism. Finally we will explore the role of SAP2 in pyrethroid avoidance behaviour using simple benchtop behavioural assays and SAP2 knockout lines.
We will then adapt our SAP2 binding assay into a screening cascade which will be used to identify the binding affinities of SAP2, and other CSPs, for insecticides from other classes and then to screen in-house and commercial libraries to identify compounds that disrupt this resistance mechanism in vitro and in vivo.

The long-term beneficiaries of this work will be in the agricultural and public health sectors and those dependent on these sectors for their health and livelihood. The work will both improve our ability to manage insecticide resistance and will, in the longer term, lead to new approaches to break existing resistance mechanisms. Effective insecticide resistance management strategies require an in depth understanding of the causes of resistance (to develop better monitoring tools), their reach (to identify alternative insecticides and/or determine when insecticides are unlikely to have the desired impact) and the impact of the resistance. The latter includes both the impact of resistance on the target organism, for example any fitness costs or pleiotropic effects affecting pathogen transmission, and the operational impact of the resistance on pest control. By characterising the molecular mechanisms by which mosquitoes (and likely agricultural pests) have co-opted chemosensory proteins to avoid the toxic effects of insecticides, we will develop tools to address many of the above key questions. For example, the transgenic lines we develop will enable us to investigate potential fitness costs of CSP based resistance under laboratory conditions which can later be translated into the field. The data on the binding affinities of mosquito CSPs for a wide range of insecticides will aide in in silico predictions of putative CSP/insecticide interactions in other organisms. Furthermore, the mosquito lines we will produce that contain two or more resistance mechanisms will be useful in the screening cascade for new insecticides to ensure these are not undermined by mechanisms already circulating in the field.

In common with antibiotics and anti-parasitic drugs, it is evident with insecticides that the evolution of resistance is outpacing our ability to produce new, safe, effective compounds. Means of mitigating the evolution of resistance are urgently required. New formulations of currently licensed insecticides, and/or the incorporation of non-toxic synergists into these insecticide formulations, are likely to have a shorter lead-time before they reach field application than the development of an entirely new insecticide. Indeed pyrethroid resistance caused by elevated expression of cytochrome P450s can be mitigated by addition of the compound piperonyl butoxide (PBO), which blocks the activity of these enzymes. Already widely used in aerosol sprays of insecticides, bednets containing pyrethroids plus PBO are now being distributed across Africa to address the threat posed by resistance. With the identification of new, more potent resistance mechanisms, such as CSPs, further resistance breaking strategies will be required. This proposal will identify compounds that inhibit binding between SAP2 and insecticides. If successful, future work, in partnership with industry, will establish whether such inhibitors can be used in combination with insecticides to circumvent pyrethroid resistance. We already have close ties with industry through various collaborative programmes and this will facilitate translating promising aspects of this research into practical product development tracks.