The role of chemosensory proteins in conferring pyrethroid resistance

Insecticide resistance is a major threat to global health and food security. Globally, vector borne diseases account for more than 17% of infectious disease annually, with over half the world's population currently at risk. Similarly, around 35% of all crops are lost to pre-harvest pests while pressure on increased agricultural output is growing due to an ever-expanding population size. Both vector control and agricultural pest management rely heavily on the use of pesticides. The efficacy of insecticide control is exemplified by the success of malaria control programmes in Africa which have been heavily dependent on the use of pyrethroid insecticides in bednets. The majority of African malaria vectors bite inside the home late at night and the massive scale up in the use of insecticide treated bednets (ITNs) has had a dramatic impact on malaria; between 2000 and 2015, ITNs are estimated to have accounted for nearly 70 % of the reduction in number of malaria cases in Africa, contributing to halving the disease burden since the turn of the century. However, with this success comes a major challenge for the future sustainability of malaria control. As with intensive use of any drug or pesticide, the target organisms (in this case Anopheles mosquitoes), have developed widespread resistance to the chemicals used to control them (in this case pyrethroid insecticides), posing a critical threat to the future of malaria control.

Understanding the mechanisms by which organisms develop resistance is critically important for two key reasons. Firstly we need quick reliable ways to assess which populations have developed resistance to which chemicals so we can select the best alternative method to use. Secondly, an understanding of existing resistance mechanisms is essential to design new drugs or pesticides that are either not affected by existing resistance mechanisms or are specifically designed to break resistance and restore susceptibility to existing chemicals.

We recently discovered a highly potent pyrethroid resistance mechanism in African Anopheles mosquitoes. An increase in the expression of a class of small proteins normally involved in chemical communications (and hence termed chemosensory proteins) in the legs of the mosquito acts as a sponge, absorbing the pyrethroid insecticides as it enters the mosquito via contact with the bednet. One specific member of this protein family, SAP2, is of key importance: mosquitoes that have elevated levels of SAP2 have a much greater chance of surviving pyrethroid exposure and, if we stop the mosquitoes producing this protein, this pyrethroid resistance largely disappears. This latter observation is remarkable as the mosquito populations tested contain additional well established resistance mechanisms including structural changes in the pyrethroid target site that reduce insecticide binding and elevated levels of enzymes that detoxify pyrethroids in the mosquito; the finding that silencing a single small protein can revert these mosquitoes to pyrethroid susceptibility opens up the exciting prospect that we may have found a way of blocking pyrethroid resistance in the mosquito, and potentially other pest species. But further investigation of the mechanism is needed to achieve this goal. In the first part of this proposal we will establish exactly how increases in expression of this SAP2 protein plays such a pivotal role in pyrethroid resistance. In the remaining sections, we will develop methods to break this resistance mechanism; we have already developed a biological test to identify chemicals that block SAP2. Here we propose to convert this to a higher throughput tool that will be used to screen existing libraries of thousands of chemicals to identify potential compounds that could be developed into additives to be used in combination with pesticides to block this resistance mechanism and restore full efficacy of pyrethroid insecticides.

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.