The role of chemosensory proteins in conferring pyrethroid resistance

抗药性是对全球健康和粮食安全的主要威胁。在全球范围内，媒介传播疾病每年占传染病的17%以上，目前世界上一半以上的人口处于危险之中。同样，约35%的作物因收获前害虫而损失，而由于人口规模不断扩大，农业产量增加的压力也在增加。病媒控制和农业虫害管理都在很大程度上依赖于农药的使用。非洲的疟疾控制方案在很大程度上依赖在蚊帐中使用拟除虫菊酯杀虫剂，其成功证明了杀虫剂控制的效力。大多数非洲疟疾病媒在深夜在家中叮咬，杀虫剂处理过的蚊帐（驱虫蚊帐）的大规模使用对疟疾产生了巨大影响; 2000年至2015年期间，驱虫蚊帐估计占非洲疟疾病例数量减少的近70%，自世纪以来，疾病负担减半。然而，随着这一成功而来的是对今后疟疾控制可持续性的重大挑战。与密集使用任何药物或杀虫剂一样，目标生物（此处为按蚊）对用于控制它们的化学品（此处为拟除虫菊酯杀虫剂）产生了广泛的抗药性，对疟疾控制的未来构成了严重威胁。了解生物体产生耐药性的机制至关重要，原因有两个。首先，我们需要快速可靠的方法来评估哪些人群已经对哪些化学品产生了抗药性，这样我们就可以选择最好的替代方法来使用。其次，了解现有的抗性机制对于设计新的药物或杀虫剂是至关重要的，这些药物或杀虫剂要么不受现有抗性机制的影响，要么专门设计用于打破抗性并恢复对现有化学品的敏感性。蚊子腿中通常参与化学通讯的一类小蛋白（因此称为化学感受蛋白）表达的增加充当海绵，当拟除虫菊酯杀虫剂通过与蚊帐接触进入蚊子时吸收拟除虫菊酯杀虫剂。这种蛋白质家族的一个特定成员SAP 2至关重要：SAP 2水平升高的蚊子有更大的机会在拟除虫菊酯暴露中幸存下来，如果我们停止蚊子产生这种蛋白质，这种拟除虫菊酯抗性基本消失。后一种观察结果是值得注意的，因为测试的蚊子种群含有额外的成熟的抗性机制，包括拟除虫菊酯靶位点的结构变化，其减少杀虫剂结合和蚊子中使拟除虫菊酯解毒的酶水平升高;沉默一个小蛋白质可以使这些蚊子恢复对拟除虫菊酯的敏感性，这一发现开辟了令人兴奋的前景，我们可能已经找到了一种阻断拟除虫菊酯敏感性的方法。蚊子对拟除虫菊酯的抗药性，以及潜在的其他害虫物种。但要实现这一目标，还需要进一步研究其机制。在本提案的第一部分中，我们将确切地确定SAP 2蛋白表达的增加如何在拟除虫菊酯抗性中发挥如此关键的作用。在剩下的部分中，我们将开发打破这种抗性机制的方法;我们已经开发了一种生物测试来识别阻断SAP 2的化学物质。在这里，我们建议将其转换为更高通量的工具，该工具将用于筛选现有的数千种化学品库，以确定可开发为添加剂的潜在化合物，这些添加剂可与杀虫剂结合使用，以阻断这种抗性机制并恢复拟除虫菊酯类杀虫剂的全部功效。

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.