Department of Entomology, College of Agriculture, University of Sargodha, Sargodha 40100, Pakistan; Department of Entomology, University of Georgia, Athens, GA 30602-2603, United States.
Department of Entomology, University of Georgia, Athens, GA 30602-2603, United States; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-2603, United States.
J Proteomics. 2020 Sep 15;227:103918. doi: 10.1016/j.jprot.2020.103918. Epub 2020 Jul 24.
Bacterial insecticidal proteins, such as the Bin toxin from Lysinibacillus sphaericus, could be used more extensively to control insecticide resistant mosquitoes. This study was aimed at identification of mosquito cell proteins binding Bin toxin. Results showed that purified toxin was toxic to Anopheles gambiae larvae and Ag55 cultured cells. Clathrin heavy chain (an endocytosis protein) and glycolytic enzymes such as pyruvate kinase, enolase and dihydrolipoamide dehydrogenase were identified as binders of Bin toxin. The viability of Ag55 cells in the presence of endocytosis inhibitor, pitstop2, was significantly decreased upon Bin treatment, while the inhibitor chlorpromazine did not affect Bin toxicity. Bin toxin treatment decreased ATP production and mitochondrial respiration in Ag55 cells, whereas non-mitochondrial oxygen consumption significantly increased after Bin toxin treatment. These findings are steps towards understanding how Bin toxin kills mosquitoes. SIGNIFICANCE: Mosquitoes are vectors of pathogens causing human diseases such as dengue fever, yellow fever, zika virus and malaria. An insecticidal toxin from Lysinibacillus sphaericus called Binary, or Bin, toxin could be used more extensively to control insecticide resistant mosquitoes. Bin toxin enter cells in susceptible mosquitoes and induces apoptosis or autophagy. In the current research, we used the malaria mosquito Anopheles gambiae Ag55 cell line as a model. A proteomic-based approach identified proteins that interact with Bin toxin. Interacting proteins include clathrin heavy chain (endocytosis protein) and glycolysis enzymes such as pyruvate kinase, enolase and dihydrolipoamide dehydrogenase. In Ag55 cell toxicity assays, an endocytosis inhibitor, pitstop2, increased Bin toxicity. Real time assays with a Seahorse™ flux analyzer showed that Bin significantly affects mitochondrial respiration, a result consistent with cell death via apoptosis or autophagy. These research findings add insights into how an unusual binary protein exploits cellular machinery to kill mosquitoes.
细菌杀虫蛋白，如球形芽孢杆菌的 Bin 毒素，可更广泛地用于控制对杀虫剂有抗性的蚊子。本研究旨在鉴定与 Bin 毒素结合的蚊子细胞蛋白。结果表明，纯化的毒素对冈比亚按蚊幼虫和 Ag55 培养细胞具有毒性。网格蛋白重链（一种内吞作用蛋白）和糖酵解酶，如丙酮酸激酶、烯醇酶和二氢硫辛酰胺脱氢酶被鉴定为 Bin 毒素的结合物。在存在内吞作用抑制剂 pitstop2 的情况下，Ag55 细胞的活力在用 Bin 处理时显著降低，而抑制剂氯丙嗪对 Bin 的毒性没有影响。Bin 毒素处理降低了 Ag55 细胞中的 ATP 产生和线粒体呼吸，而在用 Bin 毒素处理后，非线粒体耗氧量显著增加。这些发现是了解 Bin 毒素杀死蚊子的途径的步骤。意义：蚊子是引起人类疾病（如登革热、黄热病、寨卡病毒和疟疾）的病原体的载体。一种名为 Binary 或 Bin 的球形芽孢杆菌杀虫毒素可更广泛地用于控制对杀虫剂有抗性的蚊子。Bin 毒素进入敏感蚊子的细胞，并诱导细胞凋亡或自噬。在当前的研究中，我们使用疟蚊冈比亚按蚊 Ag55 细胞系作为模型。基于蛋白质组学的方法鉴定了与 Bin 毒素相互作用的蛋白质。相互作用的蛋白质包括网格蛋白重链（内吞作用蛋白）和糖酵解酶，如丙酮酸激酶、烯醇酶和二氢硫辛酰胺脱氢酶。在 Ag55 细胞毒性测定中，内吞作用抑制剂 pitstop2 增加了 Bin 的毒性。使用 Seahorse™通量分析仪的实时测定表明，Bin 显著影响线粒体呼吸，这一结果与通过细胞凋亡或自噬导致的细胞死亡一致。这些研究结果为了解一种不寻常的二元蛋白如何利用细胞机制杀死蚊子提供了新的见解。
