Mishra Ruchir, Guo Ya, Kumar Pavan, Cantón Pablo Emiliano, Tavares Clebson S, Banerjee Rahul, Kuwar Suyog, Bonning Bryony C
Department of Entomology and Nematology, University of Florida, PO Box 110620, Gainesville, FL 32611, USA.
Curr Res Insect Sci. 2021 Feb 18;1:100012. doi: 10.1016/j.cris.2021.100012. eCollection 2021.
Phage display libraries have been used to isolate insect gut binding peptides for use as pathogen transmission blocking agents, and to provide artificial anchors for increased toxicity of bacteria-derived pesticidal proteins. Previously, phage clones displaying enriched peptides were sequenced by Sanger sequencing. Here we present a streamlined protocol for identification of insect gut binding peptides, using insect-appropriate feeding strategies, with next generation sequencing and tailored bioinformatics analyses. The bioinformatics pipeline is designed to eliminate poorly enriched and false positive peptides, and to identify peptides predicted to be stable and hydrophilic. In addition to developing streamlined protocols, we also sought to address whether candidate gut binding peptides can bind to insects from more than one order, which is an important consideration for safe, practical use of peptide-modified pesticidal proteins. To this end, we screened phage display libraries for peptides that bind to the gut epithelia of two pest insects, the Asian citrus psyllid, (Hemiptera) and beet armyworm, (Lepidoptera), and one beneficial insect, the western honey bee, (Hymenoptera). While unique peptide sequences totaling 13,427 for , 89,561 for and 69,053 for were identified from phage eluted from the surface of the insect guts, final candidate pools were comprised of 53, 107 and 1423 peptides respectively. The benefits of multiple rounds of biopanning, along with peptide binding properties in relation to practical use of peptide-modified pesticidal proteins for insect pest control are discussed.
噬菌体展示文库已被用于筛选昆虫肠道结合肽，用作病原体传播阻断剂，并为提高细菌来源的杀虫蛋白的毒性提供人工锚定。此前，通过桑格测序对展示富集肽的噬菌体克隆进行测序。在此，我们提出了一种简化方案，利用适合昆虫的喂食策略、下一代测序和定制的生物信息学分析来鉴定昆虫肠道结合肽。该生物信息学流程旨在消除富集不足和假阳性肽，并鉴定预测为稳定且亲水性的肽。除了开发简化方案外，我们还试图探讨候选肠道结合肽是否能与多个目的昆虫结合，这是肽修饰的杀虫蛋白安全、实际应用的一个重要考虑因素。为此，我们筛选噬菌体展示文库，寻找能与两种害虫（半翅目的亚洲柑橘木虱和鳞翅目的甜菜夜蛾）以及一种益虫（膜翅目的西方蜜蜂）的肠道上皮结合的肽。虽然从昆虫肠道表面洗脱的噬菌体中分别鉴定出了针对亚洲柑橘木虱的总共13427个、针对甜菜夜蛾的89561个和针对西方蜜蜂的69053个独特肽序列，但最终的候选肽库分别由53个、107个和1423个肽组成。文中讨论了多轮生物淘选的益处，以及肽结合特性与肽修饰的杀虫蛋白在害虫防治实际应用中的关系。
