Targeting Multiple Toxin Proteins in Persistent Bacteria with mRNA Display

在整个生物学中，蛋白质的功能是通过与其他生物分子（包括与其他蛋白质）的相互作用来介导的。由此产生的蛋白质-蛋白质相互作用（PPI）的复杂阵列介导了所有生命形式的每个细胞途径。目前非靶向PPI的一个主要类别是毒素-抗毒素相互作用，这是细菌持久性的基础，是抗生素治疗失败和复发性感染的常见原因。持久性是指细菌形成持久性的能力，持久性是一种对抗生素耐受的休眠的非生长表型，并且在抗生素去除后重新启动生长，导致感染复发。细菌毒素-抗毒素（TA）操纵子在表达时，形成无害的毒素-抗毒素PPI。在环境或抗生素胁迫下，抗毒素迅速降解，释放毒素，然后灭活细菌生长，导致形成持久性。沙门氏菌中的单TA模块敲除显示出显著降低的持久性水平。然而，细菌中存在多个TA对，例如沙门氏菌中存在14个，并且针对单个PPI不太可能足以完全根除持久性。肽筛选技术正在迅速成为快速产生高亲和力PPI抑制剂的有效方法。在这些方法中，mRNA展示提供了筛选多达1013个大环肽的文库的能力，通过引入天然和非天然氨基酸覆盖了大量的化学空间。该方法尚未用于发现多蛋白靶点的泛毒素抑制剂。本项目旨在利用mRNA展示技术产生泛毒素抑制剂，最初靶向沙门氏菌中的三个相关TA对。这项工作有可能提高我们预防感染性细菌持久性和逆转抗生素耐受性的能力。

Throughout biology, protein functions are critically mediated through interactions with other biomolecules, including with other proteins. The resulting complex array of protein-protein interactions (PPIs) mediates every cellular pathway across all forms of life. A major class of currently untargeted PPIs are toxin-antitoxin interactions which underlie bacterial persistence, a frequent cause of antibiotic treatment failure and relapsing infections. Persistence denotes the ability of bacteria to form persisters, a dormant non-growing phenotype tolerant to antibiotics, and to reinitiate growth after antibiotic removal causing infection relapse.Bacterial toxin-antitoxin (TA) operons, when expressed, form a harmless toxin-antitoxin PPI. Under environmental or antibiotic stress, the antitoxin is rapidly degraded, releasing the toxin which then inactivates bacterial growth, leading to the formation of persisters. Single TA module knockouts in Salmonella have shown markedly reduced levels of persistence. However, multiple TA pairs exist within bacteria, 14 within Salmonella for example, and targeting a single PPI is unlikely to be enough to completely eradicate persisters. Peptide screening technologies are fast emerging as effective methods to rapidly generate high-affinity PPI inhibitors. Of these methods, mRNA display offers the ability to screen libraries of up to 1013 macrocyclic peptides, covering a vast array of chemical space through the introduction of both natural and unnatural amino acids. This method has yet to be employed for the discovery of pan-inhibitors of multiple protein targets.This project will aim to generate pan-toxin inhibitors using mRNA display technology targeting initially three related TA pairs in Salmonella. This work has the potential to increase our ability to prevent persistence and reverse antibiotic tolerance in infectious bacteria.