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的相互作用来调节的，包括与其他蛋白质的相互作用。由此产生的复杂的蛋白质-蛋白质相互作用(PPI)阵列调节着所有形式生命的每一条细胞途径。目前未靶向的一类主要PPI是毒素-抗毒素相互作用，它是细菌持久性的基础，是抗生素治疗失败和复发感染的常见原因。持久性是指细菌形成持续体的能力，这是一种对抗生素耐受的休眠不生长的表型，并在抗生素移除后重新开始生长，导致感染复发。细菌毒素-抗毒素(TA)操纵子在表达时形成一种无害的毒素-抗毒素PPI。在环境或抗生素压力下，抗毒素迅速降解，释放毒素，然后抑制细菌的生长，导致持久菌的形成。沙门氏菌中单一的TA模块敲除已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TA对，针对单个PPI不太可能足以完全根除顽固性细菌。多肽筛选技术正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，通过引入天然和非天然氨基酸，mRNA Display提供了筛选多达1013个大环肽文库的能力，覆盖了大量的化学空间。该方法还没有被用于发现多个蛋白质靶点的PAN抑制物。本项目的目标是利用针对沙门氏菌中最初相关的三个TA对的mRNA展示技术来产生PAN毒素抑制物。这项工作有可能提高我们预防感染细菌的持久性和逆转抗生素耐药性的能力。

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.