Uncovering aryl polyene biology to identify new drug targets in Gram-negative bacterial pathogens

摘要 在一个抗生素使用量上升的时代， 耐药细菌病原体。新抗生素的发现速度跟不上这种迫切的需求 因此，我们必须确定替代药物靶点，并制定补充消除策略。这是 对于革兰氏阴性菌引起的感染，这是一项特别具有挑战性的任务，因为它们的外膜 对许多化合物是不可渗透的。我们发现了一种芳基多烯（APE）毒力因子，广泛分布于 革兰氏阴性病原体，并在这里提出表征其生物合成，并将其用作革兰氏阴性病原体的靶标。 制定消除战略。APE是生物合成基因家族中最丰富的产物 在我们的全球细菌基因组计算机分析中，我们使用了BGC。它们存在于多种药物中- 我们的初步数据显示，APE被酯化到一个尚未识别的锚上， 革兰氏阴性外膜中的分子。此外，我们发现APE作为毒性 保护其生产者免受宿主先天免疫清除机制影响的因子。我们的长期目标是 在分子、功能和进化水平上了解APE生物学。 目前的建议是集中在假设，APE生物合成和细胞包膜定位提供 针对革兰氏阴性病原体的特异性抗毒力策略的靶点。我们先来测试一下 表征APE外膜定位途径内单个基因的功能， 鉴定含APE的锚分子。接下来，我们将确定环境条件， 使用正向遗传学方法控制APE表达的调节。最后，我们将开发干扰 使用生物合成途径中起始酶的靶向抑制剂抑制APE产生的策略。 我们提出的项目将产生对APE生物学的机理见解，以及识别小分子 工具化合物，可用于研究APE生物学和实现替代病原体根除策略。

ABSTRACT We are rapidly running out of options to combat infectious diseases in an era that is marked by a rise in antibiotic resistant bacterial pathogens. The discovery rate of novel antibiotics is not keeping up with this pressing need and thus we have to identify alternative drug targets and develop complementary elimination strategies. This is a particularly challenging task for infections caused by Gram-negative bacteria because their outer membrane is impermeable to many compounds. We discovered an aryl polyene (APE) virulence factor, widespread among Gram-negative pathogens, and here propose to characterize its biosynthesis and use it as a target for the development of elimination strategies. APEs are the product of the most abundant family of biosynthetic gene clusters (BGCs) in our global in silico analysis of bacterial genomes. They are present in several multidrug- resistant pathogens, and our preliminary data shows that APEs are esterified to a yet unidentified anchor molecule in the Gram-negative outer membrane. Furthermore, we discovered that APEs serve as virulence factors that protect their producers from host innate immune clearance mechanisms. Our long-term goal is to understand APE biology on a molecular, functional and evolutionary level. The current proposal is centered on the hypothesis that APE biosynthesis and cell envelope localization provide targets for specific antivirulence strategies against Gram-negative pathogens. We will test this by first characterizing the function of individual genes within the APE outer membrane localization pathway and identifying the APE-containing anchor molecule. Next, we will determine the environmental conditions that control regulation of APE expression using a forward genetics approach. Finally, we will develop interference strategies to inhibit APE production, using a targeted inhibitor of the initiation enzyme in the biosynthetic pathway. Our proposed project will yield mechanistic insights into the biology of APEs, as well as identify small molecule tool compounds that can be used to study APE biology and enable alternative pathogen eradication strategies.