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

摘要 在一个以抗生素增加为标志的时代，我们正在迅速耗尽对抗传染病的选择 耐药细菌病原体。新抗生素的发现率跟不上这种迫切的需求。 因此，我们必须确定替代药物目标，并制定配套的消除战略。这是 对于革兰氏阴性菌引起的感染来说，这是一个特别具有挑战性的任务，因为它们的外膜 对许多化合物都是不透水的。我们发现了一种芳基多烯(APE)毒力因子，广泛存在于 革兰氏阴性病原菌，并在这里建议表征其生物合成，并将其用作 制定淘汰战略。类人猿是最丰富的生物合成基因家族的产物 在我们的全球细菌基因组的电子分析中的簇(BGC)。它们存在于几种多种药物中- 抵抗病原体，我们的初步数据显示，类人猿被酯化为一种尚未识别的锚 革兰氏阴性外膜中的分子。此外，我们还发现，类人猿作为一种毒力 保护其生产者免受宿主先天免疫清除机制影响的因素。我们的长期目标是 在分子、功能和进化水平上理解类人猿生物学。 目前的建议是基于这样的假设，即猿的生物合成和细胞膜定位提供了 针对革兰氏阴性病原体的特定抗毒力策略的靶点。我们将首先对此进行测试 表征类人猿外膜定位途径中单个基因的功能 鉴定含有类人猿的锚分子。接下来，我们将确定 使用正向遗传学方法控制类人猿表达的调控。最后，我们将开发干扰 在生物合成途径中使用有针对性的起始酶抑制剂来抑制类人猿生产的策略。 我们提议的项目将产生对类人猿生物学的机械性见解，以及识别小分子。 可用于研究类人猿生物学和实现替代病原体根除策略的工具化合物。

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.