Department of Microbial Bioactive Compounds, Interfaculty Institute of Microbiology and Infection Medicine, University of Tuebingen, Tuebingen, Germany.
M. G. DeGroote Institute for Infectious Disease Research, Department of Biochemistry and Biomedical Sciences, David Braley Centre for Antibiotic Discovery, McMaster University, Hamilton, Ontario, Canada.
Appl Environ Microbiol. 2019 Oct 1;85(20). doi: 10.1128/AEM.01292-19. Print 2019 Oct 15.
The increasing threat posed by multiresistant bacterial pathogens necessitates the discovery of novel antibacterials with unprecedented modes of action. ADEP1, a natural compound produced by NRRL 15010, is the prototype for a new class of acyldepsipeptide (ADEP) antibiotics. ADEP antibiotics deregulate the proteolytic core ClpP of the bacterial caseinolytic protease, thereby exhibiting potent antibacterial activity against Gram-positive bacteria, including multiresistant pathogens. ADEP1 and derivatives, here collectively called ADEP, have been previously investigated for their antibiotic potency against different species, structure-activity relationship, and mechanism of action; however, knowledge on the biosynthesis of the natural compound and producer self-resistance have remained elusive. In this study, we identified and analyzed the ADEP biosynthetic gene cluster in NRRL 15010, which comprises two NRPSs, genes necessary for the biosynthesis of (4,2)-4-methylproline, and a type II polyketide synthase (PKS) for the assembly of highly reduced polyenes. While no resistance factor could be identified within the gene cluster itself, we discovered an additional homologous gene (named ) located further downstream of the biosynthetic genes, separated from the biosynthetic gene cluster by several transposable elements. Heterologous expression of ClpP in three ADEP-sensitive species proved its role in conferring ADEP resistance, thereby revealing a novel type of antibiotic resistance determinant. Antibiotic acyldepsipeptides (ADEPs) represent a promising new class of potent antibiotics and, at the same time, are valuable tools to study the molecular functioning of their target, ClpP, the proteolytic core of the bacterial caseinolytic protease. Here, we present a straightforward purification procedure for ADEP1 that yields substantial amounts of the pure compound in a time- and cost-efficient manner, which is a prerequisite to conveniently study the antimicrobial effects of ADEP and the operating mode of bacterial ClpP machineries in diverse bacteria. Identification and characterization of the ADEP biosynthetic gene cluster in NRRL 15010 enables future bioinformatics screenings for similar gene clusters and/or subclusters to find novel natural compounds with specific substructures. Most strikingly, we identified a cluster-associated homolog (named ) as an ADEP resistance gene. ClpP constitutes a novel bacterial resistance factor that alone is necessary and sufficient to confer high-level ADEP resistance to across species.
越来越多的多药耐药细菌病原体构成的威胁，促使人们去发现具有前所未有作用模式的新型抗菌药物。ADEP1 是一种天然化合物，由 NRRL 15010 产生，它是新型酰基二肽（ADEP）抗生素的原型。ADEP 抗生素会使细菌组织蛋白酶的蛋白水解核心 ClpP 失活，从而对包括多药耐药病原体在内的革兰氏阳性菌表现出强大的抗菌活性。ADEP1 和衍生物，在这里统称为 ADEP，以前曾因其对不同物种的抗生素效力、结构-活性关系和作用机制而被研究过；然而，关于天然化合物的生物合成和产生菌自身抗性的知识仍然难以捉摸。在这项研究中，我们鉴定并分析了 NRRL 15010 中的 ADEP 生物合成基因簇，该基因簇包含两个 NRPSs、（4,2）-4-甲基脯氨酸生物合成所必需的基因，以及一个用于组装高度还原多烯的 II 型聚酮合酶（PKS）。虽然在基因簇本身中没有发现抗性因子，但我们在生物合成基因的下游发现了一个额外的同源基因（命名为），它与生物合成基因簇由几个转座元件隔开。在三种对 ADEP 敏感的物种中异源表达 ClpP 证明了它在赋予 ADEP 抗性方面的作用，从而揭示了一种新型的抗生素抗性决定因子。酰基二肽抗生素（ADEPs）是一类很有前途的新型强效抗生素，同时也是研究其靶标分子功能的有价值的工具，ClpP 是细菌组织蛋白酶的蛋白水解核心。在这里，我们提出了一种简单的 ADEP1 纯化程序，该程序以高效、省时的方式产生大量纯化合物，这是方便研究 ADEP 的抗菌作用和不同细菌中细菌 ClpP 机器的工作模式的前提。鉴定和表征 NRRL 15010 中的 ADEP 生物合成基因簇，使未来能够进行类似基因簇和/或亚簇的生物信息学筛选，以发现具有特定亚结构的新型天然化合物。最引人注目的是，我们鉴定出一个与簇相关的同源基因（命名为）作为 ADEP 抗性基因。ClpP 构成了一种新型的细菌抗性因子，单独使用就足以赋予不同物种对 ADEP 的高度抗性。
