Emerging antibiotic resistance in Gram-negative pathogens

项目总结 多食伯克霍尔德氏菌是一种成功的病原菌，是洋葱杆菌复合体(BCC)的成员 导致患有囊性纤维化等潜在肺部疾病的免疫功能低下的人患肺炎 (CF)和慢性肉芽肿性疾病(CGD)。BCC由17个密切相关的革兰氏阴性菌组成 具有极端遗传能力和代谢多样性的细菌。几个BCC成员可触发慢性呼吸道 在CF患者中发生感染，并已成为机会性肺部病原体。B.多食性和B. 盲肠吸虫是两个最常见的隔离物种，它们是暴发的威胁。慢性阻塞性肺疾病中的基底细胞癌感染 患者与发病率和死亡率的增加有关。它们也有能力引起快速的临床 败血症恶化，导致死亡。多食杆菌的几次暴发造成了严重的发病率 CF和非CF患者的死亡率均已发生。 基底细胞癌病原体对多种抗菌药具有内在抗药性，包括β-内酰胺类、 氟喹诺酮类、氨基糖苷类、多粘菌素和阳离子多肽，对 BCC肺部感染的治疗。类霍普诺素在支持外膜稳定性方面起着主要作用 和多食杆菌的屏障功能，从而参与对多粘菌素B和粘菌素的抗性。类霍普诺斯 是五环三萜类脂类，能够插入细菌的细胞膜，并有助于其 稳定性和刚性。类霍普阿诺德帮助细胞膜抵御破坏性的压力条件，包括高 温度、低pH值和抗生素的存在。重要的是，类胡萝卜素的生产在 中华盲囊线虫的生理和致病机理。 尽管类霍帕诺在细菌中很重要，但细胞内类霍帕诺转运的机制已经 没有被探索过。我们建议以多食性芽孢杆菌HpnN(类霍帕尼亚生物合成相关抗性)为靶标。 结节-细胞分裂(RND)转运蛋白，这是革兰氏阴性菌细胞壁重塑所必需的 细菌。我们的工作假设是，HpnN在芽孢杆菌的内在耐药性中起着重要作用。 通过将类胡萝卜素从细胞质膜穿梭到外膜来加强细胞 墙。细胞内类霍帕尼素的运输过程也可能需要周质 亲脂蛋白HpnM。我们将阐明多食杆菌多药耐药的分子机制。 由HpnN和HpnM介导。 我们将定义多食性假单胞菌HpnN的晶体结构，在没有和存在Hopanoid的情况下都是如此。 基于结构信息，我们将识别重要的残基，用于霍帕尼类化合物的识别和运输。 我们的初步数据有力地表明，HpnN从内部的外叶穿梭类Hopanid分子 从膜到外膜。模拟已经显示了通过HpnN的双戊烯的确切路径， 表明该类何首乌分子是如何通过HpnN转运体形成的通道输出的。我们会 确定HpnM在类胡萝卜素贩运中的作用。我们还将应用噬菌体展示方法来鉴定小说 与HpnN或HpnM强相互作用的多肽，抑制其转运类胡萝卜素的功能。我们 假设我们将能够生产出独特的抑制剂，使多食性芽孢杆菌对抗生素敏感。 抑制HpnN功能的多肽将被用来与该转运蛋白共结晶。这些结构将 让我们了解抑制的机制。此外，梅隆格列氏菌和小鼠感染模型 将用于测试这些以多肽为基础的抑制剂的疗效。这些多肽不会抑制肿瘤细胞的生长 伯克霍尔德氏菌细胞在没有抗生素的情况下。然而，它们会使细菌对抗生素和 充当治疗感染的“抗生素佐剂”。如果成功，我们的战略可能会转移到 其他细菌病原体，这将提供一个额外的机制来治疗感染。

PROJECT SUMMARY Burkholderia multivorans is a successful pathogen and a member of the B. cepacia complex (Bcc) that causes pneumonia in immunocompromised individuals with underlying lung diseases, such as cystic fibrosis (CF) and chronic granulomatous disease (CGD). Bcc consists of a group of 17 closely related Gram-negative bacteria with extreme genetic capacity and metabolic diversity. Several Bcc members can trigger chronic airway infections in CF patients and have emerged as opportunistic pulmonary pathogens. B. multivorans and B. cenocepacia are the two most commonly isolated species, which are threats for outbreaks. Bcc infections in CF patients are associated with enhanced morbidity and mortality. They also have the capacity to cause rapid clinical deterioration with septicemia that leads to death. Several outbreaks of B. multivorans causing severe morbidity and mortality in both CF and non-CF patients have occurred. Bcc pathogens are intrinsically resistant to a broad range of antimicrobials, including b-lactams, fluoroquinolones, aminoglycosides, polymyxins and cationic peptides, creating a major challenge to the treatment of Bcc pulmonary infections. Hopanoids play a predominant role in supporting outer membrane stability and barrier function in B. multivorans, thus participating in the resistance to polymyxin B and colistin. Hopanoids are pentacyclic triterpenoid lipids that are capable of inserting in bacterial membranes and contributing to their stability and stiffness. Hopanoids help membranes withstand damaging stress conditions, including high temperature, low pH and the presence of antibiotics. Importantly, hopanoid production plays an important role in the physiology and pathogenesis of B. cenocepacia. In spite of the importance of hopanoids in bacteria, the mechanism of intracellular hopanoid trafficking has not been explored. We propose to target the B. multivorans HpnN (hopanoid biosynthesis-associated resistance- nodulation-cell division (RND)) transporter, which is essential for cell wall remodeling in this Gram-negative bacterium. Our working hypothesis is that HpnN plays a major role in the intrinsic antimicrobial resistance of B. multivorans by shuttling hopanoids from the cytoplasmic membrane to outer membrane, strengthening the cell wall. The process of intracellular hopanoid trafficking may also require the participation of the periplasmic lipophilic protein HpnM. We will elucidate the molecular mechanisms of multidrug resistance in B. multivorans mediated by HpnN and HpnM. We will define crystal structures of B. multivorans HpnN both in the absence and presence of hopanoids. Based on the structural information, we will identify important residues for hopanoid recognition and transport. Our preliminary data strongly suggest that HpnN shuttles hopanoid molecules from the outer leaflet of the inner membrane to the outer membrane. Simulations have shown the exact pathway through HpnN for diploptene, indicating how this hopanoid molecule is exported through the channel formed by the HpnN transporter. We will ascertain the role of HpnM in hopanoid trafficking. We will also apply phage display methodology to identify novel peptides that strongly interact with HpnN or HpnM, inhibiting their function to transport hopanoids. We hypothesize that we will be able to produce unique inhibitors that render B. multivorans susceptible to antibiotics. Peptides that inhibit the function of HpnN will be used to co-crystallize with this transporter. The structures will allow us to understand the mechanism of inhibition. In addition, Galleria mellonella and mouse models of infection will be used to test the efficacy of these peptide-based inhibitors. These peptides would not inhibit the growth of Burkholderia cells in the absence of antibiotics. However, they can render bacteria susceptible to antibiotics and act as “antibiotic adjuvants” for the treatment of infections. If successful, our strategy could be transferred to other bacterial pathogens, which would provide an added mechanism to treat infections.