Emerging antibiotic resistance in Gram-negative pathogens

项目摘要 多食伯克霍尔德氏菌是一种成功的病原体，是B的成员。洋葱复合体（BCC）， 导致免疫功能低下的人患有肺部疾病，如囊性纤维化， (CF)和慢性肉芽肿病（CGD）。BCC由一组17个密切相关的革兰氏阴性杆菌组成。 具有极高的遗传能力和代谢多样性的细菌。几个BCC成员可以触发慢性气道 CF患者中的感染，并已成为机会性肺部病原体。B。multivorans和B. 新洋葱属是两种最常见的孤立物种，它们是爆发的威胁。CF中的Bcc感染 患者的发病率和死亡率增加。它们也有能力引起快速的临床 败血症恶化导致死亡。几次爆发B。导致严重发病率的多食动物 CF和非CF患者均发生死亡。 BCC病原体本质上对广泛的抗菌剂具有耐药性，包括β-内酰胺类， 氟喹诺酮类、氨基糖苷类、多粘菌素类和阳离子肽类药物，对 治疗BCC肺部感染。Hopanoids在支持外膜稳定性方面发挥着主导作用 和B中的屏障功能。多粘菌素，从而参与对多粘菌素B和粘菌素的抗性。藿烷 是五环三萜类脂质，其能够插入细菌膜中并有助于它们的 稳定性和刚度。Hopanoids帮助膜承受破坏性的应力条件，包括高 温度、低pH值和抗生素的存在。重要的是，hopanoid的产生在 B. cenocepacia。 尽管类霍帕烷在细菌中的重要性，但细胞内类霍帕烷运输的机制已被证实。 没有被探索过。我们建议以B为目标。hopanoid biosynthesis associated resistance- 结瘤细胞分裂（RND））转运蛋白，这是至关重要的细胞壁重塑，在这种革兰氏阴性 细菌。我们的工作假设是HpnN在B的内在耐药性中起主要作用。 通过穿梭于细胞质膜到外膜的类霍帕酸，加强细胞 墙细胞内类阿片的运输过程可能也需要周质的参与， 亲脂蛋白HpnM。我们将阐明B多药耐药的分子机制。多鸟 由HpnN和HpnM介导。 我们将定义B的晶体结构。多噬菌HpnN的存在和不存在的hopanoids。 基于结构信息，我们将确定重要的残基的hopanoid识别和运输。 我们的初步数据有力地表明，HpnN穿梭的hopanoid分子从外小叶的内 膜外膜。模拟已经显示了通过HpnN的确切途径， 这表明该hopanoid分子如何通过HpnN转运蛋白形成的通道输出。我们将 确定HpnM在hopanoid运输中的作用。我们还将应用噬菌体展示方法来鉴定新的 与HpnN或HpnM强烈相互作用的肽，抑制其转运类霍帕酸的功能。我们 假设我们将能够产生独特的抑制剂，使B。对抗生素敏感的多食动物。 抑制HpnN功能的肽将用于与该转运蛋白共结晶。这些结构将 帮助我们理解抑制机制。此外，大蜡螟和小鼠感染模型 将用于测试这些基于肽的抑制剂的功效。这些肽不会抑制 伯克霍尔德氏菌细胞在没有抗生素的情况下。然而，它们可以使细菌对抗生素敏感， 作为治疗感染的“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.