Emerging antibiotic resistance in Gram-negative pathogens

革兰氏阴性病原体中新出现的抗生素耐药性

• 批准号: 10328513
• 负责人: HERBERT P. SCHWEIZER
• 金额: $ 66.71万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10328513
• 关键词: Adjuvant; Affect; Affinity; Aminoglycosides; Anabolism; Antibiotics; Antimicrobial Resistance; Bacteria; Bacterial Infections; Binding; Biogenesis; Burkholderia; Burkholderia cepacia; Burkholderia cepacia complex; Cations; Ceftazidime; Cell Membrane Permeability; Cell Wall; Cell division; Cell membrane; Cells; Cessation of life; Chloramphenicol; Chronic; Chronic Granulomatous Disease; Ciprofloxacin; Clinical; Colistin; Crystallization; Cystic Fibrosis; Cytosol; Czech Republic; Data; Deterioration; Disease; Disease Outbreaks; Exhibits; Fluorescence Polarization; Fluoroquinolones; Future; Genetic; Gram-Negative Bacteria; Growth; Health; High temperature of physical object; Hypersensitivity; Immunocompromised Host; Individual; Infection; Lipids; Lipoproteins; Lung diseases; Lung infections; Mediating; Membrane; Membrane Proteins; Membrane Transport Proteins; Metabolic; Methodology; Modeling; Molecular; Morbidity - disease rate; Motion; Multi-Drug Resistance; Mus; Nodule; Novobiocin; Pathogenesis; Pathway interactions; Patients; Peptides; Phage Display; Physiology; Play; Pneumonia; Polymyxin B; Polymyxin Resistance; Polymyxins; Process; Production; Proteins; Reporting; Resistance; Respiratory Tract Infections; Roentgen Rays; Role; Septicemia; Site-Directed Mutagenesis; Stress; Structure; Technology; Testing; Tetracyclines; Thigh structure; United States; antimicrobial; antimicrobial drug; base; beta-Lactams; clinical practice; combat; computer studies; cystic fibrosis infection; cystic fibrosis patients; efficacy testing; emerging antibiotic resistance; experimental study; inhibitor; insight; lipophilicity; lung pathogen; member; molecular dynamics; mortality; mouse model; mutant; necrotizing pneumonia; network models; novel; novel antibiotic class; pathogen; pathogenic bacteria; periplasm; resistant strain; screening; simulation; trafficking

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.

项目概要 多食伯克霍尔德菌是一种成功的病原体，也是洋葱伯克霍尔德菌复合体 (Bcc) 的成员， 在患有潜在肺部疾病（例如囊性纤维化）的免疫功能低下的个体中引起肺炎 (CF) 和慢性肉芽肿病 (CGD) 由 17 种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的一些 Bcc 成员可以引发慢性气道。 CF 患者感染，并已成为机会性肺部病原体。 新洋葱是两种最常见的分离物种，是 CF 爆发的威胁。 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症状。 败血症恶化，导致死亡。 CF 和非 CF 患者均发生死亡。 Bcc 病原体本质上对多种抗菌药物具有耐药性，包括 β-内酰胺、 氟喹诺酮类、氨基糖苷类、多粘菌素和阳离子肽，对 治疗 Bcc 肺部感染。 Hopanoid 在支持外膜稳定性方面发挥主要作用。 和 B. multivorans 的屏障功能，从而参与对多粘菌素 B 和粘菌素的抗性。 是五环三萜类脂质，能够插入细菌膜并有助于其 稳定性和刚度有助于膜承受破坏性的应力条件，包括高应力条件。 重要的是，温度、低 pH 值和抗生素的存在，霍烷酸的产生起着重要作用。 B. cenocepacia 的生理学和发病机制。 尽管藿类化合物在细菌中很重要，但细胞内藿类化合物运输的机制已经 我们建议针对 B. multivorans HpnN（藿烷类生物合成相关耐药性）。 结节细胞分裂（RND））转运蛋白，对于革兰氏阴性细胞壁重塑至关重要 我们的工作假设是，HpnN 在芽孢杆菌的内在抗菌素耐药性中发挥着重要作用。 multivorans 通过将hopanoids从细胞质膜穿梭到外膜，强化细胞 细胞内hopanoid运输的过程可能也需要周质的参与。 我们将阐明 B. multivorans 的多药耐药性的分子机制。 由 HpnN 和 HpnM 介导。 我们将定义在不存在和存在藿类化合物的情况下 B. multivorans HpnN 的晶体结构。 根据结构信息，我们将鉴定霍烷类化合物识别和运输的重要残基。 我们的初步数据强烈表明，HpnN 从内部小叶的外部小叶中穿梭霍帕类分子。 模拟显示了二肽通过 HpnN 的确切路径， 表明这种hopanoid分子如何通过HpnN转运蛋白形成的通道输出。 确定 HpnM 在hopanoid 贩运中的作用我们还将应用噬菌体展示方法来识别新的。 与 HpnN 或 HpnM 强烈相互作用的肽，抑制其转运藿类化合物的功能。 我们将能够生产出独特的抑制剂，使 B. multivorans 对抗生素敏感。 抑制 HpnN 功能的肽将用于与该转运蛋白共结晶。 此外，让我们了解大蜡螟和小鼠感染模型的抑制机制。 将用于测试这些基于肽的抑制剂的功效这些肽不会抑制生长。 然而，在没有抗生素的情况下，伯克霍尔德氏菌细胞会使细菌对抗生素敏感。 作为治疗感染的“抗生素佐剂”如果成功的话，我们的策略可以转移到。 其他细菌病原体，这将提供治疗感染的额外机制。