Targeting intrinsic resistance to combat multi-drug resistant P. aeruginosa

针对内在耐药性对抗多重耐药铜绿假单胞菌

• 批准号: 9206984
• 负责人: BARBARA I KAZMIERCZAK
• 金额: $ 25.06万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9206984
• 关键词: Achievement; Affinity; Animal Model; Antibiotic Resistance; Antibiotics; Antigens; Antimicrobial susceptibility; Attenuated; Bacteria; Bacterial Proteins; Bacteriophages; Binding; Biological Assay; Biology; Biotechnology; Cell Wall; Chronic Obstructive Airway Disease; Clinical; Complex; Crystallization; Cystic Fibrosis; Cytoplasm; Development; Enterobacteriaceae; Essential Genes; Exhibits; Future; Genes; Genetic; Genetic Screening; Goals; High-Throughput Nucleotide Sequencing; Homeostasis; Horizontal Gene Transfer; Human; Immune; Immunocompromised Host; Immunoglobulin G; In Vitro; Infection; Intention; Intrabody; Lead; Libraries; Lung; Maps; Measures; Mediating; Medical Device; Mutation; Phenotype; Play; Pseudomonas aeruginosa; Reagent; Resistance; Resources; Role; Route; Stress; Structure; Surface; Testing; Therapeutic; Work; active method; antimicrobial; bacterial fitness; base; cell envelope; combat; derepression; design; drug development; fitness; gene product; genetic information; genetic manipulation; genome-wide; improved; in vivo; interest; killings; member; multi-drug resistant pathogen; multidrug-resistant Pseudomonas aeruginosa; mutant; novel; overexpression; pathogen; periplasm; public health relevance; resistance mechanism; response; small molecule; tool; trait

 DESCRIPTION (provided by applicant): Antibiotic resistance is often thought of as a trait acquired by previously susceptible bacteria, either by horizontal gene transfer or by de novo mutations in chromosomal genes, and selected for by antibiotic exposure. However, the spectrum of antibiotic activity is even more limited by intrinsic antibiotic resistance, a phenotyp mediated by many gene products that limit access of antibiotics to their bacterial targets. I propose to identify intrinsic resistance mechanisms that limit utility of multiple antibiotic classs against the human pathogen Pseudomonas aeruginosa. This study will increase our basic understanding of mechanisms that contribute to P. aeruginosa cell envelope homeostasis under stress conditions, which are often distinct from those used by Enterobacteriaceae. These studies are significant, as intrinsic resistance mechanisms represent new targets for the development of antimicrobials against a clinically important and often-multidrug resistant pathogen. Two independent and complementary approaches will be employed to achieve this goal. First, I will use the novel tool of intrabodies. These heavy-chain-only IgGs of camelid origi will be expressed in the bacterial cytoplasm and periplasm, assayed for their ability to attenuate intrinsic antibiotic resistance or cell wall integrity, and then used to identify and purify their bacterial targets. In parallel, I will construct an InSeq library in P. aeruginosa and use this resource to identify and map transposon insertions that diminish bacterial fitness in the presence of antibiotics. Information obtained through these two separate approaches will be combined to generate a comprehensive list of P. aeruginosa gene products that contribute to intrinsic resistance. Bacterial proteins that play critical roles in maintaining intrinsic antibiotc resistance have not been heavily exploited in antibiotic development to date, though this approach has many advantages. Intrinsic resistance mechanisms are conserved among all members of a species, not just the subset that have acquired antibiotic resistance through mutation or acquisition of new genetic information; they promote bacterial homeostasis and survival in response to multiple antibiotic- or immune-mediated stresses, not just a single antimicrobial; they are often the substrate for acquired resistance, and their derepression or overexpression can lead to high level acquired antibiotic resistance. Thus, identifying and disarming mechanisms of intrinsic resistance is likely to have a broad impact on antimicrobial susceptibility of P. aeruginosa, making this course of study highly significant.

 描述（由申请人提供）：抗生素耐药性通常被认为是先前敏感细菌通过水平基因转移或染色体基因从头突变获得的性状，并通过抗生素暴露进行选择。然而，抗生素活性谱甚至更受内在抗生素抗性的限制，这是一种由许多基因产物介导的表型，其限制了抗生素对其细菌靶标的访问。我建议确定内在的耐药机制，限制效用的多种抗生素类对人类病原体铜绿假单胞菌。这项研究将增加我们对压力条件下铜绿假单胞菌细胞被膜稳态机制的基本理解，这些机制通常与肠杆菌科所使用的机制不同。这些研究意义重大，因为内在耐药机制代表了针对临床重要且通常具有多重耐药病原体的抗菌药物开发的新靶点。为实现这一目标，将采用两种独立和互补的方法。首先，我将使用胞内抗体这一新工具。骆驼科动物来源的这些仅重链IgG将在细菌细胞质和周质中表达，测定其减弱内在抗生素抗性或细胞壁完整性的能力，然后用于鉴定和纯化其细菌靶标。与此同时，我将在铜绿假单胞菌中构建一个InSeq文库，并使用此资源来识别和映射在抗生素存在下降低细菌适应性的转座子插入。通过这两种不同的方法获得的信息将被合并，以产生一个全面的清单铜绿假单胞菌基因产物，有助于内在耐药。迄今为止，在维持内在抗生素抗性方面发挥关键作用的细菌蛋白质尚未在抗生素开发中大量利用，尽管这种方法具有许多优点。内在耐药机制在一个物种的所有成员中是保守的，而不仅仅是通过突变或获得新的遗传信息获得抗生素耐药性的子集;它们促进细菌的稳态和生存，以响应多种抗生素或免疫介导的应激，而不仅仅是单一的抗菌剂;它们通常是获得性耐药性的底物，它们的去阻遏或过表达可导致高水平的获得性抗生素耐药性。因此，识别和消除内在耐药机制可能对铜绿假单胞菌的抗菌药物敏感性产生广泛影响，使本研究过程非常重要。