Mutators and Pseudomonas Genome Evolution in the CF-lung Enviornment

CF-肺环境中的突变体和假单胞菌基因组进化

• 批准号: 7522479
• 负责人: Raphael F Rosenzweig
• 金额: $ 21.23万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-10 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7522479
• 关键词: Accounting; Acute; Address; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Appearance; Architecture; Area; Bacteria; Base Pairing; Cataloging; Catalogs; Cessation of life; Characteristics; Chronic; Clinical; Collection; Condition; Cystic Fibrosis; DNA; DNA Repair; DNA Repair Gene; DNA-Directed DNA Polymerase; Data; Defect; Disease; Disease Outcome; Disease Progression; Evolution; Face; Fellowship; Fingerprint; Frequencies; Gene Duplication; Generations; Genes; Genetic; Genetic Recombination; Genetic Variation; Genome; Genomics; Goals; Growth; Horizontal Gene Transfer; Immune system; Infection; Institution; Lead; Lesion; Life; Localized; Location; Lung; Medical Surveillance; Mentors; Metabolism; Microbe; Mission; Modeling; Multi-Drug Resistance; Mutation; National Institute of Allergy and Infectious Disease; Numbers; Paris, France; Patients; Pediatric Hospitals; Phenotype; Phylogenetic Analysis; Phylogeny; Play; Population; Population Density; Process; Pseudomonas; Pseudomonas Infections; Pseudomonas aeruginosa; Public Health; Quality of life; Rate; Regulation; Reproduction; Research; Research Project Grants; Resolution; Respiratory Failure; Respiratory Tract Infections; Rifampicin resistance; Role; Screening procedure; Series; Site; Structure; Testing; Therapeutic; Thinking; Time; Variant; Work; base; comparative genomic hybridization; cystic fibrosis patients; density; improved; mathematical model; member; mortality; mucoid; mutant; pathogen; pressure; symposium

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) patients are highly susceptible to persistent endobronchial infection by Pseudomonas aeruginosa, a common environmental microbe that readily adapts to the CF-lung. Evolutionary adaptations that lead to chronic infection arise from large-scale changes in genome architecture such as insertion, translocation, duplication and deletion, as well as small-scale changes such as single-base pair substitutions. Among clinical strains, small- and large- scale changes are thought to be more commonly transmitted vertically than horizontally. How then do Pseudomonas populations generate sufficient variation to evolve rapidly under the selective pressures of constant immune system surveillance and intermittent antibiotic treatment? (PARAGRAPH) "Mutators," bacteria that have high background mutation rates, are frequently observed in chronic, but not acute, infections, suggesting that clones defective in DNA repair may play an important role in producing genetic variation. We will address this possibility via a two-pronged approach. We will conduct a longitudinal genomic study of strains isolated from multiple CF patients at the Necker Children's Hospital in Paris, France. We will compare the tempo and mode of genome evolution in "mutator" and "non-mutator" lineages. Concurrently, we will develop and test mathematical models that predict the conditions under which mutators emerge and persist. To date, we have produced genomic fingerprints for each clinical isolate, phenotyped each with respect to antibiotic resistance, mucoid status, and frequency of spontaneous rifampicin-resistant mutants. We define "mutators" as clones that produce RifR mutants at a frequency ten-fold greater than the population median. Our preliminary data have led us to hypothesize that: (1) Pseudomonas in the CF-lung evolves by periodic selection of adaptively favored clones, (2) specific defects in different DNA repair result in different background mutation rates, (3) "mutators" increase genetic variation over what we might expect in non-mutator populations, (4) mutator-containing lineages evolve more rapidly than non-mutator lineages. We will test these hypotheses by: performing microarray-based comparative genomic hybridization (a-CGH) to detect strain-specific deletions and duplications and localize breakpoints to single-gene resolution; using multilocus sequence typing (MLST) to estimate small-scale genomic change and infer clonal phylogeny; screening DNA repair gene sequences to determine the basis for each "mutator;" and developing and testing continuous mathematical models that predict conditions for emergence and persistence of mutators. Specifically, we will extend continuous models we have developed to include competition and asynchronous generations, then estimate mutation and reversion rates under conditions where we can control for generation time, population density, and levels of antibiotic. (PARAGRAPH) We will be assisted in these efforts by undergraduates engaged in mentored research. Undergraduate team members will work through an annual cycle supported by fellowships, receive upper-division credit, give formal presentations at local and national conferences, and help bring key aspects of this project into the classroom. Our Specific Aims advance the overall NIH-AREA mission to support pilot, health-related research projects at predominantly undergraduate institutions, as well as specific National Institute of Allergy and Infectious Diseases objectives to support meritorious research in the areas of pathogen genomics and evolution. PUBLIC HEALTH RELEVANCE: Cystic fibrosis (CF) patients are highly susceptible to chronic respiratory tract infection by the common environmental microbe, Pseudomonas aeruginosa. Chronic infections greatly diminish patient quality of life, and respiratory failure, often attributable to Pseudomonas infection, accounts for >90% CF mortality. Early in life, CF patients seem to acquire the bacterial strain to which they succumb years later. Because thousands of bacterial generations elapse between initial infection and death, disease progression is an evolutionary process. We seek to better understand how these bacteria evolve in the face of constant immune system surveillance and intermittent antibiotic treatment. A characteristic feature of chronic infections is the emergence of "mutator" strains, bacteria that have high mutation rates and seemingly facilitate acquisition of multiple antibiotic resistance. We aim to investigate how "mutators" alter the pace and trajectory of evolution: we will genetically analyze strains isolated from multiple patients over time, and model conditions for mutators to arise and persist. Our goal is to help clinicians devise antibiotic therapies that minimize the likelihood that multi-drug resistance emerges during chronic infections.

描述(申请人提供)：囊性纤维化(CF)患者非常容易受到铜绿假单胞菌持续的支气管内感染，铜绿假单胞菌是一种常见的环境微生物，很容易适应囊性纤维化肺。导致慢性感染的进化适应源于基因组结构的大规模变化，如插入、易位、复制和缺失，以及小范围的变化，如单碱基对替换。在临床菌株中，小规模和大规模的变化被认为更容易垂直传播，而不是水平传播。那么，在持续的免疫系统监测和间歇性抗生素治疗的选择性压力下，假单胞菌种群如何产生足够的变异以快速进化？(段落)“突变者”是一种具有高背景突变率的细菌，经常在慢性但不急性感染中观察到，这表明在DNA修复方面有缺陷的克隆可能在产生遗传变异方面发挥重要作用。我们将通过双管齐下的方法解决这一可能性。我们将在法国巴黎的Necker儿童医院对从多名CF患者中分离出的菌株进行纵向基因组研究。我们将比较“突变者”和“非突变者”谱系中基因组进化的节奏和模式。同时，我们将开发和测试数学模型，预测突变菌出现和持续存在的条件。到目前为止，我们已经为每个临床分离株制作了基因组指纹，根据抗生素耐药性、粘液状态和自发利福平耐药突变的频率对每个分离株进行表型鉴定。我们将“突变者”定义为产生RIFR突变体的克隆，其频率比种群中位数高出十倍。我们的初步数据使我们假设：(1)CFL中的假单胞菌是通过周期性地选择适应性有利的克隆来进化的，(2)不同DNA修复中的特定缺陷导致不同的背景突变率，(3)“突变者”增加了我们在非突变者群体中可能预期的遗传变异，(4)含有突变者的谱系比非突变者谱系进化得更快。我们将通过以下方式检验这些假设：进行基于微阵列的比较基因组杂交(a-CGH)，以检测特定菌株的缺失和复制，并将断点定位到单基因分辨率；使用多位点序列分型(MLST)来估计小规模基因组变化并推断克隆系统；筛选DNA修复基因序列，以确定每个“突变子”的基础；以及开发和测试预测突变子出现和持续条件的连续数学模型。具体地说，我们将扩展我们开发的连续模型，以包括竞争和异步世代，然后在我们可以控制世代时间、种群密度和抗生素水平的条件下估计突变和返回率。在这些努力中，我们将得到从事指导研究的本科生的协助。本科生团队成员将完成由奖学金支持的年度周期，获得高级学分，在地方和国家会议上进行正式演讲，并帮助将该项目的关键方面带入课堂。我们的具体目标是推进NIH地区的总体使命，以支持以本科为主的机构的试点、与健康相关的研究项目，以及支持病原体基因组学和进化领域中有价值的研究的国家过敏和传染病研究所的具体目标。公共卫生相关性：囊性纤维化(CF)患者非常容易受到常见环境微生物铜绿假单胞菌的慢性呼吸道感染。慢性感染极大地降低了患者的生活质量，呼吸衰竭，通常可归因于假单胞菌感染，占&gt；90%的CF死亡率。在生命的早期，CF患者似乎获得了几年后屈从于他们的细菌菌株。因为从最初的感染到死亡经历了数千代细菌，疾病的发展是一个进化的过程。我们试图更好地了解这些细菌在面对持续的免疫系统监测和间歇性抗生素治疗时是如何进化的。慢性感染的一个特征是出现“变种”菌株，这种细菌具有很高的突变率，似乎有助于获得多重抗生素耐药性。我们的目标是研究“突变者”如何改变进化的速度和轨迹：我们将对从多个患者中分离出来的菌株进行遗传学分析，并建立突变者出现和持续的模型条件。我们的目标是帮助临床医生设计抗生素疗法，将慢性感染期间出现多药耐药的可能性降至最低。