Genetic Analysis in Chlamydia

衣原体遗传分析

• 批准号: 7773385
• 负责人: Raphael H Valdivia
• 金额: $ 23.4万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7773385
• 关键词: Adherent Culture; Agar; Alkanesulfonates; Antibiotic Resistance; Antibiotics; Applications Grants; Backcrossings; Bacteria; Biology; Cells; Chemicals; Chlamydia; Chlamydia Infections; Chlamydia trachomatis; Chromosomes; Chronic; Clinical; Collection; Comparative Genomic Analysis; Complement; DNA; DNA Sequence; Disease; Epithelial; Essential Genes; Experimental Genetics; Frequencies; Genes; Genetic; Genetic Recombination; Genitourinary system; Genome; Genomics; Genotype; Individual; Infection; Infectious Agent; Infertility; Inflammation; Lead; Maps; Membrane Proteins; Methodology; Methods; Molecular; Monitor; Mutagenesis; Mutagens; Mutation; Nonsense Mutation; Pathogenesis; Pelvic Inflammatory Disease; Phenotype; Proteins; Public Health; Recombinant DNA; Relative (related person); Research; Rifampin; Role; Sexually Transmitted Diseases; Single Nucleotide Polymorphism; Solid; Species Specificity; Spectinomycin; Surface; System; Technology; Testing; Therapeutic Intervention; Time; Tissues; Trachoma; Translations; Trimethoprim; Vaccine Design; Variant; Virulence Factors; base; genetic analysis; genome sequencing; high risk; loss of function mutation; microorganism; mutant; novel; pathogen; pathogenic bacteria; public health relevance; reproductive development; research study; resistant strain; segregation; theories; tissue/cell culture; tool

DESCRIPTION (provided by applicant): The obligate intracellular bacterium Chlamydia trachomatis is a widely disseminated pathogen that infects epithelial surfaces of the conjuctiva and urogenital tract. Chronic inflammation from repeated and persistent infections can lead to severe complications such as blinding trachoma, pelvic inflammatory disease and infertility. Despite the clinical and public health importance of chlamydial diseases, the bacterium's intractability to mutational analysis has significantly hindered a molecular understanding of its pathogenesis. In this grant application, we propose to develop a methodology using chemical mutagens and DNA deep sequencing technologies to perform genetic analysis in Chlamydia. In Aim 1, we will identify mutations responsible for the formation of a "small plaque" (Spq) phenotype by backcrossing mutants to reference strains and following the co-segregation of single nucleotide polymorphisms (SNP) with the Spq phenotype. Putative causative mutations will be then identified by sequencing the minimal region of DNA that co-segregates with the Spq phenotype. In Aim2, we will define the essential gene set required for Chlamydia replication in tissue culture cells. Chromosomal DNA from a large set of independently derived, heavily mutagenized Chlamydia isolates will be sequenced in pools. We will assess the relative contribution of individual genes to Chlamydia viability by monitoring their ability to tolerate loss-of-function mutations. The proposed approaches will significantly accelerate the translation of Chlamydia genomic sequences into functional information and establish a blueprint for genetic analysis in other "genetically-intractable" pathogens. PUBLIC HEALTH RELEVANCE: Chlamydial infections are a significant public health burden and a high risk for the development of reproductive diseases. Our understanding of their biology and how they cause disease is limited by the lack of tools to perform genetic analysis. Here we propose to develop methods to perform such an analysis based on new DNA sequencing technologies. Results from this research will identify factors important for Chlamydia survival in the host and provide new targets for therapeutic intervention and vaccine design.

描述（由申请人提供）：专性细胞内性衣原体沙眼性是一种广泛传播的病原体，可感染结肠和泌尿生殖道的上皮表面。反复感染和持续感染引起的慢性炎症会导致严重的并发症，例如盲，骨盆炎症性疾病和不育。尽管衣原体疾病具有临床和公共健康的重要性，但该细菌的突变分析的棘手性显着阻碍了对其发病机理的分子理解。在此赠款应用中，我们建议使用化学诱变剂和DNA深度测序技术开发一种方法，以在衣原体中进行遗传分析。在AIM 1中，我们将通过将突变体反向引用菌株并随着单核苷酸多态性（SNP）与SPQ表观型的共隔离来确定导致“小斑块”（SPQ）表型形成的突变。然后，将通过测序与SPQ表型共隔离的DNA的最小区域来确定推定的致病突变。在AIM2中，我们将定义组织培养细胞中衣原体复制所需的必需基因集。来自一大批独立衍生的，重诱变的衣原体分离株的染色体DNA将在池中测序。我们将通过监测耐受功能丧失突变的能力来评估单个基因对衣原体生存能力的相对贡献。所提出的方法将显着加速衣原体基因组序列到功能信息中的翻译，并在其他“遗传学可减轻”病原体中建立用于遗传分析的蓝图。 公共卫生相关性：衣原体感染是巨大的公共卫生负担，也是生殖疾病发展的高风险。由于缺乏进行遗传分析的工具，我们对它们的生物学以及如何引起疾病的理解受到限制。在这里，我们建议开发基于新的DNA测序技术进行此类分析的方法。这项研究的结果将确定对宿主中衣原体生存重要的因素，并为治疗干预和疫苗设计提供了新的目标。