Genetic Transformation of Chlamydia

衣原体的遗传转化

• 批准号: 6227846
• 负责人: HARLAN D CALDWELL
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6227846
• 关键词: Chlamydia trachomatis; bacterial genetics; cell transformation; electroporation; gene targeting; genetic markers; genetic techniques; genetic transduction; molecular cloning; plaque assay; technology /technique development

Chlamydial trachomatis are obligate intracellular bacteria and are the most common cause of sexually transmitted diseases (STD) of humans. A common sequelae of genital infection of women is salpingitis that can lead to pelvic inflammatory disease (PID) and infertility. Treatment of chlamydial STDs is estimated to cost 5 billion dollars annually. Although much progress has been made in understanding the pathogenesis of chlamydial STD there remains key unanswered questions in the pathophysiology and immunology of infection that have hampered advances in the development of much needed preventive therapies. A major obstacle toward this end has been the inability to use molecular genetic approaches to study this important human pathogen. It is likely that significant progress in the control of chlamydial STD will only come when genetic manipulation of the pathogen is possible. Recent advances in genomics have greatly increased the feasibility of developing new strategies for the study of many pathogenic microorganisms. This is indeed true for Chlamydia. For example, the availability of the complete C. trachomatis genomic sequence shows that the organism possess a reasonably complete complement of genes necessary for genetic recombination and repair. This strongly suggests that it should be feasible to develop a genetic transformation system for chlamydiae. The purpose of this project is to begin the process of developing a genetic system for Chlamydia. Once the basic tools of genetics are developed the project will address other challenges aimed at defining methods for optimum DNA introduction and transformation. These will include the targeting of both the infectious extracellular and non-infectious intracellular life forms of the organism using electroporation and particle bombardment technologies. Once conditions for stable genetic transformation have been developed experiments will be done to mutate or knock out targeted genes identified from the genome sequence as potentially important virulence determinants, and introduce new genes into the chlamydial genome that might allow the organism to be grown outside cells. The results of these studies should significantly advance the understanding of chlamydial pathogenic mechanism(s) and disease processes, as well as possibly produce insights for the development of novel live-attenuated organisms that can be tested as vaccine candidates in pre-clinical models of chlamydial infection. To date we have used the recently described plaque cloning procedure to isolate spontaneous rifampicin resistant mutants of C. trachomatis. The development of stable genetic resistance markers will enable us to test the ability of C. trachomatis to incorporate DNA following introduction by electroporation or particle bombardment means. - Plaque assay, cloning, antibiotic resistance, recombination, electroporation, particle bombardment, and DNA mutagenesis

衣原体气管性是务实的细胞内细菌，是人类性传播疾病（STD）的最常见原因。女性生殖器感染的常见后遗症是盐酸炎，可能导致骨盆炎性疾病（PID）和不育。估计每年耗资50亿美元的衣原体性病治疗。尽管在理解衣原体性病的发病机理方面取得了很多进展，但在感染的病理生理学和免疫学中，仍然取得了关键的未解决问题，这些问题阻碍了急需的预防疗法的发展。这一目的的主要障碍是无法使用分子遗传学方法研究这种重要的人类病原体。只有在可能对病原体的遗传操纵时，才可能在控制衣原体性病的控制方面取得重大进展。基因组学的最新进展大大提高了开发许多致病微生物研究的新策略的可行性。衣原体确实如此。例如，完整的沙眼基因组序列的可用性表明，该生物具有相当完整的遗传重组和修复所必需的基因补体。这强烈表明，为衣原体开发遗传转化系统应该是可行的。该项目的目的是开始为衣原体开发遗传体系的过程。一旦开发了遗传学的基本工具，该项目将解决旨在定义最佳DNA引入和转化方法的其他挑战。这些将包括使用电穿孔和粒子轰击技术的传染性细胞外和非感染细胞内生命形式的靶向。一旦开发出稳定的遗传转化条件，将进行实验，以突变或淘汰从基因组序列鉴定为潜在重要的毒力决定簇的靶向基因，并将新基因引入衣原体基因组中，这些基因组可能使生物体可以生长在外部细胞外部。这些研究的结果应显着提高人们对衣原体致病机制和疾病过程的理解，并可能为开发新型的生物衰减生物的开发提供见解，这些生物可以在衣原体感染前临床模型中作为疫苗候选者进行测试。迄今为止，我们已经使用了最近描述的斑块克隆程序来隔离沙眼梭状芽孢杆菌的自发利福平突变体。稳定的遗传抗性标记的发展将使我们能够测试沙眼梭状芽孢杆菌在通过电穿孔或颗粒轰击均值引入后结合DNA的能力。 - 斑块测定，克隆，抗生素耐药性，重组，电穿孔，颗粒轰击和DNA诱变