Genetic Transformation of Chlamydia

沙眼衣原体是专性细胞内细菌，是人类性传播疾病(STD)的最常见原因。女性生殖器感染的一个常见后遗症是输卵管炎，可能导致盆腔炎(PID)和不孕。据估计，衣原体性病的治疗每年要花费50亿美元。尽管在了解衣原体性病的发病机制方面已经取得了很大的进展，但在感染的病理生理学和免疫学方面仍然存在一些关键的未回答的问题，这些问题阻碍了急需的预防性治疗的发展。实现这一目标的一个主要障碍是无法使用分子遗传学方法来研究这种重要的人类病原体。很可能只有在对病原体进行基因操作的情况下，才能在控制衣原体STD方面取得重大进展。基因组学的最新进展极大地增加了为许多病原微生物研究开发新策略的可行性。衣原体确实是这样的。例如，完整的沙眼衣原体基因组序列的可获得性表明，生物体拥有相当完整的基因，这是基因重组和修复所必需的。这有力地表明，建立衣原体遗传转化系统是可行的。该项目的目的是开始开发衣原体基因系统的进程。一旦开发出遗传学的基本工具，该项目将解决旨在确定最佳DNA引进和转化方法的其他挑战。这些措施将包括利用电穿孔和粒子轰击技术瞄准具有感染性的细胞外生命形式和非传染性的细胞内生命形式。一旦开发出稳定遗传转化的条件，就会进行实验，从基因组序列中突变或敲除被确定为潜在重要毒力决定因素的目标基因，并将新基因引入衣原体基因组，这可能会使有机体在细胞外生长。这些研究的结果将大大促进对衣原体致病机制(S)和疾病过程的理解，并可能为开发新型减毒活菌提供洞察力，这些生物可以作为疫苗候选在衣原体感染的临床前模型中进行测试。到目前为止，我们已经使用最近描述的空斑克隆程序来分离沙眼衣原体自发的利福平耐药突变株。稳定的遗传抗性标记的开发将使我们能够测试沙眼衣原体通过电穿孔或基因轰击的方法导入DNA的能力。-空斑分析、克隆、抗生素耐药性、重组、电穿孔、粒子轰击和DNA突变

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