Genetic transformation system for Chlamydia suis

描述(申请人提供)：衣原体的研究因缺乏遗传转化系统而变得复杂。这种系统的开发可能会给这一领域的研究带来革命性的变化。这项建议描述了一种利用猪衣原体作为基因转移目标来解决这一问题的方法。这些实验是基于最近在生产设施中从猪的猪链球菌分离株的基因组中发现了一个稳定的四环素耐药岛。这个岛在结构上与革兰氏阴性细菌的质粒有关，很可能是通过一个未知细菌物种的水平传播进入这个物种的。岛内的基因编码质粒动员和复制功能、四环素外排泵和候选转座酶。替代系统中的实验数据表明，其中一个转座酶负责将基因组岛作为质粒引入猪链霉菌中。推测编码的转座酶和tet(C)基因可以作为工具分别用于将基因导入猪衣原体和筛选可能的阳性。这项提案描述了将探索的不同方法来检验这一假说，最终目标是为衣原体开发一个可行的遗传系统。首先，我们实验室正在开发的一种新的筛选技术将被优化，以从感染四环素敏感猪链霉菌的细胞群体中分离出候选的四环素耐药转化子。下一步，将构建代表可能的供体质粒的重组质粒，该质粒负责将DNA自然导入猪链霉菌。这些质粒将用于几种不同的技术，旨在将DNA导入细菌。在这些尝试中将使用几种可能的转化和接合介导的方法，所有这些方法都基于这样的前提，即引入的DNA将用于将tet(C)岛传递到衣原体染色体。如果开发出一种衣原体转化技术，我们将把这项工作扩大到一项为期五年的提案，该提案将涉及该技术的应用和将议定书转让给外地调查人员的问题。 拟议研究的成功将为我们对衣原体生物学的理解带来快速发展的机会。这将有助于识别和分析新的候选疫苗，并更清楚地了解这些重要病原体的毒力机制。

DESCRIPTION (provided by applicant): The study of Chlamydia is complicated by the lack of a genetic transformation system. The development of such a system will likely revolutionize research in this area. This proposal describes an approach to solving this problem using Chlamydia suis as a target for gene transfer. The experiments are based on the recent discovery of a stable tetracycline resistance island within the genome of recent isolates of C. suis from pigs in production facilities. This island is structurally related to plasmids of Gram-negative bacteria, and was likely introduced to this species via horizontal transmission from an unknown bacterial species. Genes within the island encode plasmid mobilization and replication functions, tetracycline efflux pump, and candidate transposases. Experimental data in a surrogate system suggests that one of these transposases was responsible for the integration of the genomic island into C. suis following its introduction as a plasmid. It is hypothesized that the encoded transposase and the tet(C) gene can be used as tools to introduce genes into Chlamydia suis and for screening possible positives, respectively. This proposal describes different approaches that will be explored to test this hypothesis, with the ultimate goal of developing a workable genetic system for the chlamydiae. First, a novel screening technique under development in our laboratory will be optimized for isolating candidate tetracycline-resistant transfromants from a population of cells infected with tetracycline-sensitive C. suis. Next, recombinant plasmids will be constructed that represent the likely donor plasmid responsible for the natural introduction of DNA into C. suis. These plasmids will be used in several different techniques designed to introduce the DNA into the bacterium. Several variations of possible transformation and conjugation-mediated approaches will be used in these attempts, all based on the premise that introduced DNA will serve to deliver the tet(C) island to the chlamydial chromosome. If a technique for chlamydial transformation is developed, we will expand the work into a five-year proposal that will address the application of the technology and the transfer of the protocol to investigators in the field. Success in the proposed studies will lead to opportunities for rapid developments in our understanding of chlamydial biology. This will assist in the identification and analysis of novel vaccine candidates and to a clearer understanding of the mechanism of virulence in these important pathogens.