Single Cell Expression Profiling of Chlamydia Genes

描述（由申请人提供）：沙眼衣原体是一种细胞内病原体，在世界范围内造成重大的人类发病率。在与本提案相关的基金资助下（5 R01 AI039558-07），我们一直在鉴定和测试一些T细胞抗原在沙眼原体保护性免疫中的作用。通过本探索性/发育性应用中描述的工作，我们建议利用已发表的基因组序列和荧光原位杂交（FISH）技术，根据其在发育周期中相关时间的表达以及在相关组织中的表达来鉴定候选沙眼衣原体T细胞抗原。由于衣原体感染难以同步，因此对这些生物在其发育周期中的基因表达知之甚少。FISH技术的使用规避了非同步感染的问题，因为我们将能够在单个细菌的水平上分析衣原体基因的表达。在第一个特异性目标中，我们提出使用FISH来探索沙眼衣原体在培养细胞中的发育周期。使用FISH，我们应该能够检测到以前使用其他技术无法检测到的基因的诱导或抑制。在优化探针设计和杂交条件后，我们将使用FISH使用不同荧光染料标记的探针同时监测生物体内多个基因的表达。然后，我们将编制一个数据库，其中包含许多沙眼衣原体基因在发育周期的不同时期的相对表达水平。这些信息将应用于基于细胞内发育早期表达的候选T细胞抗原的合理鉴定，此时T细胞反应将是最有效的。该数据库还将有助于破译存在于每个发育阶段的途径和调控网络。在第二个特定目标中，我们提出使用FISH研究小鼠感染过程中生殖道组织中衣原体基因的表达。特别地，我们将研究衣原体基因在这些组织中表达的时空差异。我们还建议分析在不同宿主免疫或化疗压力下衣原体基因表达的变化。这些数据还将允许在T细胞识别最有效的时候识别在适当组织类型中表达的候选抗原。

DESCRIPTION (provided by applicant): Chlamydia trachomatis is an intracellular pathogen that is responsible for significant human morbidity throughout the world. Under the funded grant linked to this proposal (5 R01 AI039558-07) we have been identifying and testing a number of T cell antigens for their role in protective immunity to C. trachomatis. Through the work described in this Exploratory/Developmental application, we propose to use the published genome sequence and fluorescence in situ hybridization (FISH) technology to identify candidate C. trachomatis T cell antigens based on their expression at the relevant time in the developmental cycle and their expression in the relevant tissues. Little is known about gene expression in these organisms during their developmental cycle because of the difficulty in synchronizing Chlamydia infections. The use of FISH technology circumvents the problem of asynchronous infections because we will be able to analyze the expression of Chlamydia genes at the level of individual bacteria. In the first Specific Aim, we propose to use FISH to explore the developmental cycle of C. trachomatis in cultured cells. Using FISH, we should be able to detect induction or repression of genes that were previously undetectable using other techniques. After optimizing probe design and hybridization conditions, we will use FISH to simultaneously monitor the expression of multiple genes within an organism using probes tagged with different fluorescent dyes. We will then compile a database containing the relative levels of expression of many C. trachomatis genes at various times in the developmental cycle. This information will be applied to the rational identification of candidate T cell antigens based on their expression early in intracellular development, when a T cell response would be most effective. The database will also aid in deciphering the pathways and regulatory networks present at each developmental stage. In the second Specific Aim, we propose to use FISH to study Chlamydia gene expression in genital tissues during murine infection. In particular, we will look at the temporal and spatial differences in Chlamydia gene expression in these tissues. We also propose to analyze changes in Chlamydia gene expression under various host immunological or chemotherapeutic pressures. These data will also allow for the identification of candidate antigens expressed in appropriate tissue types at times when T cell recognition would be most effective.