Single Cell Expression Profiling of Chlamydia Genes

描述（由申请方提供）：沙眼衣原体是一种细胞内病原体，是导致全球范围内重大人类发病率的原因。根据与该提案相关的资助赠款（5 R 01 AI 039558 -07），我们已经鉴定并测试了许多T细胞抗原在对C.沙眼通过本探索性/开发性申请中描述的工作，我们建议使用已发表的基因组序列和荧光原位杂交（FISH）技术来鉴定候选C。根据沙眼T细胞抗原在发育周期中相关时间的表达及其在相关组织中的表达，确定沙眼T细胞抗原。由于衣原体感染难以同步，因此对这些生物体在发育周期中的基因表达知之甚少。FISH技术的使用避免了异步感染的问题，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。在第一个具体目标中，我们建议使用FISH来探索C。培养细胞中的沙眼衣原体。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，我们将使用荧光原位杂交技术，使用标记有不同荧光染料的探针同时监测生物体内多个基因的表达。然后，我们将编译一个数据库，其中包含许多C语言的相对表达水平。沙眼衣原体基因在发育周期的不同时期。这些信息将被应用于候选T细胞抗原的合理鉴定，基于它们在细胞内发育早期的表达，此时T细胞应答将是最有效的。该数据库还将有助于破译每个发育阶段存在的途径和调控网络。在第二个具体目标中，我们建议使用FISH来研究衣原体在小鼠感染期间生殖器组织中的基因表达。特别是，我们将看看在这些组织中的衣原体基因表达的时间和空间差异。我们还建议分析衣原体基因表达的变化下，各种主机的免疫或化疗压力。这些数据还将允许在T细胞识别最有效时鉴定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.