Defining the persistence associated transcriptome in Chlamydia trachomatis

项目摘要 沙眼衣原体是一种革兰氏阴性、专性胞内细菌，仍是世界上最 常见性传播细菌性感染年复一年。高无症状率的组合 病例、对自然清除的抗药性以及疫苗开发的缓慢进展使C. 沙眼衣原体，以维持其作为重大公共卫生威胁的地位。沙眼衣原体作为一种胞内病原体 与许多宿主免疫效应器隔绝。此外，干扰素的下游效应，一种由 宿主免疫细胞对抗感染，导致衣原体转变为持久状态--一种可行但非 复制和非传染性状态，专注于生存而不是增长。在没有增长的情况下， 免疫压力消散，使沙眼衣原体恢复到其发育能力状态，并 继续感染循环。考虑到衣原体的无症状性质和发展的难度 有效的疫苗，一种预防性的药物治疗可能是最好的选择-一种专门设计的抑制 衣原体的持久性。不幸的是，开发一种针对持续性反应的预防性药物目前还不是 这似乎是合理的，因为进入、维持和退出持久性的分子机制在很大程度上 未知。目前的知识仅限于持续反应中的广泛观察，例如 转录失调和翻译减少。我们假设翻译结果的减少 在转录抑制物的丧失中，允许西格玛因子在启动转录时变得过度活跃。 本提案旨在描述与启动和维持基因相关的转录变化。 通过联合RNA测序和染色质免疫沉淀和测序(CHIP)实现持续状态 SEQ)接近。干扰素和另外两种诱导持续性的模型将被用来观察 通过RNA序列与持续性相关的转录变化。为了更深入地了解 转录变化，芯片序列将在持续期间使用多个沙眼衣原体菌株进行， 每一个都在不同的西格玛因子上含有内源性多组氨酸标签。除了提供第一个 沙眼衣原体标准生长和持续生长过程中Sigma因子活性的全基因组分析， RNA序列观察到的转录变化将与特定的西格玛因子有关。这张高分辨率地图 持续期间发生的转录变化的研究将指导未来旨在揭示 持续反应涉及的分子机制。在该奖学金下的培训将提供广泛的 在最先进的衣原体分子技术、下一代测序、大型 数据集，以及科学写作和演示。基因组学核心设施和生物信息学和 系统生物学核心设施，内布拉斯加大学医学中心为支持这些目标做好了充分的准备 在这项提案中概述了。此外，衣原体领域的几位专家是UNMC的教员， 每一项都能够提供与提案相关的各种观点和专门知识。

Project Summary Chlamydia trachomatis is a Gram-negative, obligate intracellular bacterium that continues to be the world’s most common bacterial sexually transmitted infection year after year. A combination of a high rate of asymptomatic cases, resistance to natural clearance, and slow progress in the development of a vaccine has allowed C. trachomatis to maintain its status as a significant public health threat. As an intracellular pathogen, C. trachomatis is insulated from many host immune effectors. Additionally, downstream effects of IFN, a cytokine released by host immune cells to combat infection, cause Chlamydia to transition into a persistent state – a viable yet non- replicative and non-infectious state that specializes in survival rather than growth. In the absence of growth, immune pressure dissipates, allowing C. trachomatis to revert back to its developmentally competent state and continue the infection cycle. Considering Chlamydia’s asymptomatic nature and the difficulty in developing an effective vaccine, a prophylactic drug treatment may be the best alternative – one specifically designed to inhibit chlamydial persistence. Unfortunately, developing a prophylactic to target the persistent response is not currently plausible since the molecular mechanisms involved in entering, maintaining, and exiting persistence are largely unknown. Current knowledge is limited to broad observations in the persistent response, such as the dysregulation of transcription and a decrease in translation. We hypothesize that a decrease in translation results in the loss of transcriptional repressors, allowing sigma factors to become overactive in initiating transcription. This proposal aims to characterize transcriptional changes associated with the initiation and maintenance of the persistent state through combined RNA sequencing and chromatin immunoprecipitation and sequencing (ChIP seq) approaches. IFN and two additional models shown to induce persistence will be used to observe transcriptional changes associated with persistence via RNA seq. To gain further insight into the dynamics of transcriptional changes, ChIP seq will be performed during persistence using multiple strains of C. trachomatis, each harboring an endogenous polyhistidine tag on a different sigma factor. In addition to providing the first genome wide analysis of sigma factor activity in C. trachomatis during standard growth and persistence, transcriptional changes observed by RNA seq will be linked to specific sigma factors. This high resolution map of transcriptional changes that occur during persistence will direct future investigations aimed at uncovering the molecular mechanisms involved in the persistent response. Training under this fellowship will provide extensive experience in state-of-the-art chlamydial molecular techniques, next generation sequencing, analysis of large data sets, and scientific writing and presentation. Home to both a Genomics Core Facility and Bioinformatics and Systems Biology Core Facility, the University of Nebraska Medical Center is fully equipped to support the goals outlined in this proposal. Furthermore, several experts in the chlamydial field are faculty members at UNMC, each capable of providing various perspectives and expertise relevant to the proposal.