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， 常见的细菌性传播感染年复一年。高比例的无症状 病例、对自然清除的抵抗以及疫苗开发的缓慢进展使得C. 沙眼保持其作为重大公共卫生威胁的地位。C.沙眼衣原体 与许多宿主免疫效应物绝缘。此外，IFN γ的下游效应，一种细胞因子释放， 宿主免疫细胞对抗感染，导致衣原体转变为持久状态--一种可行但非持久的状态， 复制和非传染性的状态，专门用于生存而不是生长。在没有增长的情况下， 免疫压力消散，使C.沙眼患者恢复到其发育能力状态， 继续感染循环。考虑到衣原体的无症状性质和开发一种 有效的疫苗，预防性药物治疗可能是最好的选择-一个专门设计来抑制 衣原体持续存在不幸的是，开发针对持续反应的预防剂目前还没有 这是合理的，因为参与进入，维持和退出持久性的分子机制在很大程度上是 未知目前的知识仅限于对持续反应的广泛观察，例如 转录失调和翻译减少。我们假设翻译的减少会导致 在转录抑制因子的损失，使σ因子成为过度活跃在启动转录。 该建议旨在描述与启动和维持转录相关的转录变化， 通过联合RNA测序和染色质免疫沉淀和测序（ChIP）持续状态 seq）方法。IFN γ和另外两种显示诱导持久性的模型将用于观察 通过RNA测序与持久性相关的转录变化。为了更深入地了解 转录变化，ChIP seq将在持续期间使用多株C.沙眼， 每一个在不同的σ因子上携带内源性多组氨酸标签。除了提供第一个 在C.沙眼衣原体在标准生长期和持续期， 通过RNA测序观察到的转录变化将与特定的σ因子相关联。这张高分辨率地图 转录变化发生在持久性将指导未来的调查，旨在揭示 参与持续反应的分子机制。该研究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，科学写作和演示。拥有基因组学核心设施和生物信息学， 系统生物学核心设施，内布拉斯加大学医学中心是完全装备，以支持这些目标 在这份提案中。此外，衣原体领域的几位专家是联合国医学中心的教员， 每个人都能够提供与提案相关的各种观点和专业知识。

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.