In vivo Transcriptional Analysis of Latent Tuberculosis

描述(申请人提供)：我们对结核病慢性阶段在分子水平上的理解是不完整的，肯定需要改进。比较分析结核病早期和晚期的基因表达谱将丰富我们对结核病不同阶段的理解。该项目的主要目标是确定在小鼠中建立慢性结核病的基因调控网络。基于一组强有力的初步结果，我们假设结核杆菌正在积极转录特定的基因集，以调节它们在宿主内的代谢活动，尽管在疾病的慢性阶段保持在一个恒定的水平。为了确定慢性感染期间分枝杆菌基因调控网络的关键组件，我们将使用体内微阵列分析来： 1.鉴定结核分枝杆菌。导致结核病进入慢性期的基因。我们将使用慢性结核病的小鼠模型结合微阵列分析来确定小鼠在感染的前220天以及在体外培养期间的差异表达基因。小鼠结核病的再激活模型也将被评估为在免疫抑制期间模拟人类结核病。 2.确定持续基因在建立慢性结核病中的作用。我们将跟踪结核分枝杆菌的器官定植。BALB/c小鼠气溶胶感染后持久性基因缺陷株(等位基因交换产生)与野生型结核分枝杆菌的比较。H37Rv.此外，在确定的体外应激条件下生长的突变体的转录图谱将使用DNA微阵列进行评估。 我们相信，本提案中采用的方法将产生与结核分枝杆菌代谢状态有关的丰富信息。在感染的慢性阶段。拟议的研究将进一步提高我们对宿主-病原体相互作用的性质的理解，这些相互作用可以为药物开发提供潜在的靶点。该项目的成果将为利用人体组织对特定的基因组进行更详细和重点的研究提供必要的基础。可能导致结核病慢性期和复活期的转录调控因子将是未来分析的目标。

DESCRIPTION (provided by applicant): Our understanding of the chronic stages of tuberculosis on a molecular level is incomplete and definitely needs improvement. Comparative analysis of gene expression profiled during early vs. late stages of tuberculosis will enrich our understanding of various stages of tuberculosis. The main objective of this project is to identify gene regulatory networks responsible for establishing chronic tuberculosis in mice. Based on a strong set of preliminary results, we hypothesize that the tuberculous bacilli are actively transcribing specific sets of genes to regulate their metabolic activity inside the host despite remaining at a constant level during the chronic stase of the disease. To identify key components of the mycobacterial gene regulatory network during chronic infection, we will employ the in vivo microarray analysis to: 1. Identify M.tb. genes responsible for entering into the chronic stage of tuberculosis. We will employ the mouse model of chronic tuberculosis combined with microarray analysis to identify differentially expressed genes in mice during the first 220 days of infection as well as during in vitro cultures. A reactivation model of mouse tuberculosis also will be assessed to mimic human tuberculosis during immune suppression. 2. Characterize the role of putative persistence genes in establishing chronic tuberculosis. We will follow organ colonization of M.tb. strains with defective persistence genes (generated by allelic exchange) after aerosol infection of BALB/c mice in comparison to the wild type strain of M.tb. H37Rv. Also, the transcriptional profile of mutants growing under defined in vitro stress conditions will be assessed using DNA microarrays. We believe that the approaches exploited in this proposal will generate a wealth of information related to the metabolic states of M.tb. during chronic stages of infection. The proposed studies will further improve our understanding of the nature of the host-pathogen interactions that can provide potential targets for drug development. Outcomes from this project will provide the necessary foundation for more detailed and focused studies on specific sets of genes utilizing human tissues. Transcriptional regulators that could contribute to both chronic and reactivation stages of tuberculosis will be the target for future analysis.