A Novel Clock to Monitor M. tuberculosis in vivo

描述(申请人提供)：结核病(TB)仍然是一种全球祸害，尽管有化疗，估计每年仍有200万人死亡。绝大多数病例都是临床潜伏期，仍然很难诊断。全球疫情的控制取决于我们识别和治疗这一庞大的潜伏感染人群的能力；因此，至关重要的是，我们对慢性结核分枝杆菌(MTB)感染的基本观点是准确的。然而，尽管经过了一个世纪的研究，但对持续感染期间结核分枝杆菌的生理状态仍然知之甚少。为了监测MTB在宿主内的复制，我们开发了一种不稳定的质粒，它在没有抗生素选择的情况下以稳定的、可量化的速度从细胞分裂中丢失，用作复制时钟。通过这个工具，我们最近确定了MTB在小鼠慢性感染过程中继续复制，并受到宿主免疫系统的抑制。这一结果支持对当前假设抑菌的结核病持久性模型进行彻底的重新评估。此外，还提出了重大的实际问题，因为结核病药物发现工作越来越注重消除非复制体，以此作为改进治疗和治愈潜伏感染的关键。因此，尽可能快速和完整地评估不稳定的质粒复制时钟和我们关于MTB潜伏期的假设是重要的。在这个提案中，我们概述了一系列实验，以测试我们对MTB复制动力学的发现，并扩展我们对宿主内感染的看法。首先，我们将分析体外和体内质粒行为的关键参数，以完善我们技术的应用。这将涉及到使用环境应激源以及有条件地复制的突变株来产生一个健壮的质粒分离模型。其次，我们将结合复制时钟使用荧光蛋白技术来可视化和量化肉芽肿水平上的结核分枝杆菌动力学。这一创新将使我们能够表征细菌复制在单个感染器官内不同病变中的异质性。我们建议的研究的完成将牢固地建立一个独特的工具，用来评估宿主内的MTB行为。这项技术不仅对结核分枝杆菌潜伏期的关键方面有重要的洞察力，而且在治疗发现和疫苗开发的更大努力中提供了应用。公共卫生相关性：目前对引起潜伏性结核病(TB)的宿主和细菌因素的了解严重不足。该项目将产生新的工具和见解，以提高我们对活体内结核分枝杆菌动力学的理解。其结果将是对结核病潜伏期和发病机制的更准确的描绘，这反过来将引发与这一困扰当今世界20亿人的祸害作斗争的努力。

DESCRIPTION (provided by applicant): Tuberculosis (TB) retains its place as a global scourge, killing an estimated 2 million people per year despite the availability of chemotherapy. The huge majority of cases are clinically latent and remain difficult to diagnose. Control of the global epidemic hinges on our ability to identify and treat this vast population of latently infected individuals; as such, it is critical that our fundamental view of chronic Mycobacterium tuberculosis (MTB) infection is accurate. However, despite a century of research, the physiologic state of MTB during persistent infection remains poorly understood. To monitor MTB replication within hosts, we have developed an unstable plasmid that is lost at a stable, quantifiable rate from dividing cells in the absence of antibiotic selection for use as a replication clock. With this tool, we have recently determined that MTB continues to replicate throughout chronic infection of mice and is restrained by the host immune system. This result argues for a thorough re-appraisal of current TB persistence models that postulate bacteriostasis. In addition, significant practical questions are raised, since TB drug discovery efforts increasingly focus on eliminating non-replicating organisms as the key to improving therapy and curing latent infection. It is therefore important to evaluate both the unstable plasmid replication clock and our assumptions about MTB latency as quickly and completely as possible. In this proposal, we outline a series of experiments to test our findings of MTB replication dynamics and expand our view of infection within a host. First, we will analyze key parameters of plasmid behavior both in vitro and in vivo to refine the application of our technique. This will involve use of environmental stressors as well as a conditionally replicating mutant strain to produce a robust model of plasmid segregation. Second, we will employ fluorescent protein technology in conjunction with the replication clock to visualize and quantify MTB dynamics at the level of the granuloma. This innovation will allow us to characterize the heterogeneity of bacterial replication in different lesions within a single infected organ. Completion of our proposed studies will firmly establish a unique tool with which to evaluate MTB behavior within a host. This technique promises not only significant insight into key aspects of MTB latency, but offers application within larger efforts of therapeutic discovery and vaccine development. PUBLIC HEALTH RELEVANCE: Current knowledge of the host and bacterial factors that are involved in latent tuberculosis (TB) are woefully inadequate. This project will generate novel tools and insights to improve our understanding of M. tuberculosis dynamics in vivo. The result will be a vastly more accurate picture of TB latency and pathogenesis, which will in turn spark efforts to combat this scourge that afflicts two billion people in the world today.