A Novel Clock to Monitor M. tuberculosis in vivo

描述（由申请人提供）：结核病（TB）仍然是一个全球性的祸害，尽管有化疗，每年仍有大约200万人死于结核病。绝大多数病例是临床潜伏的，仍然难以诊断。控制全球流行病取决于我们是否有能力识别和治疗这一庞大的潜在感染者群体；因此，我们对慢性结核分枝杆菌（MTB）感染的基本看法是准确的，这一点至关重要。然而，尽管经过一个世纪的研究，MTB在持续感染期间的生理状态仍然知之甚少。为了监测宿主内MTB的复制，我们开发了一种不稳定的质粒，在没有抗生素选择的情况下，它以稳定的、可量化的速率从分裂的细胞中丢失，用作复制时钟。有了这个工具，我们最近确定MTB在小鼠的慢性感染中继续复制，并受到宿主免疫系统的抑制。这一结果主张彻底重新评估目前假定细菌抑制作用的结核病持久性模型。此外，由于结核病药物发现工作日益侧重于消除非复制生物体作为改进治疗和治愈潜伏感染的关键，因此提出了重大的实际问题。因此，尽快和完整地评估不稳定的质粒复制时钟和我们对MTB延迟的假设是很重要的。在这个建议中，我们概述了一系列的实验来测试我们的MTB复制动力学的发现，并扩大了我们对宿主内感染的看法。首先，我们将分析质粒在体外和体内行为的关键参数，以完善我们技术的应用。这将涉及使用环境压力源以及有条件地复制突变株，以产生一个健壮的质粒分离模型。其次，我们将采用荧光蛋白技术与复制时钟相结合，在肉芽肿水平上可视化和量化MTB动态。这一创新将使我们能够表征单个感染器官内不同病变中细菌复制的异质性。完成我们提出的研究将牢固地建立一个独特的工具来评估宿主的结核分枝杆菌行为。这项技术不仅有望对结核分枝杆菌潜伏期的关键方面有重要的了解，而且可以在治疗发现和疫苗开发的更大努力中得到应用。公共卫生相关性：目前关于潜伏性结核病（TB）的宿主和细菌因素的知识严重不足。该项目将产生新的工具和见解，以提高我们对体内结核分枝杆菌动力学的理解。其结果将大大提高对结核病潜伏期和发病机制的准确了解，从而推动努力防治这一当今世界上20亿人遭受的祸害。

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.