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，估计每年仍有200万人死亡。绝大多数病例在临床上是潜伏的，仍然难以诊断。全球流行病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，我们对慢性结核分枝杆菌（MTB）感染的基本观点是准确的，这一点至关重要。然而，尽管经过了世纪的研究，MTB在持续感染期间的生理状态仍然知之甚少。为了监测宿主内的MTB复制，我们开发了一种不稳定的质粒，其在不存在抗生素选择的情况下以稳定的、可量化的速率从分裂细胞中丢失，用作复制时钟。有了这个工具，我们最近确定MTB在小鼠的慢性感染过程中继续复制，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结核病的持久性模型，假设抑菌作用。此外，由于结核病药物发现工作越来越多地将重点放在消除非复制生物体上，将其作为改善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潜伏期的假设是很重要的。在这个建议中，我们概述了一系列的实验，以测试我们的发现MTB复制动力学和扩大我们的观点感染内的主机。首先，我们将分析质粒在体外和体内行为的关键参数，以完善我们的技术的应用。这将涉及使用环境应激物以及条件复制突变株来产生质粒分离的稳健模型。其次，我们将采用荧光蛋白技术结合复制时钟来可视化和量化肉芽肿水平的MTB动态。这一创新将使我们能够表征单一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，以评估MTB在宿主中的行为。该技术不仅有望对MTB潜伏期的关键方面有重要的了解，而且还可以在治疗发现和疫苗开发的更大努力中提供应用。公共卫生相关性：目前对潜伏性结核病（TB）中涉及的宿主和细菌因素的认识非常不足。这个项目将产生新的工具和见解，以提高我们对M。体内结核动力学。其结果将是对结核病潜伏期和发病机制的更加准确的描述，这反过来将激发人们努力对抗这一困扰当今世界20亿人的祸害。

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.