Non-invasive Fluorescent Imaging Mycobacterium tuberculosis Extrapulmonary Infect

描述（由申请人提供）：结核病（TB）仍然是世界范围内主要的公共卫生问题。大约5-20%的结核病患者还会发展为肺外结核（EPTB），这使诊断复杂化，死亡率高，而且难以治疗。关于分枝杆菌侵入和扩散到肺外组织的机制，我们目前的知识是不完整的。利用动物模型进行EPTB的研究由于结核分枝杆菌在琼脂平板上生长缓慢而受到阻碍。非侵入性体内光学成像可以补充经典的解剖病理学研究，有助于揭示EPTB的复杂性，特别是入侵的时间方面和初始肺部细菌数量。我们开发了一种体内成像系统，称为β -内酰胺酶报告酶荧光（REF），用于活体小鼠结核分枝杆菌肺部感染的实时成像。它是基于β -内酰胺酶的近红外（NIR）荧光底物，这种酶由结核杆菌自然表达，而不是由真核宿主表达。底物由近红外染料通过β -内酰胺环连接到猝灭剂组成。一旦-内酰胺环被结核分枝杆菌-内酰胺酶（BlaC）水解，荧光染料就从猝灭剂中释放出来，并产生荧光。我们建议REF可以用于研究EPTB的时间动力学和活体动物的特定器官参与，并研究涉及的细菌基因和宿主因素。我们将研究两种底物在未感染和感染小鼠肺和肺外器官中的配置和动力学；检查每种底物的阈值，以检测小鼠器官中的细菌，特别是肺外器官；评估细菌菌落形成单位（CFU）与荧光信号的相关性；并研究EPTB在各器官中发展的时间顺序。为了验证REF在研究EPTB相关细菌基因和宿主因子中的作用，我们将选择一个已知参与EPTB发展的细菌基因hbhA和一个宿主基因IFN-g。对感染hbhA突变株、补体株和野生亲本株的小鼠进行成像。比较各组间定量荧光水平和CFU。对于IFN-g，我们将对感染的IFN-g敲除小鼠和野生亲代小鼠进行成像，并比较荧光水平。两个实验都将收集CFU来验证成像结果。这项研究的成功可以促进结核病研究的进展，使研究人员能够直接监测结核分枝杆菌肺外感染并量化活动物的细菌活力。这项研究将首次在活体动物模型中获得对该过程和器官特异性事件序列的时间理解。该系统将为研究在EPTB发展中起重要作用的细菌基因和宿主因子提供优雅的策略。确定影响EPTB的细菌和宿主因素将有助于开发改进的诊断工具、抗结核疗法和疫苗。

DESCRIPTION (provided by applicant): Tuberculosis (TB) continues to be a leading public health problem worldwide. Approximately 5-20% of TB patients also develop extrapulmonary tuberculosis (EPTB), which complicates diagnosis, has high mortality rate, and is difficult to treat. Our current knowledge regarding the mechanisms by which mycobacteria invade and spread into extrapulmonary tissues is incomplete. Research on EPTB using animal models is hampered by slow growth of M. tuberculosis on agar plates. Non-invasive in vivo optical imaging can complement classical anatomopathological studies, helping to unravel the intricacies of EPTB, especially the temporal aspects of invasion and initial pulmonary bacterial numbers. We have developed an in vivo imaging system, designated beta-lactamase reporter enzyme fluorescence (REF), for real-time imaging of M. tuberculosis pulmonary infection of live mice. It is based on near infrared (NIR) fluorogenic substrates for beta-lactamase, an enzyme naturally expressed by tubercle bacilli but not by their eukaryotic hosts. Substrates are composed of a NIR dye connected to a quencher through the beta-lactam ring. Once the beta-lactam ring is hydrolyzed by M. tuberculosis beta-lactamase, BlaC, the fluorescent dye is freed from the quencher, and produces fluorescence. We propose that REF can be used to study the temporal kinetics of EPTB and specific organ- involvement in live animals and to investigate bacterial genes and host factors involved. We will investigate dispositions and kinetics of two substrates for REF imaging in lungs and extrapulmonary organs in uninfected and infected mice; examine threshold of each substrate for detection of bacteria in mouse organs, especially extrapulmonary organs; evaluate the correlation between bacterial colony forming units (CFU) and fluorescent signal; and investigate the temporal sequence of EPTB development in various organs. To validate REF in investigating bacterial genes and host factors involved in EPTB, we will select one bacterial gene, hbhA, and one host gene, IFN-g, that have known involved in EPTB development. Mice infected with hbhA mutant strain, complement strain and wild parental strain will be imaged. The quantified fluorescent level and CFU will be compared among groups. For IFN-g, we will image infected IFN-g knock-out mice and wild parental mice, and compare the fluorescent levels. Both experiments will have CFU collected to validate imaging results. The success of this proposed study could facilitate tuberculosis research progress by allowing investigators to directly monitor M. tuberculosis extrapulmonary infection and quantify bacterial viability in live animals. This study will obtain the first temporal understanding of the process ad an organ-specific sequence of events in a live animal model. This system will allow elegant strategies for investigation of bacterial genes and host factors that play important roles in EPTB development. Identifying bacterial and host factors affecting EPTB would help in the development of improved diagnostic tools, anti-tuberculosis therapies and vaccines. PUBLIC HEALTH RELEVANCE: Extrapulmonary tuberculosis complicates diagnosis, is hard to treat, and has high rate of mortality. We propose application of a non-invasive optical imaging method to investigating extrapulmonary tuberculosis pathogenesis in live animals. Success of this study will greatly facilitate tuberculosis research and help in development of diagnostic tools, anti-tuberculosis therapies and vaccines.