Non-invasive Fluorescent Imaging Mycobacterium tuberculosis Extrapulmonary Infect

描述（由申请人提供）：结核病（TB）仍然是全球主要的公共卫生问题。大约5-20%的TB患者还发展为肺外结核（EPTB），其使诊断复杂化，具有高死亡率，并且难以治疗。我们目前关于分枝杆菌侵入和扩散到肺外组织的机制的知识是不完整的。利用动物模型进行EPTB的研究受到M生长缓慢的阻碍。琼脂平板上的结核病。非侵入性体内光学成像可以补充经典的解剖病理学研究，有助于解开错综复杂的EPTB，特别是入侵和初始肺部细菌数量的时间方面。我们已经开发了一个在体内成像系统，指定β-内酰胺酶报告酶荧光（REF），实时成像的M。活鼠肺结核感染。它基于β-内酰胺酶的近红外（NIR）荧光底物，β-内酰胺酶是一种由结核杆菌天然表达但不由其真核宿主表达的酶。底物由通过β-内酰胺环连接至猝灭剂的NIR染料组成。一旦β-内酰胺环被M水解，在结核病β-内酰胺酶BlaC中，荧光染料从猝灭剂中释放，并产生荧光。我们建议REF可用于研究EPTB的时间动力学和活体动物的特定器官参与，并调查细菌基因和宿主因素。我们将研究未感染和感染小鼠肺和肺外器官中REF成像的两种底物的处置和动力学;检查用于检测小鼠器官尤其是肺外器官中细菌的每种底物的阈值;评估细菌菌落形成单位（CFU）与荧光信号之间的相关性;并研究EPTB在各种器官中发展的时间顺序。为了验证REF在研究细菌基因和宿主因素参与EPTB，我们将选择一个细菌基因，hbhA，和一个宿主基因，IFN-g，已知参与EPTB的发展。将对感染hbhA突变株、补体株和野生亲本株的小鼠进行成像。将比较各组之间的定量荧光水平和CFU。对于IFN-g，我们将对受感染的IFN-g敲除小鼠和野生亲本小鼠进行成像，并比较荧光水平。两项实验都将采集CFU以验证成像结果。这项拟议研究的成功可以通过允许研究人员直接监测M.肺外结核感染和定量活动物中的细菌活力。这项研究将获得第一个时间的理解的过程中广告的器官特异性序列的事件在活的动物模型。该系统将允许优雅的策略，调查细菌基因和宿主因素，发挥重要作用，在EPTB的发展。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.