Non-invasive Fluorescent Imaging Mycobacterium tuberculosis Extrapulmonary Infect

无创荧光成像结核分枝杆菌肺外感染

• 批准号: 8508307
• 负责人: Ying Kong
• 金额: $ 2.07万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-10 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8508307
• 关键词: Acute; Affect; Agar; Animal Model; Animals; Antitubercular Agents; Bacillus (bacterium); Bacteria; Bacterial Genes; Cell Wall; Cells; Chronic; Colony-forming units; Complement; Cytoplasm; Data; Detection; Development; Diagnosis; Diagnostic; Disease; Dose; Dyes; Enzymes; Event; Fluorescence; Fluorescent Dyes; Fluorogenic Substrate; Genes; Genus Mycobacterium; Growth; Histopathology; Image; Imaging technology; Immune; Infection; Integration Host Factors; Interferon Type II; Invaded; Investigation; Kinetics; Knockout Mice; Knowledge; Lactamase; Lactams; Life; Lung; Measures; Metabolic; Metabolism; Methods; Monitor; Mus; Mycobacterium tuberculosis; Organ; Pathogenesis; Patients; Play; Process; Public Health; Recombinants; Reporter; Research; Research Personnel; Role; Route; Side; Signal Transduction; Site; Specificity; Staging; Surface; System; Time; Tissues; Tuberculosis; Tuberculosis Vaccines; base; beta-Lactamase; beta-Lactams; cost; cytokine; fluorescence imaging; imaging modality; improved; in vivo; mortality; mutant; optical imaging; research study; success; tool; tuberculosis treatment

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.

描述（由申请人提供）：结核病（TB）仍然是全球领先的公共卫生问题。大约5-20％的结核病患者还会出现肺外结核病（EPTB），这使诊断复杂化，死亡率高且难以治疗。我们目前关于分枝杆菌入侵并扩散到肺外组织的机制的知识是不完整的。使用动物模型对EPTB进行的研究受到琼脂板上结核分枝杆菌的生长缓慢的影响。非侵入性体内光学成像可以补充经典的解剖学研究，有助于揭示EPTB的复杂性，尤其是入侵的时间方面和初始的肺细菌数量。我们已经开发了一个体内成像系统，该系统指定为β-内酰胺酶报道酶荧光（REF），用于实时成像活小鼠肺部感染的实时成像。它基于β-内酰胺酶的近红外（NIR）荧光底物，这是一种由结核杆菌自然表达的酶，而不是由其真核宿主表达的。底物由通过β-内酰胺环连接到淬火剂的NIR染料组成。一旦β-内酰胺环被结核分枝杆菌β-内酰胺酶（Blac）水解水解，荧光染料将从淬火剂中释放出来，并产生荧光。我们建议REF可用于研究EPTB的时间动力学以及活动物中的特定器官参与，并研究细菌基因和所涉及的宿主因素。我们将研究两种底物的处置和动力学，用于在未感染和感染小鼠的肺和肺外器官中进行参考成像。检查每个底物的阈值，以检测小鼠器官，尤其是肺外器官中的细菌；评估细菌菌落形成单元（CFU）和荧光信号之间的相关性；并研究各种器官中EPTB发育的时间序列。为了验证REF研究EPTB中涉及的细菌基因和宿主因素，我们将选择一个已知参与EPTB发育的细菌基因，HBHA和一个宿主基因IFN-G。将成像被HBHA突变株，补体菌株和野生父母菌株感染的小鼠。将在组之间比较定量的荧光水平和CFU。对于IFN-G，我们将对感染的IFN-G基因敲除小鼠和野生父母小鼠进行成像，并比较荧光水平。这两个实验都将收集CFU以验证成像结果。这项拟议的研究的成功可以通过允许研究人员直接监测结核分枝杆菌外感染并量化活动物中细菌生存能力来促进结核病研究的进度。这项研究将获得对该过程的第一个时间理解，在活动物模型中，一个器官特定的事件序列。该系统将允许研究细菌基因的优雅策略和在EPTB开发中起重要作用的宿主因素。识别影响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。