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) 仍然是全球主要的公共卫生问题。约5-20%的结核病患者同时发展为肺外结核（EPTB），该病诊断复杂、死亡率高、治疗困难。我们目前对分枝杆菌侵入和扩散到肺外组织的机制的了解还不完整。使用动物模型进行 EPTB 的研究因琼脂平板上结核分枝杆菌生长缓慢而受到阻碍。无创体内光学成像可以补充经典的解剖病理学研究，有助于阐明 EPTB 的复杂性，特别是侵袭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。它基于 β-内酰胺酶的近红外 (NIR) 荧光底物，β-内酰胺酶是结核杆菌天然表达的酶，但真核宿主不表达。底物由通过 β-内酰胺环连接至猝灭剂的近红外染料组成。一旦 β-内酰胺环被结核分枝杆菌 β-内酰胺酶 BlaC 水解，荧光染料就会从猝灭剂中释放出来，并产生荧光。我们建议 REF 可用于研究 EPTB 的时间动力学和活体动物特定器官受累，并研究所涉及的细菌基因和宿主因素。我们将研究未感染和感染小鼠的肺部和肺外器官中用于 REF 成像的两种底物的分布和动力学；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）和荧光信号之间的相关性；并研究不同器官中 EPTB 发展的时间顺序。为了验证 REF 研究与 EPTB 相关的细菌基因和宿主因素的能力，我们将选择一种已知参与 EPTB 发展的细菌基因 hbhA 和一种宿主基因 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。定量荧光水平和 CFU 将在组间进行比较。对于 IFN-g，我们将对感染的 IFN-g 敲除小鼠和野生亲本小鼠进行成像，并比较荧光水平。两个实验都将收集 CFU 以验证成像结果。这项拟议研究的成功可以让研究人员直接监测结核分枝杆菌肺外感染并量化活体动物中的细菌活力，从而促进结核病研究进展。这项研究将首次了解活体动物模型中器官特异性事件序列的过程。该系统将为研究在 EPTB 发展中发挥重要作用的细菌基因和宿主因素提供优雅的策略。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。