Sharan Riti, Yang Hee-Jeong, Sule Preeti, Cirillo Jeffrey D
Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center.
Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health.
J Vis Exp. 2018 Feb 26(132):56801. doi: 10.3791/56801.
Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.
报告酶荧光（REF）利用对目标生物体中存在的酶具有特异性的底物，通过荧光或生物发光进行成像或检测。我们使用BlaC，一种由所有结核分枝杆菌菌株组成性表达的酶。REF能够快速定量感染小鼠肺部的细菌。同一组小鼠可以在多个时间点进行成像，大大降低成本，更快地计数细菌，在宿主-病原体相互作用中进行新的观察，并提高统计效力，因为每组可以更容易地饲养更多动物。由于BlaC酶报告基因的催化性质，REF极其灵敏，并且由于使用了定制的荧光共振能量转移（FRET）或荧光底物而具有特异性。REF不需要重组菌株，确保了正常的宿主-病原体相互作用。我们描述了使用在800nm处具有最大发射的FRET底物对结核分枝杆菌感染进行成像。该底物的波长允许在哺乳动物中进行灵敏的深部组织成像。我们将概述用结核分枝杆菌对小鼠进行气溶胶感染、小鼠麻醉、REF底物给药以及光学成像。该方法已成功应用于评估宿主-病原体相互作用以及针对结核分枝杆菌的抗生素的疗效。
