Imaging tuberculosis with endogenous β-lactamase reporter enzyme fluorescence in live mice
Imaging tuberculosis with endogenous β-lactamase reporter enzyme fluorescence in live mice
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DOI：
10.1073/pnas.1000643107
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发表时间：
2010-07-06
影响因子：
11.1
通讯作者：
Cirillo, Jeffrey D.
中科院分区：
文献类型：
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作者：
Kong, Ying;Yao, Hequan;Cirillo, Jeffrey D.
结核杆菌生长速度缓慢，遗传上难以控制，这阻碍了对结核病的认识，结核病是世界上最常见的死亡原因之一。我们通过开发β-内酰胺酶的近红外（NIR）荧光底物克服了这一障碍，β-内酰胺酶是一种由结核杆菌表达的酶，但不是由其真核宿主表达的酶，可以实时成像肺部感染和快速定量。通过一种称为报告酶荧光（REF）的策略，在活体动物中对细菌进行阳离子化。该策略在体外孵育仅24小时的NIR底物CNIR 5中具有6 +/- 2 × 10（2）菌落形成单位（CFU）的细菌的检测限，并且在活小鼠的肺中具有少至10（4）CFU的检测限。REF也可用于通过使用共聚焦显微镜和荧光激活细胞分选来区分感染的巨噬细胞和未感染的巨噬细胞。结核分枝杆菌和卡介苗可以直接在活小鼠的肺中追踪，而不需要牺牲动物。还可以通过直接在活小鼠中使用体外全细菌测定在治疗后24小时内REF信号的损失来评估治疗功效。我们期待快速的量化。在活宿主的组织内和在实验室中细菌的阳离子对于结核病毒力研究、治疗剂的评价和候选疫苗的功效具有潜在的变革性。这是内源性细菌酶探针检测和成像结核杆菌的独特用途，表明REF可能用于许多细菌感染的研究。
The slow growth rate and genetic intractability of tubercle bacilli has hindered progress toward understanding tuberculosis, one of the most frequent causes of death worldwide. We overcame this roadblock through development of near-infrared (NIR) fluorogenic substrates for beta-lactamase, an enzyme expressed by tubercle bacilli, but not by their eukaryotic hosts, to allow real-time imaging of pulmonary infections and rapid quanti. cation of bacteria in living animals by a strategy called reporter enzyme fluorescence (REF). This strategy has a detection limit of 6 +/- 2 x 10(2) colony-forming units (CFU) of bacteria with the NIR substrate CNIR5 in only 24 h of incubation in vitro, and as few as 10(4) CFU in the lungs of live mice. REF can also be used to differentiate infected from uninfected macrophages by using confocal microscopy and fluorescence activated cell sorting. Mycobacterium tuberculosis and the bacillus Calmette-Guerin can be tracked directly in the lungs of living mice without sacrificing the animals. Therapeutic efficacy can also be evaluated through loss of REF signal within 24 h posttreatment by using in vitro whole-bacteria assays directly in living mice. We expect that rapid quanti. cation of bacteria within tissues of a living host and in the laboratory is potentially transformative for tuberculosis virulence studies, evaluation of therapeutics, and efficacy of vaccine candidates. This is a unique use of an endogenous bacterial enzyme probe to detect and image tubercle bacilli that demonstrates REF is likely to be useful for the study of many bacterial infections.