Department of Microbial and Molecular Pathogenesis, Texas A&M Health Sciences Center, College Station, TX 77843, USA.
Proc Natl Acad Sci U S A. 2010 Jul 6;107(27):12239-44. doi: 10.1073/pnas.1000643107. Epub 2010 Jun 21.
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 quantification 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-Guérin 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 quantification 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.
结核分枝杆菌的生长缓慢和遗传难操作性阻碍了对结核病的理解，结核病是全球最常见的死亡原因之一。我们通过开发近红外（NIR）荧光β-内酰胺酶底物克服了这一障碍，该酶由结核分枝杆菌表达，但不是其真核宿主表达，从而允许通过称为报告酶荧光（REF）的策略实时成像肺部感染并快速定量活动物中的细菌。该策略在体外孵育 24 小时时，使用 NIR 底物 CNIR5 的检测限为 6 +/- 2 x 10(2)个细菌菌落形成单位（CFU），在活小鼠的肺部中低至 10(4)个 CFU。REF 还可以通过共聚焦显微镜和荧光激活细胞分选来区分感染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部中追踪，而无需牺牲动物。通过在活小鼠中直接进行体外全细菌测定，在治疗后 24 小时内通过 REF 信号丢失，还可以评估治疗效果。我们预计，在活宿主组织内和实验室中对细菌进行快速定量，可能会对结核病毒力研究、治疗评估和疫苗候选物的功效产生变革性影响。这是一种利用内源性细菌酶探针来检测和成像结核分枝杆菌的独特方法，表明 REF 可能对许多细菌感染的研究有用。
