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Imaging B. anthracis heme catabolism in mice using the IFP1.4 gene reporter

机译：使用IFP1.4基因报告基因对小鼠炭疽杆菌血红素分解代谢进行成像

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摘要

B. anthracis is a gram-positive, spore-forming bacterium which likes all pathogenic bacteria, survive by sequestering heme from its host. To image B. anthracis heme catabolism in vivo, we stably transfect new red excitable fluorescent protein, IFP1.4, that requires the heme catabolism product biliverdin (BV). IFP1.4 reporter has favorable excitation and emission characteristics, which has an absorption peak at 685 nm and an emission peak at 708 nm. Therefore, IFP1.4 reporter can be imaged deeply into the tissue with less contamination from tissue autofluorescence. However, the excitation light "leakage" through optical filters can limit detection and sensitivity of IFP1.4 reporter due to the small Stoke's shift of IFP1.4 fluorescence. To minimize the excitation light leakage, an intensified CCD (ICCD) based infrared fluorescence imaging device was optimized using two band pass filters separated by a focus lens to increase the optical density at the excitation wavelength. In this study, a mouse model (DBA/J2) was first injected with B. anthracis bacteria expressing IFP1.4, 150 μl s.c, on the ventral side of the left thigh. Then mouse was given 250 μl of a lmM BV solution via I.V. injection. Imaging was conducted as a function of time after infection under light euthanasia, excised tissues were imaged and IFP1.4 fluorescence correlated with standard culture measurements of colony forming units (CFU). The work demonstrates the use of BFP1.4 as a reporter of bacterial utilization of host heme and may provide an important tool for understanding the pathogenesis of bacterial infection and developing new anti-bacterial therapeutics.
机译：炭疽芽孢杆菌是一种革兰氏阳性，可形成孢子的细菌，它与所有病原菌一样，都通过从其宿主中隔离血红素而存活下来。为了在体内成像炭疽杆菌血红素分解代谢，我们稳定地转染了需要血红素分解代谢产物biliverdin（BV）的新的红色可激发荧光蛋白IFP1.4。 IFP1.4报告分子具有良好的激发和发射特性，在685 nm处有一个吸收峰，在708 nm处有一个发射峰。因此，IFP1.4报告基因可以在组织内深入成像，而组织自发荧光的污染较少。但是，由于IFP1.4荧光的斯托克位移较小，因此通过滤光片的激发光“泄漏”会限制IFP1.4报告分子的检测和灵敏度。为了使激发光泄漏最小化，使用了两个聚焦透镜分开的带通滤光片来优化基于增强CCD（ICCD）的红外荧光成像设备，以增加激发波长处的光密度。在这项研究中，首先在左大腿腹侧向小鼠模型（DBA / J2）注射表达IFP1.4的炭疽芽孢杆菌细菌（150μls.c）。然后，通过静脉注射给小鼠250μllmM BV溶液。注射。轻度安乐死感染后随时间变化进行成像，对切除的组织进行成像，并将IFP1.4荧光与菌落形成单位（CFU）的标准培养物测量值相关。这项工作证明了BFP1.4作为宿主血红素细菌利用情况的报道者的用途，并可能为理解细菌感染的发病机理和开发新的抗菌疗法提供重要的工具。

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