非侵入性 体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植
Summary
提出了一种利用生物发光标记细菌对肠出血性大肠杆菌 (大肠杆菌) 进行殖民化的小鼠模型的详细协议。在活体动物中, 通过非侵入性的体内成像系统来检测这些生物发光细菌, 可以提高我们目前对大肠杆菌定植的认识。
Abstract
肠出血性大肠杆菌 (肠出血性大肠杆菌) O157:H7, 是 causesdiarrhea、出血性结肠炎 (HS) 和溶血尿毒症综合征 (溶血尿毒症) 的食源病原体, 可殖民到人类肠道。为了研究肠内大肠杆菌定植的详细机制在体内,必须有动物模型来监测和量化大肠杆菌的定植。通过将生物发光表达质粒转化为肠出血性大肠杆菌来监测和定量活寄主中大肠杆菌的定植, 我们在这里展示了一种小鼠大肠杆菌的定植模型。用生物发光标记的大肠杆菌接种的动物, 通过非侵入性的体内成像系统检测, 显示了小鼠强烈的生物发光信号。感染后1和2天后, 生物发光信号仍可在受感染的动物身上检测到, 这表明在寄主中至少有2天发生了大肠杆菌的殖民。我们还表明, 这些发光的肠外大肠杆菌定位到小鼠肠道, 特别是在盲肠和结肠, 从前体图像。这种鼠肠大肠杆菌定植模型可以作为一种工具, 以提高目前的知识, 肠出血性大肠杆菌的殖民机制。
Introduction
大肠杆菌 O157:H7 是一种病原体, 引起腹泻1, HS2, 溶血症3, 甚至急性肾功能衰竭4通过污染的水或食物。肠出血性大肠杆菌是一个致病性 enterobacterium 和殖民的胃肠道的人1。当出血性大肠杆菌第一次坚持宿主肠道上皮, 他们通过 III. 型分泌系统 (T3SS) 将定植因子注入宿主细胞, 该类型作为分子注射器诱导附加和谦逊 (a/E) 病变随后执行附着力 (殖民化)5。enterocyte 避嫌 (李) 致病性岛5的轨迹编码了这些涉及 A/E 病变形成的基因。
生物发光是一种光产生化学反应, 荧光素酶催化其基底荧光素生成可见光 6.这种酶法通常要求存在氧或三磷酸腺苷 (ATP)6。生物发光成像 (BLI) 允许研究人员的可视化和量化的寄主病原体相互作用的活动物7。BLI 可以通过以下生物发光细菌来表征活体动物的细菌感染周期, 它们迁移到并侵入不同的组织7;这揭示了感染的动态发展。此外, 动物的细菌负荷与生物发光信号8有关;因此, 简单直接地估计实验动物的病理状况是一种方便的指标。
这里使用的质粒包含荧光素酶操纵, luxCDABE, 这是从细菌Photorhabdus luminescens编码其自己的荧光素酶基板7,9。通过将这种荧光素酶表达质粒转化为细菌, 可以通过观察活动物中的这些生物发光细菌来监测其定植和感染过程。总体而言, BLI 和生物发光标记的细菌允许研究人员监测细菌数量和位置, 细菌的生存能力与抗生素/治疗治疗, 和细菌基因表达感染/殖民化6,7. 报告了许多致病细菌, 表达了luxCDABE操纵检查其感染周期和/或感染中的基因表达。这些细菌, 包括致肾盂肾炎大肠杆菌 10, 出血性大肠杆菌8, 11, 12, 13,致病大肠杆菌 (EPEC)8 , 柠檬酸杆菌rodentium14,15, 沙门氏菌伤寒16, 李斯特氏李斯特氏增生17, 耶尔森氏菌小肠结肠炎 18, 19,和霍乱弧菌20已被记录在案。
已开发了几个实验模型, 以促进研究肠大肠杆菌的殖民化在体外和体内21,22,23。然而, 缺乏适当的动物模型来研究肠内大肠杆菌的定植在体内, 从而导致缺乏细节。为促进对肠内大肠杆菌定植机制的研究,在体内, 建立动物模型来观察和量化活体动物的肠外培养是一种无创的方法是很有价值的。
这篇手稿描述了一个老鼠大肠杆菌的殖民模型, 使用一个生物发光表达系统来监测大肠杆菌在活寄主时间的殖民化。小鼠灌接种生物发光标记的大肠杆菌, 并在小鼠身上检测到无侵入性的体内成像系统13中的发光信号。感染了生物发光标记的大肠杆菌病的小鼠在2天后的肠道感染后显示出明显的生物发光信号, 这表明这些细菌在感染后2天后在宿主肠道内被殖民。前体图像数据表明, 这种殖民化在小鼠的盲肠和结肠中特别存在。利用该模型, 通过体内成像系统, 在活体宿主中检测出生物发光大肠杆菌的定植, 研究肠道细菌定植的详细机制, 从而促进对大肠杆菌诱发的生理和病理改变。
Protocol
注意: 根据疾病控制和预防中心 (CDC) 生物安全指导 (https://www.cdc.gov/), 大肠杆菌 O157:H7 是生物安全等级 2 (BSL-2) 病原体。因此, 所有涉及肠出血性大肠杆菌的实验程序都必须在 BSL-2 设施中进行。在进行实验时, 要穿上实验室的大衣和手套。在认证的生物安全柜 (BSC) 工作。对实验台进行70% 乙醇消毒实验。所有接触 (或潜在接触) 肠出血性大肠杆菌的仪器或设备应使用70% 乙醇或漂白剂进行消毒。污染 (?…
Representative Results
我们通过口服饲对6周大的雌性 C57BL/6 小鼠进行生物发光标记的肠内毒素 (~ 109细菌细胞) 的管理。在1小时内对小鼠进行口服大肠杆菌疫苗接种后, 通过体内成像系统检查动物的荧光信号, 如图 7所示。结果表明, 饲小鼠具有生物发光标记的大肠杆菌的强烈发光信号。我们检查了2天后感染的信号。如图 8A所示, 接种了?…
Discussion
据报道, 用荧光素酶质粒转化出的肠出血性大肠杆菌已被用于检查其在宿主或基因表达式中的定位在体内8,11,12。在这里展示的小鼠模型也被报道来检测在小鼠宿主8中的肠大肠杆菌的殖民时间和定位。然而, 我们提供了如何管理肠内大肠杆菌疫苗接种小鼠灌的详细协议, 以及如何精心准备口腔饲的生物发?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们从医学研究部、美梅医疗中心 (台湾台南) 等地认识到, 帮助小鼠感染, 以及国立诚武大学实验动物中心的支持。这项工作得到了科技部长 (多数) 赠款 (多数 104-2321-006-019、105-2321-b-006-011、and106-2321-B-006-005) 的支持。
Materials
| Shaker incubator | YIH DER | LM-570R | bacteria incubation |
| Orbital shaking incubator | FIRSTEK | S300 | bacteria incubation |
| pBSL180 | source of nptII gene | ||
| pAKlux2 | source of luxCDABE operon | ||
| T&A Cloning Kit | Yeastern Biotech | FYC001-20P | use for TA cloning |
| Nsi I | NEB | R0127S | use for plasmid cloning |
| Sca I | NEB | R0122S | use for plasmid cloning |
| Spe I-HF | NEB | R0133S | use for plasmid cloning |
| Sma I | NEB | R0141S | use for plasmid cloning |
| T4 ligase | NEB | M0202S | use for plasmid cloning |
| Ex Taq | TaKaRa | RR001A | use for PCR amplification |
| 10X Ex Taq Buffer | TaKaRa | RR001A | use for PCR amplification |
| dNTP Mixture | TaKaRa | RR001A | use for PCR amplification |
| PCR machine | applied Biosystem | 2720 thermal cycler | for PCR amplification |
| Glycerol | SIGMA | G5516-1L | use for bacteria stocking solution |
| NaCl | Sigma | 31434-5KG-R | chemical for making LB medium, 10 g/L |
| Tryptone | CONDA pronadisa | Cat 1612.00 | chemical for making LB medium, 10 g/L |
| Yeast Extract powder | Affymetrix | 23547-1 KG | chemical for making LB medium, 5 g/L |
| Agar | CONDA pronadisa | Cat 1802.00 | chemical for making LB agar |
| kanamycin | Sigma | K4000-5G | antibiotics, use for seleciton |
| streptomycin | Sigma | S6501-100G | antibiotics, eliminate the microbiota in mice |
| EDL933 competent cell | Homemade | method is on supplemental document | |
| Electroporator | MicroPulser | for electroporation | |
| Electroporation Cuvettes | Gene Pulser/MicroPulser | 1652086 | for electroporation |
| High-speed centrifuge | Beckman Coulter | Avanti, J-26S XP | use for centrifuging bacteria |
| Fixed-Angle Rotor | Beckman Coulter | JA25.5 | use for centrifuging bacteria |
| Fixed-Angle Rotor | Beckman Coulter | JLA10.5 | use for centrifuging bacteria |
| centrifuge bottles | Beckman Coulter | REF357003 | use for centrifuging bacteria |
| centrifuge bottles | Thermo Fisher scientific | 3141-0500 | use for centrifuging bacteria |
| eppendorf biophotometer plus | eppendorf | AG 22331 hamburg | for measuring the OD600 value of bacteria |
| C57BL/6 mice | Laboratory Animal Center of NCKU | ||
| lab coat, gloves | for personnel protection | ||
| isoflurane | Panion & BF Biotech Inc. | G-8669 | for mice anesthesia, pharmaceutical grade |
| 1ml syringe | use for oral gavage of mice | ||
| Reusable 22 G ball-tipped feeding needle | φ0.9 mm X L 50 mm | use for oral gavage of mice | |
| surgical scissors | use for mice experiment | ||
| Xenogen IVIS 200 imaging system | Perkin Elmer | IVIS spectrum | use for bioluminescent image capture |
| Living Image Software | Perkin Elmer | version 4.1 | use for quantifying the image data |
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Cite This Article
Kuo, C., Wang, S., Chen, C. Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System. J. Vis. Exp. (134), e56169, doi:10.3791/56169 (2018).