金黄色葡萄球菌生长使用人血红蛋白作为铁源
Summary
在这里，我们描述一个生长实验<em>金黄色葡萄球菌</em使用血红蛋白作为有效养分铁的唯一来源。此法规定参与血红蛋白衍生的铁收购的细菌因素的作用。
Abstract
金黄色葡萄球菌是一种致病细菌，需要铁来进行重要的代谢功能，导致疾病的。储层人类宿主内的铁是最丰富的血红素，这是血红蛋白的辅因子。为了获得铁血红蛋白，S.金黄色葡萄球菌利用一个复杂的系统，被称为铁规管表面的行列式（ISD）系统1。 ISD系统首次绑定主机血红蛋白的组成部分，然后提取并导入血红素，和，终于解放血红素铁在细菌胞浆2,3。通过这条途径已解剖众多的体外研究4-9。此外，ISD系统感染的贡献已被反复证明在小鼠模型中8,10-14。建立血红蛋白衍生的铁收购和增长的贡献ISD系统已被证明是更具挑战性的。生长测定血红蛋白作为唯一的铁源是复杂的by是不稳定市售血红蛋白，在生长培养基中的游离铁污染，并与铁螯合剂毒性。在这里，我们提出了一种方法，该方法克服了这些限制。从新鲜血液中制备高品质的血红蛋白，并储存于液氮中。纯化血红蛋白补充铁消耗介质模仿铁所遇到的脊椎动物宿主病原体内的恶劣环境。饥饿S.黄色葡萄球菌的最小操纵形式的血红蛋白诱导生长的方式，结合血红蛋白，提取血红素，通过血红素通过细菌的细胞壁，并降低在细胞质中的血红素的能力完全依赖于铁和补充。此法将有助于研究人员寻找阐明机制的hemoglobin-/heme-derived铁收购S.金黄色葡萄球菌和其他可能的细菌病原体。
Protocol
1。从新鲜血液中的血红蛋白纯化收购辅以抗凝剂的人新鲜血液。血液在冰上或4℃下保持整个纯化过程。 20分钟，于1500×g离心血液。的红血细胞（红细胞）将在试管底部。小心地吸出上清液，并轻轻将沉淀重悬于冰冷的0.9％（重量/体积）NaCl溶液。重复离心和洗涤3次。 在1倍体积的冰冷却的10mM Tris-HCl（pH值8.0）中重悬沉淀。这将诱导的红细胞由于渗透压的裂解。添加甲苯〜20…
Representative Results
我们人体血红蛋白溶血纯化与HPLC（协议1.7步）。 图1显示了记录洗脱液在280和410 nm波长的吸光度。收集的级分5，以及其它馏分被丢弃。五到十五毫克每毫升洗脱液血红蛋白，产量的影响通常是获得。纯化血红蛋白，一式两份，并通过SDS-PAGE分析，凝胶或蛋白质的染色，或转移到硝酸纤维素和免疫印迹（议定书步骤1.10， 图2）。 我们已评估纯化的人血?…
Discussion
铁是人体必需的营养物质，生物王国的寿命15。在脊椎动物中，铁被隔离，以避免造成此元素的毒性。封存也掩盖铁入侵的微生物的过程被称为营养免疫16。对此，病原体已经发展策略，规避营养的免疫力。一种这样的机制依赖于血红蛋白，这是最丰富的铁源内的主机17。血红蛋白被包含在红血细胞。血红蛋白受损的红血细胞释放的约束的主机结合珠蛋白，这标志着快速去?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究是由美国公共卫生服务补助AI69233和AI073843从国家过敏和传染病研究所的支持。 EPS是一个巴勒斯惠康研究员，传染病的发病机制。 KPH是由细胞与分子微生物学培训补助计划5 T32 A107611-10。
Materials
| Name of Reagent/Material | Company | Catalogue Number | Comments |
| HPLC anion exchange column | Varian | PL1551-3802 | |
| Drabkin’s reagent | Sigma | D5941-6VL | |
| Hemoglobin standard | Pointe Scientific | H7506-STD | |
| RPMI | HyClone | SH30011.02 | |
| Chelex 100 sodium form | Sigma | C7901 | |
| EDDHA | LGC Standards GmbH | ANC 001 | |
| Hemoglobin a antibody | Santa Cruz Biotechnology, Inc | SC-21005 | |
| Tryptic soy agar | BD | 236920 |
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Citer Cet Article
Pishchany, G., Haley, K. P., Skaar, E. P. Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source. J. Vis. Exp. (72), e50072, doi:10.3791/50072 (2013).