铜绿假单胞菌产生铁载体的定性和定量分析
Summary
该方案提供来自 铜绿假单胞菌的总铁载体，pyoverdine和脓霉素的定性和定量分析。
Abstract
铜绿假单胞 菌（铜绿假单胞菌）以其产生多种毒力因子以在宿主中建立感染而闻名。其中一种机制是通过铁载体的产生来清除铁。 铜绿假单 胞菌产生两种不同的铁载体：铁螯合亲和力较低的脓脓苷和铁螯合亲和力较高的脓苷。本报告表明，吡啶可以直接从细菌上清液中定量，而脓皮单在定量前需要从上清液中提取。
定性分析铁载体产生的主要方法是蓝铝磺酸铬 （CAS） 琼脂平板测定。在该测定中，Fe3+-染料复合物中释放的 CAS 染料导致颜色从蓝色变为橙色，表明铁载体的产生。为了定量总铁载体，将细菌上清液与微量滴定板中的CAS染料等比例混合，然后在630nm处进行分光光度分析。通过将Pyoverdine与50 mM Tris-HCl等比例混合，直接从细菌上清液中定量，然后进行分光光度法分析。380 nm处的峰证实了pyoverdine的存在。至于Pyochelin，不可能从细菌上清液中直接定量，因此必须首先提取。随后的分光光度分析显示存在 pyochelin，峰值为 313 nm。
Introduction
生物体需要铁来执行各种重要功能，例如电子传输和 DNA 复制1。 铜绿假单胞菌是一种革兰氏阴性机会性病原体，已知具有多种毒力因子在宿主中建立感染，其中一种机制是铁载体形成2。在缺铁条件下， 铜绿假单 胞菌会释放出称为铁载体的特殊分子，这些分子会从周围环境中淬灭铁。铁载体在细胞外螯合铁，产生的铁载体复合物被主动转运回细胞3。
已知铜绿假单胞菌产生两种铁载体，pyoverdine 和 pyochelin。已知 Pyoverdine 具有较高的铁螯合亲和力 （1：1），而已知 pyochelin 具有较低的铁螯合亲和力 （2：1）4。脓蘑菇素也被称为次级铁载体，因为它具有较低的铁螯合亲和力5。铜绿假单胞菌 6 中铁载体的产生和调节受到群体感应 （QS） 系统的主动控制。
除铁淬火外，铁载体还参与调节毒力因子，并在生物膜形成中发挥积极作用7。铁载体还发挥着其他关键作用，包括参与细胞信号传导、防御氧化应激和促进微生物群落之间的相互作用8.铁载体通常根据它们螯合铁的特定官能团进行分类。该分类中的三种主要双酸配体是儿茶酚酸盐、羟肟酸盐和α-羟基羧酸盐3。Pyoverdines 是荧光假单胞菌属物种的标志，例如铜绿假单胞菌和荧光假单胞菌 5。它们由与含有 6-12 个氨基酸的寡肽偶联的混合绿色荧光发色团组成。几种非核糖体肽合成酶 （NRP） 参与其合成9.参与吡啶产生和调控的四个基因是 pvdL、pvdI、pvdJ 和 pvdD10。Pyoverdine 还负责哺乳动物的感染和毒力11.铜绿假单胞菌在中度限铁条件下产生脓蘑菇素，而吡脓草定在严格的限铁环境中产生12。参与 pyochelin 生产的两种操纵子是 pchDCBA 和 pchEFGHI13。值得注意的是，在碧蓝蛋白存在下，脓脓苷（儿茶酚酸酯）诱导氧化损伤和炎症，并产生羟基自由基，其对宿主组织有害11。
与微生物测定相比，蓝铝磺酸铬 （CAS） 测定因其全面、高灵敏度和更大的便利性而被广泛采用，微生物测定虽然灵敏，但可能过于特异性14.CAS测定可以在琼脂表面或溶液中进行。它依赖于当三价铁离子从其强烈的蓝色络合物转变为橙色时发生的颜色变化。CAS 比色法定量了 Fe-CAS 表面活性剂三元络合物中铁的消耗。这种特殊的络合物由金属、有机染料和表面活性剂组成，呈蓝色，并在 630 nm 处表现出吸收峰。
本报告提出了一种定性检测铁载体产生的方法，其中可以检测琼脂平板上铁载体的产生。还提供了一种定量估计微量滴定板中总铁载体产生的方法，以及来自 铜绿假单胞菌的两种铁载体pyoverdine和脓脓明的检测和定量分析方法。
Protocol
Representative Results
Discussion
该方案使研究人员能够从细菌游离上清液中定量铜绿假单胞菌的总铁载体和两种不同的铁载体，即pyoverdine和pyochelin。在CAS琼脂平板测定中，CAS染料和Fe3+离子形成络合物。当细菌产生铁载体时，它们会淬灭 CAS-Fe3+ 复合物中的 Fe3+ 离子，导致细菌生长周围的颜色发生变化。这种变化导致细菌生长周围出现明显的橙色光晕14,15…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢 DBT（生物技术教学计划）、DBT（BUILDER 计划）和 FIST 的资助。MR 感谢 SHODH 的奖学金。惠普感谢CSIR的奖学金。
Materials
| Agar Agar, Type I | HIMEDIA | GRM666 | |
| 8-Hydroxyquinoline | Loba Chemie | 4151 | |
| Casamino Acid | SRL Chemicals | 68806 | |
| Cetyltrimethyl Ammonium Bromide (CTAB) | HIMEDIA | RM4867-100G | |
| Chloroform | Merck | 1070242521 | |
| Chrome azurol sulfonate | HIMEDIA | RM336-10G | |
| Citric acid | Merck | 100241 | |
| Dextrose monohydrate | Merck | 108342 | |
| Dichloromethane | Merck | 107020 | |
| Ferric chloride hexahydrate | HIMEDIA | GRM6353 | |
| Glass Flasks | Borosil | 5100021 | |
| Glass Test-tubes | Borosil | 9820U05 | |
| Hydrochloric acid | SDFCL | 20125 | |
| King's medium B base | HIMEDIA | M1544-500G | |
| M9 Minimal Medium Salts | HIMEDIA | G013-500G | |
| Magnesium Sulphate | Qualigens | 10034 | |
| MultiskanGO UV Spectrophotometer | Thermo Scientific | 51119200 | |
| Peptone Type I, Bacteriological | HIMEDIA | RM667-500G | |
| PIPES free acid | MP Biomedicals | 190257 | |
| Potassium dihydrogen phosphate | Merck | 1048731000 | |
| Proteose peptone | HIMEDIA | RM005-500G | |
| Shimadzu UV-Vis Spectrophotometer | Shimadzu | 2072310058 | |
| Sigma Laborzentrifuge | Sigma-Aldrich | 3-18K | |
| Sodium chloride | Qualigens | 15915 |
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