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Immunologia e infezioni

内塞里亚淋病的金属有限生长，用于金属反应基因特征和从宿主配体中获取金属

Published: March 04, 2020
doi:
10.3791/60903
,
1Institute for Biomedical Sciences,Georgia State University

Summary

我们在这里描述了一种在金属受限液体介质中生长淋病的方法，以促进金属吸走基因的表达。我们还概述了下游实验，以表征在这些条件下生长的淋球菌的表型。这些方法可以适应其他细菌中金属反应基因的表征。

Abstract

微量金属，如铁和锌，是已知在原核过程（包括基因调控、催化和蛋白质结构）中起关键作用的重要营养物质。主机的金属封存通常会导致细菌的金属限制。这种限制诱导细菌基因表达，其蛋白质产品允许细菌克服其金属有限的环境。这种基因的特性具有挑战性。细菌必须在精心准备的培养基培养中生长，使营养金属能够充分获得，从而允许细菌生长，同时保持有利于表达上述基因的金属轮廓。因此，必须为这些金属的浓度建立微妙的平衡。生长一种营养挑剔的有机体，如奈瑟里亚淋病，这种有机体进化到只在人类宿主中生存，增加了一层复杂性。在这里，我们描述了一种定义的金属有限介质的制备，该介质足以允许淋球菌生长和所需的基因表达。这种方法允许调查员从不需要的来源中分离铁和锌，同时用定义的铁或锌来源补充介质，其制备也描述。最后，我们概述了利用这种介质帮助描述金属调节性淋球菌基因的蛋白质产物的三个实验。

Introduction

淋病导致常见的性传播感染淋病。在感染期间，致病性Neisseria表达一系列金属反应基因，使细菌能够克服人类宿主1、2、3的金属限制。铁和锌等微量金属在许多细胞过程中起着关键作用，如与催化位点的酶结合、参与氧化还原反应以及各种蛋白质4、5的结构因素。在金属有限条件下，金属反应位位被压减，其产生的蛋白质可以帮助获得这些营养物质。这些基因和蛋白质的特性对研究者来说是一个独特的技术挑战。金属离子必须从细菌中预扣，才能诱导这些基因从原生位点转录，但是这些离子从含金属介质中的有效结合可能难以优化。源水的不同金属轮廓和粉末成分固有的批次到批次变化6意味着从富介质中去除特定金属所需的夹子数量会因不同位置、成分供应商而异，甚至在更换化学品库存时，在单个实验室中随时间而变化。

为了规避这一挑战，我们描述了在制备过程中用Chelex-100树脂处理的已定义介质的制备，以去除溶液中的微量金属。这种介质营养密集，允许淋巴球菌的生长，这种生长在人类宿主之外很难培养，并且允许调查员通过添加自己定义的来源和浓度来引入特定的金属轮廓。金属。将所需金属控制添加到耗尽介质的方法提高了实验的一致性，并允许进行可靠、可复制的实验，而不考虑水源和化学批次数等因素。此外，这种介质可以部署为液体或固体，只需稍作修改，使其相当通用。

为了证明这种介质的效用，我们概述了用于淋球菌生长的协议，并描述了三个成功的实验来描述金属反应性奈塞利亚基因。首先，我们从金属耗尽或补充培养物制备淋球菌全细胞解结物，并展示来自金属响应位点的蛋白质生产水平。然后，我们概述锌限制生长测定，其中淋球菌生长通过补充特定的，可使用的锌源控制。最后，我们展示了结合测定，证明整个淋球细胞表达金属反应性表面受体结合到各自的含金属配体。这些受体的成功表面呈现需要金属耗尽介质的生长。

本方案是专门针对淋病的，但许多其他细菌病原体在感染7期间采用金属采集策略，因此该协议可以适用于研究其他细菌的金属平衡。优化此介质和用于其他细菌的这些实验方案可能需要对 Chelex-100 的金属包合剂浓度和/或处理时间进行轻微修改，因为其他细菌的金属要求可能与淋球菌略有不同。铁和锌是上述调查关注的主要金属，但其他金属（如锰）已被证明对细菌至关重要，包括Neisseria8、9、10、11、12。此外，还描述了真核细胞培养工作中金属表征的类似方法，也可以考虑。13

Protocol

1. Chelex 处理的定介质 （CDM） 库存解决方案的制备 库存解决方案 I 将 NaCl （233.8 g）、K2SO4 （40.0 g）、NH4Cl （8.8 g）、K2HPO4 （13.9 g） 和 KH2PO4 （10.9 g） 结合到去离子水中，最终体积为 1 L。 过滤溶液消毒，并等分成50 mL锥形管。 储存在-20°C。 库存解决方案 2 将硫胺 HCl （0.2 g）、硫胺热?…

Representative Results

在没有微量金属的情况下，开发和实施了一种用于对金属反应基因及其基因产物进行表征的特异性介质。在优化的协议中，介质的金属轮廓通过由调查人员酌情添加金属来控制，而不是通过金属目标的定位夹层来控制，从而增加了实验室到实验室的控制和一致性，并进行了实验实验。这种介质可用于液体和固态，使其在许多实验设置中具有通用性。 <p class="jove_co…

Discussion

生长介质在微生物研究中发挥着多种作用。专用介质用于选择、浓缩以及各种其他应用，用于许多独特的研究类型。其中一种应用是诱导金属响应基因，这通常是通过添加针对特定金属子的特异性包剂来完成的。这种方法是有限的，因为由于含有独特金属型材的不同水源和含有不同金属浓度的两批相同介质成分，各种微量金属所需的包合量可能是可变的。为了避免这种固有的缺?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了 NIH 授予 R01 AI125421、R01 AI127793 和 U19 AI144182 的支持。写作作者要感谢所有为验证和审查此方法做出贡献的实验室成员。

Materials

125 mL sidearm flasks Bellco 2578-S0030 Must be custom ordered
2-Mercaptoethanol VWR M131 Open in fume hood
3MM Paper GE Health 3030-6461 Called "filter paper" in text
Agarose Biolone BIO-41025 Powder
Ammonium chloride Sigma-Aldrich A9434 Powder
Biotin Sigma-Aldrich B4501 Powder
Blotting grade blocker Bio-Rad 170-6404 Nonfat dry milk
Bovine serum albumin Roche 3116964001 Powder
Bovine transferrin Sigma-Aldrich T1428 Powder
Calcium chloride dihydrate Sigma-Aldrich C5080 Powder
Calcium pantothenate Sigma-Aldrich C8731 Powder
Calprotectin N/A N/A We are supplied with this by a collaborator
Chelex-100 Resin Bio-Rad 142-2832 Wash with deionized water prior to use
Cotton-tipped sterile swab Puritan 25-806 Cotton is better than polyester for this application
Deferoxamine Sigma-Aldrich D9533 Powder
D-glucose Sigma-Aldrich G8270 Powder
Dialysis cassette Thermo 66380 Presoak in buffer prior to use
Dot blot apparatus Schleicher & Schwell 10484138 Lock down lid as tightly as possible before sample loading
Ethanol Koptec V1016 Flammable liquid, store in flammables cabinet
Ferric chloride Sigma-Aldrich F7134 Irritant, do not inhale
Ferric nitrate nonahydrate Sigma-Aldrich F1143 Irritant, do not inhale
GC medium base Difco 228950 Powder, already contains agar
Glycine Sigma-Aldrich G8898 Powder
HEPES Fisher L-15694 Powder
Human transferrin Sigma-Aldrich T2030 Powder
Hypoxanthine Sigma-Aldrich H9377 Powder
Klett colorimeter Manostat 37012-0000 Uses color transmission to assess culture density
L-alanine Sigma-Aldrich A7627 Powder
L-arginine Sigma-Aldrich A5006 Powder
L-asparagine monohydrate Sigma-Aldrich A8381 Powder
L-aspartate Sigma-Aldrich A9256 Powder
L-cysteine hydrochloride Sigma-Aldrich C1276 Powder
L-cystine Sigma-Aldrich C8755 Powder
L-glutamate Sigma-Aldrich G1251 Powder
L-glutamine Sigma-Aldrich G3126 Powder
L-histidine monohydrochloride Sigma-Aldrich H8125 Powder
L-isoleucine Sigma-Aldrich I2752 Powder
L-leucine Sigma-Aldrich L8000 Powder
L-lysine Sigma-Aldrich L5501 Powder
L-methionine Sigma-Aldrich M9625 Powder
L-phenylalanine Sigma-Aldrich P2126 Powder
L-proline Sigma-Aldrich P0380 Powder
L-serine Sigma-Aldrich S4500 Powder
L-threonine Sigma-Aldrich T8625 Powder
L-tryptophan Sigma-Aldrich T0254 Powder
L-tyrosine Sigma-Aldrich T3754 Powder
L-valine Sigma-Aldrich V0500 Powder
Magnesium sulfate Sigma-Aldrich M7506 Powder
Methanol VWR BDH1135-4LP Flammable liquid, store in flammables cabinet
Nitrocellulose GE Health 10600002 Keep in protective sheath until use
Potassium phosphate dibasic Sigma-Aldrich 60356 Powder
Potassium phosphate monobasic Sigma-Aldrich P9791 Powder
Potassium sulfate Sigma-Aldrich P0772 Powder
Potato starch Sigma-Aldrich S4251 Powder
Reduced glutathione Sigma-Aldrich G4251 Handle carefully. Can oxidize easily.
S100A7 N/A N/A We are supplied with this by a collaborator
Sodium bicarbonate Sigma-Aldrich S5761 Powder
Sodium chloride VWR 470302 Powder
Sodium citrate Fisher S279 Powder
Sodium hydroxide Acros Organics 383040010 Highly hygroscopic
Thiamine hydrochloride Sigma-Aldrich T4625 Powder
Thiamine pyrophosphate Sigma-Aldrich C8754 Also called cocarboxylase
TPEN Sigma-Aldrich P4413 Powder
Tris VWR 497 Powder
Uracil Sigma-Aldrich U0750 Powder
Zinc sulfte heptahydrate Sigma-Aldrich 204986 Irritant, do not inhale
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Tags

Metal-limited GrowthNeisseria GonorrhoeaeMetal-responsive GenesMetal AcquisitionHost LigandsNutritional ImmunityMetal Restricted ConditionsBacterial GrowthCDMKlett ColorimeterMetal Loaded ProteinsTransferrinDialysis96 Well MicroplateOptical Density
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Maurakis, S., Cornelissen, C. N. Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands. J. Vis. Exp. (157), e60903, doi:10.3791/60903 (2020).
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