Vitronectin 在细菌黏附和血清耐药性中作用的实验研究
Summary
本报告描述了用于表征细菌外膜蛋白与人体补体调节器 vitronectin 之间相互作用的协议。该协议可用于研究 vitronectin 在任何细菌物种中的结合反应和生物学功能。
Abstract
细菌利用补充调节器作为逃避宿主免疫反应的手段。在这里, 我们描述了评估在细菌细胞表面 vitronectin 获取的作用的协议, 以抵抗宿主免疫系统。流式细胞术实验确定人血浆 vitronectin 作为一种配体的细菌受体外膜蛋白 H 型流感嗜血杆菌 f。采用酶联免疫吸附法对纯化的重组蛋白 H 与 vitronectin 蛋白相互作用进行了表征, 并利用生物层干涉方法评估了结合亲和性。使用正常和 vitronectin 耗尽的人血清进行血清耐药性测定, 证实了 vitronectin 与蛋白 H 在细菌细胞表面的结合对宿主免疫应答的生物学重要性。采用无 vitronectin 涂层的玻片和革兰染色, 分析了 vitronectin 在细菌粘附中的重要性。最后, 对人肺泡上皮细胞膜的细菌黏附性进行了研究。这里描述的协议可以很容易地适应对任何细菌物种感兴趣的研究。
Introduction
Vitronectin (Vn) 是一种重要的人类糖蛋白, 通过调节纤溶系统来维持稳态。在 C5b6-7 复合形成和 C9 聚合过程中, Vn 还通过抑制末端补道来发挥补充调节作用。一些细菌病原体已被证明要招募 Vn 到细胞表面作为抵抗补体沉积1,2,3的手段。此外, Vn 在细菌和宿主上皮细胞受体之间充当 “三明治” 分子, 从而促进病原体的附着和内部化2,4,5。Vn 与细菌细胞表面的结合是由其他目前未识别的蛋白质介导的。因此, 充分阐明 vn 结合在规避软管免疫反应方面的功能作用, 将需要鉴定 vn 的蛋白质。
确定 vn 结合蛋白的最初步骤是测试是否有兴趣的病原体可以结合纯化的 vn. 流式细胞术是一种简便而直接的方法, 用于确定 vn 是否与病原细胞结合。在本研究中, 我们评估了 Vn 与不同类型的流感嗜血杆菌 (Hif) 临床分离6的结合。本文描述的方法是定量的, 可用于区分各种细菌菌株的结合能力。在以前的研究中, 我们将 Hif 的蛋白质 H (PH 值) 描述为 Vn 结合蛋白7。因此, 在本研究中, 用所述的方法比较了野生型 (WT) Hif 和 Hif M10Δlph突变体的 Vn 结合电位。
一旦确定病原体结合 Vn, 第二步是表征表面蛋白质组, 以确定潜在的 Vn 结合蛋白。可以使用多种方法来实现8、9, 但本报告没有介绍这些方法。最适合检测蛋白质-蛋白质相互作用的方法是在大肠杆菌中制备表达选择的细菌表面蛋白, 并通过亲和层析纯化。在这里, 我们使用了 PH 及其分子相互作用与 Vn 来说明该方法。重组 PH 和 Vn 之间的相互作用采用酶联免疫吸附试验 (ELISA)7和最近开发的无标签技术 (称为生物层干涉法 (BLI)10,11) 进行了表征。虽然 ELISAs 可用于确认蛋白质-蛋白质相互作用, 但 BLI 提供有关相互作用的动力学参数的详细数据。
为了研究 Vn 在细菌粘附中的功能作用, 可以使用两种不同的检测法。这里描述的第一种检测方法是直接测量对 Vn 涂层玻璃表面的细菌附着, 而第二种检测则检查附着在上皮细胞表面。对于第一种检测方法, 玻璃玻片涂有 Vn, 用革兰染色和显微显微镜评估了 WT 或突变 Hif 菌株的结合度。这种技术可以根据结合 Vn12的能力, 轻松区分细菌。然后, 通过在 II. 型肺泡上皮细胞的单层添加培养细菌, 对哺乳动物细胞的细菌黏附性进行分析;通过计数菌落形成单位 (CFUs) 的数量来评估细菌附着。附着和内化细菌可在 Vn4、13的存在或缺失时加以区分。
利用血清杀灭试验 (即血清杀菌活性) 评价了 Vn 在细菌性血清耐药性中的作用。为了评价 vn 在血清耐药性中的意义, 与正常人血清 (NHS) 的杀菌活性进行了比较。该方法可以很容易地区分 Vn 结合与非结合细菌的血清耐药性。我们用这种方法研究了 Vn 在几种细菌病原体的耐药性中的作用4,12。
许多方法已经报告了研究寄主病原体的相互作用。在这里, 我们描述了一套协议, 可以很容易地适应任何病原体的研究, 以评估 Vn 在发病机制中的作用。我们使用各种病原体测试了这些协议, 并选择了 Hif 作为本报告的示例。
Protocol
Representative Results
Discussion
细菌病原体将 Vn 招募到细胞表面, 并利用此补充调节器防止补体因子的沉积和膜攻击复杂2的完成。Vn 还充当细菌表面蛋白和宿主细胞表面受体之间的桥梁分子, 从而使病原体能够附着在上皮细胞表面并随后调解内部化。在本研究中, 我们描述了可用于估计 i 的协议: vn 与细菌细胞表面的结合, ii) 蛋白质-蛋白质相互作用和亲和常数, iii. vn 在提供血清耐药性和增强的作用附着在宿主?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了来自安娜和埃德温-伯杰基金会、Hierta、气流和 Edla 约翰逊基金会、瑞典医学研究理事会 (赠款号 K2015-57X-03163-43-4、www.vr.se)、大学癌症基金会的赠款的支持。在马尔默的医院, 地形协会 (Forssman 基金会), 和斯科讷郡议会的研究和发展基金会。
Materials
| 1.5 mL thermomixter | Eppendorf | 5355 | dry block heating and cooling shaker |
| 5 mL polystyrene round-bottom tube | BD Falcon | 60819-138 | 12 × 75 mm style |
| 5% CO2 supplied incubator | Thermo Scientific | BBD6220 | |
| 6 mL polystyrene round-bottom tube | VWR | 89000-478 | 12 × 75 mm style with cap |
| 24-well plates | BD Falcon | 08-772-1H | Cell culture grade |
| 30% Hydrogen peroxide (H2O2) solution | Sigma-Aldrich | H1009-100ML | Laboratory analysis grade |
| 75 cm2 tissue culture flask | BD Falcon | BD353136 | Vented |
| 96 well black flat bottom plate | Greiner Bio-One | 655090 | Tissue culture treated µClear black plates |
| A549 Cell Line human | Sigma-Aldrich | 86012804-1VL | |
| Cell detachment enzyme (Accutase) | Sigma-Aldrich | A6964-500ML | Cell Culture Grade |
| AR2G sensors | Pall Life Science | 18-5095 | Sensor to immobilized protein by amino coupling |
| Acetone | VWR | 97064-786 | Analysis grade |
| Bovine Serum Albumins (BSA) | Sigma-Aldrich | A2058 | Suitable for cell culture |
| Bibulous paper | VWR | 28511-007 | |
| Bio-layer interferometer | Pall Life Science | FB-50258 | Bilayer interferometry measuring equipment |
| Crystal violet solution | Sigma-Aldrich | HT90132-1L | |
| C4BP (C4b binding protein) | Complement Technology, Inc. | A109 | Bought as Frozen liquid form |
| Calcium chloride (CaCl2) | Sigma-Aldrich | C5670-500G | |
| Carbol-fuchsin solution | Sigma-Aldrich | HT8018-250ML | |
| Citric acid | Sigma-Aldrich | 251275-500G | American Chemical Society (ACS) grade |
| Decolorizing solution | Sigma-Aldrich | 75482-250ML-F | |
| E. coli host (E. Coli BL21) | Novagen | 69450-3 | Protein expression host |
| F12 medium | Sigma-Aldrich | D6421 | Cell Culture Grade |
| Flow cytometer | BD Biosciences | 651154 | Cell analysis grade for research applications |
| Fetal Calf Serum (FCS) | Sigma-Aldrich | 12003C | Suitable for cell culture |
| Normal human serum (NHS) | Complement Technology, Inc. | NHS | Pooled human serum |
| FITC-conjugated donkey anti-sheep antibodies | AbD Serotec | STAR88F | Polyclonal |
| Gentamicin | Sigma-Aldrich | G1397 | Cell culture grade |
| Glucose | Sigma-Aldrich | G8270-1KG | |
| Gelatin | Sigma-Aldrich | G9391 | Suitable for cell culture |
| Hemocytometer | Marienfeld | 640210 | |
| HRP-conjugated anti-His tag antibodies | Abcam | ab1269 | Polyclonal |
| Human factor H | Complement Technology, Inc. | A137 | Bought as Frozen liquid form |
| C4BP | Complement Technology, Inc. | A109 | Frozen solution |
| Human serum albumin | Sigma-Aldrich | A1653-10G | lyophilized powder |
| Histidine affinity resin column (HisTrap HP) | GE Health Care Life Science | 17-5247-01 | Columns prepacked with Ni Sepharose |
| His-tagged PH | Recombinantly expressed and purified in our lab | ||
| Iodine solution | Sigma-Aldrich | HT902-8FOZ | |
| Methanol | VWR | BDH1135-1LP | Analysis grade |
| Microscope | Olympus | IX73 | Inverted microscope |
| Microscope slides | Sigma-Aldrich | S8902 | plain, size 25 mm × 75 mm |
| Magnesium chloride (MgCl2) | Sigma-Aldrich | M8266-1KG | |
| Plasmid containg C terminal 6x His-tag on the backbone (pET26(b)) | Novagen | 69862-3 | DNA vector |
| Polysorb microtitre plates | Sigma-Aldrich | M9410 | For ELISA |
| Potassium hydroxide (KOH) | Sigma-Aldrich | 6009 | American Chemical Society (ACS) grade |
| Sheep anti-human Vn antibodies | AbD Serotec | AHP396 | Polyclonal |
| Shaker | Stuart Scientific | STR6 | Platform shaker |
| Tissue culture flask | BD Falcon | 3175167 | 75 cm2 |
| Thermomixer | Sigma-Aldrich | T3317 | Dry block heating and cooling shaker |
| Tetramethylbenzidine | Sigma-Aldrich | 860336-100MG | ELISA grade |
| Vitronectin (Vn) from human plasma | Sigma-Aldrich | V8379-50UG | cell culture grade |
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