抗菌肽定位的细菌 Spheroplasts 和原生质体的生产与可视化
Summary
在这里, 我们提出了一个生产革兰氏阴性大肠杆菌 (大肠杆菌) spheroplasts 和革兰氏阳性芽孢杆菌巨大(巨大) 原生质体的协议, 以清楚地形象化和快速表征多肽-细菌的相互作用。这为定义膜定位和转运肽提供了系统的方法。
Abstract
用共焦显微镜作为一种评估细菌内肽定位模式的方法, 通常被常规光学显微镜的分辨率限制所抑制。由于给定显微镜的分辨率不能很容易地增强, 我们提出了改变小棒状革兰氏阴性大肠杆菌 (大肠杆菌) 和革兰阳性芽孢杆菌巨大(巨大) 的协议 .成更大, 容易成像的球形形式称为 spheroplasts 或原生质体。这种转变使观察者能够迅速、清楚地确定肽是否进入细菌膜 (即膜定位) 或穿过细胞膜进入细胞 (即转运)。利用这种方法, 我们还提出了一种系统的方法来表征多肽作为膜定位或转运。这种方法可用于各种膜活性肽和细菌菌株, 我们通过观察 Buforin II P11A (BF2 P11A)、抗菌肽 (AMP) 与大肠杆菌的相互作用, 证明了本协议的实用性.spheroplasts 和B. 巨大原生质体。
Introduction
抗菌肽 (安培) 得到了重视, 因为他们的潜在用途作为替代传统抗生素1,2,3,4,5。安培杀死细菌的转运通过细胞膜, 并与细胞内的成分, 如核酸, 或通过 permeabilizing 膜导致细胞含量的泄漏6。除了用作抗生素外, 转运安培还可用于药物传递应用, 因为它们可以无中断地穿过不透水细胞膜7、8。因此, 我们寻求了解基本的 AMP 作用机制, 为其在药物设计中的应用打下基础。
共焦显微镜为评估细菌细胞中荧光标记安培的定位模式提供了一种方法, 提供了对其作用机制9、10、11、12的洞察力,13,14. 通过对细菌膜进行标记, 可以确定是否有荧光标记肽本地化到细胞膜或细菌细胞的细胞内空间。然而, 这种技术受细菌的小尺寸和杆型的限制, 由于常规光学显微镜的分辨率极限和15张幻灯片中细菌的变异方向, 使得成像具有挑战性。
所提出的方法的目的是通过共聚焦显微镜增强荧光标记肽定位模式的可视化。通过将小的、薄的、棒状的革兰阴性大肠杆菌 ( 大肠杆菌) 和革兰阳性芽孢杆菌巨大(B. 巨大) 细菌转化为扩大的球形形式, 将其增强, 称为spheroplasts (为革兰阴性菌株) 和原生质体 (为革兰阳性菌株)16,17,18,19,20,21。Spheroplasts 和原生质体更容易图像, 因为它们的大小和对称的形状, 这使得在幻灯片上的细菌的方向与其成像无关。此外, 我们提出了一个系统的方法来定量分析共焦显微数据, 以表征安培作为一种膜定位或转运。应用这些方法可以更容易地区分荧光标记的肽定位模式。此处提供的协议可用于评估除安培以外的各种膜活性剂的定位, 包括细胞穿透肽。
这种技术的一个明显的优点是, 它提供了对安培在单个细胞水平上的作用机制的洞察, 这可能揭示细胞对细胞的异质性15, 而不是其他常用的荧光化验方法来识别安培的作用机制, 仅提供容量估计9,22,23,24,25。使用 spheroplasts 和原生质体, 以评估 AMP 细胞进入是特别有用的26 , 因为它们比其他模型用于评估细胞进入, 如脂囊泡24的生理相关15 。
Protocol
1. 解决方案准备 注: 准备步骤1.1–1.9 和1.8–1.11 中描述的解决方案, 以便分别生产大肠杆菌spheroplasts 和巨大原生质体。 制备1米三氯, pH 7.8 通过溶解10.34 克三盐酸和 4.17 g 的三 OH 在50毫升的 dH 2O 在125毫升瓶。通过25毫米注射器过滤器进行消毒, 用0.2 µm 膜, 在室温下贮存在锥形管中。 20毫米氯化镁2, 0.7 米蔗糖, 10 毫米三氯, pH 7.8), 通过…
Representative Results
通过扩大细菌和使它们球状, 我们可以很容易地区分是否多肽定位到细菌膜或容易植物常常将跨细菌膜。常规光学显微镜的分辨率限制使它具有挑战性, 以区分是否在正常细菌的细胞膜或细胞内空间出现多肽信号, 因为在膜上定位的信号似乎会与细胞内空间 (图 3A)。相比之下, spheroplasts (2–5µm) 和原生质体 (2–3µm) 的扩大大小与正常的细菌相比, 通常只…
Discussion
这里提出的协议使研究人员能够更快地获得更大的细菌图像样本量, 因为扩大的球形细菌更容易定位、定位和图像。这种提高收集数据的能力在几个方面是有价值的。首先, 它能够对多肽定位模式进行更系统的定量分析。虽然从较小的图像集可以证明质量趋势, 但只有大量高质量图像的样本集显示了本地化模式中更微妙的趋势, 如肽 translocates 与本地化的细胞百分比膜。此外, 能够更好地解决内部荧?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
研究得到了国家过敏和传染性疾病研究所 (NIH-NIAID) 奖 R15AI079685 的支持。
Materials
| Trizma hydrocloride (Tris HCl) | Sigma | T3253 | |
| Trizma base (Tris OH) | Sigma | T1503 | |
| Magnesium chloride | Sigma | M8266 | |
| Sucrose | Sigma | S7903 | |
| Lysozyme | Sigma | L6876 | |
| Deoxyribonuclease I | Sigma | D4527 | |
| Ethylenediaminetetraacetic acid | Sigma | 106361 | Used Sigma 106361 in original protocol development; 106361 discontinued with ED2SS as replacement |
| Cephalexin hydrate | Sigma | C4895 | |
| Ampicillin | Fisher Scientific | BP1760 | |
| BBL Trypticase soy broth | Fisher Scientific | B11768 | |
| BF2 P11A FITC | NeoScientific | Custom ordered | |
| di-8-ANEPPS | Biotium | 61012 | |
| DMSO | Sigma | 34869 | Used Sigma D8779 in original protocol development; D8779 discontinued with 34869 as replacement |
| Maleic acid | Sigma | M0375 | |
| Acrodisc 25 mm Syringe Filter w/ 0.2 μm HT Tuffryn Membrane | Pall Corporation | 4192 | |
| Laser scanning confocal microscope | Leica Microsystems | TCS SP5 II | For image acquisition |
| Leica Application Suite, Advanced Fluorescence | Leica Microsystems | For image processing |
References
- Baltzer, S. A., Brown, M. H. Antimicrobial peptides: promising alternatives to conventional antibiotics. Journal of Molecular Microbiology Biotechnology. 20 (4), 228-235 (2011).
- Hancock, R. E., Sahl, H. G. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nature Biotechnology. 24 (12), 1551-1557 (2006).
- Jenssen, H., Hamill, P., Hancock, R. E. Peptide antimicrobial agents. Clinical Microbiology Reviews. 19 (3), 491-511 (2006).
- Toke, O. Antimicrobial peptides: new candidates in the fight against bacterial infections. Biopolymers. 80 (6), 717-735 (2005).
- Wang, G., et al. Antimicrobial peptides in 2014. Pharmaceuticals. 8 (1), 123-150 (2015).
- Epand, R. M., Vogel, H. J. Diversity of antimicrobial peptides and their mechanisms of action. Biochim Biophys Acta. 1462, 11-28 (1999).
- Drin, G., Rousselle, C., Scherrmann, J. -. M., Rees, A. R., Temsamani, J. Peptide Delivery to the Brain via Adsorptive-Mediated Endocytosis: Advances With SynB Vectors. AAPS PharmSciTech. 4 (4), 61-67 (2002).
- Splith, K., Neundorf, I. Antimicrobial peptides with cell-penetrating peptide properties and vice versa. European Biophysics Journal. 40 (4), 387-397 (2011).
- Bustillo, M. E., et al. Modular analysis of hipposin, a histone-derived antimicrobial peptide consisting of membrane translocating and membrane permeabilizing fragments. Biochim Biophys Acta. 1838 (9), 2228-2233 (2014).
- Koo, Y. S., et al. Structure-activity relations of parasin I, a histone H2A-derived antimicrobial peptide. Peptides. 29 (7), 1102-1108 (2008).
- Libardo, M. D., Cervantes, J. L., Salazar, J. C., Angeles-Boza, A. M. Improved bioactivity of antimicrobial peptides by addition of amino-terminal copper and nickel (ATCUN) binding motifs. ChemMedChem. 9 (8), 1892-1901 (2014).
- Park, C. B., Kim, H. S., Kim, S. C. Mechanism of Action of the Antimicrobial Peptide Buforin II: Buforin II Kills Microorganisms by Penetrating the Cell Membrane and Inhibiting Cellular Functions. Biochemical and Biophysical Research Communications. , 253-257 (1998).
- Park, C. B., Yi, K. -. S., Matsuzaki, K., Kim, M. S., Kim, S. C. Structure-activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: The proline hinge is responsible for the cell-penetrating ability of buforin II. Proceedings of the National Academy of Sciences of the United States of America. 97 (15), 8245-8250 (2000).
- Pavia, K. E., Spinella, S. A., Elmore, D. E. Novel histone-derived antimicrobial peptides use different antimicrobial mechanisms. Biochim Biophys Acta. 1818 (3), 869-876 (2012).
- Wei, L., LaBouyer, M. A., Darling, L. E., Elmore, D. E. Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides. Antimicrobial Agents and Chemotherapy. 60 (10), 6350-6352 (2016).
- Chassy, B. M., Giuffrida, A. Method for the Lysis of Gram-Positive, Asporogenous Bacteria with Lysozyme. Appl. Environ. Microbiol. 39 (1), 153-158 (1980).
- Fitz-James, P. C. Cytological and Chemical Studies of the Browth of Protoplasts of Bacillus megaterium. J. Biophysic. and Biochem. Cytol. 4 (3), 257-266 (1958).
- Martinac, B., Buechner, M., Delcour, A. H., Adler, J., Kung, C. Pressure-sensitive ion channel in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America. 84, 2297-2301 (1986).
- Martinac, B., Rohde, P. R., Cranfield, C. G., Nomura, T. Patch clamp electrophysiology for the study of bacterial ion channels in giant spheroplasts of E. coli. Methods Mol Biol. 966, 367-380 (2013).
- Nadeau, J. L. . Introduction to Experimental Biophysics: Biological Methods for Physical Scientists. , (2016).
- Sun, Y., Sun, T. L., Huang, H. W. Physical properties of Escherichia coli spheroplast membranes. Biophysical Journal. 107 (9), 2082-2090 (2014).
- Branco, P., Viana, T., Albergaria, H., Arneborg, N. Antimicrobial peptides (AMPs) produced by Saccharomyces cerevisiae induce alterations in the intracellular pH, membrane permeability and culturability of Hanseniaspora guilliermondii cells. Int J Food Microbiol. 205, 112-118 (2015).
- Kobayashi, S., et al. Membrane Translocation Mechanism of the Antimicrobial Peptide Buforin 2. Biochimie. 43 (49), 15610-15616 (2004).
- Spinella, S. A., Nelson, R. B., Elmore, D. E. Measuring peptide translocation into large unilamellar vesicles. J Vis Exp. (59), e3571 (2012).
- van der Kraan, M. I., et al. Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin. Peptides. 25 (2), 177-183 (2004).
- Sun, Y., Sun, T. L., Huang, H. W. Patch clamp electrophysiology for the study of bacterial ion channels in giant spheroplasts of E. coli. Biophys J. 111 (1), 132-139 (2016).
- Xie, Y., Fleming, E., Chen, J. L., Elmore, D. E. Effect of proline position on the antimicrobial mechanism of buforin II. Peptides. 32 (4), 677-682 (2011).
- Kobayashi, S., Takeshima, K., Park, C. B., Kim, S. C., Matsuzaki, K. Interactions of the Novel Antimicrobial Peptide Buforin 2 with Lipid Bilayers: Proline as a Translocation Promoting Factor. Biochem. 39 (29), 8648-8654 (2000).
- Decad, G. M., Nikaido, H. Outer Membrane of Gram-Negative Bacteria XII. Molecular-Sieving Function of Cell Wall. J. Bacteriol. 128 (1), 325-336 (1976).
- Choi, H., Yang, Z., Weisshaar, J. C. Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15. Proc Natl Acad Sci U S A. 112 (3), E303-E310 (2015).
- Yang, Z., Choi, H., Weisshaar, J. C. Melittin-Induced Permeabilization, Re-sealing, and Re-permeabilization of E. coli Membranes. Biophys J. 114 (2), 368-379 (2018).
- Ruthe, H. J., Adler, J. Fusion of bacterial spheroplasts by electric fields. Biochim. Biophys. Acta. 819 (1), (1985).
Citer Cet Article
Figueroa, D. M., Wade, H. M., Montales, K. P., Elmore, D. E., Darling, L. E. Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization. J. Vis. Exp. (138), e57904, doi:10.3791/57904 (2018).