Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

Moirangthem Kiran Singh; Linda J. Kenney

doi:10.3791/64382

JoVE Journal > Biology

Please note that all translations are automatically generated. Click here for the English version.

Biologia

使用遗传密码扩增对细菌分泌蛋白进行超分辨率成像

Published: February 10, 2023

doi:

10.3791/64382

Moirangthem Kiran Singh, Linda J. Kenney

¹Biochemistry & Molecular Biology,University of Texas Medical Branch

Summary

本文提供了一种简单明了的方案，使用遗传密码扩展（GCE）位点特异性标记 沙门氏菌 分泌效应子，并使用直接随机光学重建显微镜（dSTORM）对 HeLa 细胞中分泌蛋白的亚细胞定位进行成像

Abstract

三型分泌系统（T3SS）使革兰氏阴性菌能够将一系列效应蛋白直接注射到真核宿主细胞的细胞质中。进入后，注射的效应蛋白协同调节真核信号通路并重新编程细胞功能，使细菌进入并存活。在感染背景下监测和定位这些分泌的效应蛋白为定义宿主-病原体相互作用的动态界面提供了足迹。然而，在不破坏宿主细胞结构/功能的情况下标记和成像宿主细胞中的细菌蛋白在技术上具有挑战性。

构建荧光融合蛋白并不能解决这个问题，因为融合蛋白会堵塞分泌装置，因此不会被分泌。为了克服这些障碍，我们最近采用了一种使用遗传密码扩展（GCE）对细菌分泌的效应子以及其他难以标记的蛋白质进行位点特异性荧光标记的方法。本文提供了一个完整的分步方案，使用GCE位点特异性标记 沙门氏菌 分泌效应子，然后使用直接随机光学重建显微镜（dSTORM）对HeLa细胞中分泌蛋白的亚细胞定位进行成像

最近的研究结果表明，通过GCE 掺入非规范氨基酸（ncAAs），然后用含四嗪的染料进行生物正交标记，是一种可行的技术，用于选择性标记和可视化细菌分泌蛋白以及随后在宿主中进行图像分析。本文的目的是提供一个简单明了的方案，可供有兴趣使用GCE进行超分辨率成像的研究人员使用，以研究细菌和病毒中的各种生物过程，以及宿主 – 病原体相互作用。

Introduction

长期以来，细菌感染一直被视为对人体健康的严重危害。病原体使用高度进化、极其强大和复杂的防御系统，以及各种细菌毒力因子（称为效应蛋白）来逃避宿主免疫反应并建立感染¹^，².然而，由于缺乏在发病过程中直接跟踪宿主细胞中关键蛋白质成分和效应子的合适方法，这些系统背后的分子机制和单个效应蛋白的作用仍然在很大程度上是未知的。

一个典型的例子是鼠伤寒沙门氏菌，它会导致急性胃肠炎。沙门氏菌鼠伤寒使用三型分泌系统（T3SS）将各种效应蛋白直接注射到宿主细胞中。一旦沙门氏菌进入宿主细胞，它就会驻留在酸性膜结合的隔室中，称为含沙门氏菌的液泡（SCV）³^，⁴。SCV 的酸性 pH 值激活沙门氏菌致病性岛 2 （SPI-2）编码的 T3SS，并将 20 种或更多效应蛋白的凌空翻位穿过液泡膜进入宿主细胞质⁵，⁶，⁷^，⁸。在宿主内部，这些复杂的效应蛋白混合物协调操纵宿主细胞信号通路，导致形成高度动态、复杂的管状膜结构，从 SCV 沿微管延伸，称为沙门氏菌诱导的细丝（SIF），使沙门氏菌能够在宿主细胞内存活和复制⁹，¹⁰^，¹¹。

可视化、跟踪和监测细菌效应子定位以及检查它们在宿主细胞内的运输和相互作用的方法，为支撑细菌发病机制提供了重要的见解。宿主细胞内沙 门氏菌 分泌的T3SS效应蛋白的标记和定位已被证明是一项技术挑战¹²^，¹³;尽管如此，基因编码荧光蛋白的发展已经改变了我们研究和可视化生命系统中蛋白质的能力。然而，荧光蛋白的大小（~25-30 kDa）15通常与目标蛋白的大小相当甚至更大（POI;例如，SsaP为^13.65 kDa，SifA为37.4 kDa）。事实上，效应器的荧光蛋白标记通常会阻断标记效应子的分泌并堵塞T3SS¹⁴。

此外，荧光蛋白稳定性较差，在光漂白前发射的光子数量很少，限制了它们在超分辨率显微技术中的使用¹⁶，¹⁷，¹⁸^，特别是在光活化定位显微镜（PALM），STORM和受激发射耗尽（STED）显微镜中。虽然有机荧光染料的光物理性质优于荧光蛋白，但诸如CLIP/SNAP¹⁹，20，Split-GFP 21，ReAsH / FlAsH²²，23和HA-Tags ^24，²⁵等方法/技术需要额外的蛋白质或肽附属物^，这些附件可能通过干扰翻译后修饰或运输来损害目标效应蛋白的结构功能一种最大限度地减少必要蛋白质修饰的替代方法涉及在通过GCE翻译期间将ncAA掺入POI中。ncAA要么是荧光的，要么可以通过点击化学¹²，¹³^，^26，27^，²⁸制成荧光。

使用GCE，具有微小的、功能性的生物正交基团（如叠氮化物、环丙烯或环辛炔基团）的ncAA几乎可以在靶蛋白的任何位置引入。在该策略中，在POI基因的指定位置将天然密码子与稀有密码子（例如琥珀色（TAG）终止密码子）交换。修饰的蛋白质随后与正交氨酰基-tRNA合成酶/tRNA对一起在细胞中表达。tRNA合成酶活性位点被设计为仅接收一个特定的ncAA，然后将其共价连接到识别琥珀色密码子的tRNA的3’末端。ncAA被简单地引入生长培养基中，但它必须被细胞吸收并到达细胞质，其中氨酰基-tRNA合成酶（aaRS）可以将其连接到正交tRNA;然后将其合并到指定位置的POI中（见图1）¹²。因此，GCE能够将大量的生物正交反应基团（如酮，叠氮化物，炔烃，环辛炔，转环辛烯，四嗪，去甲骨烯，α，β-不饱和酰胺和双环[6.1.0]-壬炔）掺入POI中，可能克服传统蛋白质标记方法的局限性¹²，²⁶，²⁷^，²⁸。

超分辨率成像技术的最新新兴趋势为在分子水平上研究生物结构开辟了新的途径。特别是，STORM是一种基于定位的单分子超分辨率技术，已成为可视化低至~20-30纳米的细胞结构的宝贵工具，并且能够一次研究一个分子的生物过程，从而发现细胞内分子的作用，这些作用在传统的集合平均研究中尚不为人知¹³.单分子和超分辨率技术需要带有明亮、光稳定的有机荧光团的小标签，以获得最佳分辨率。我们最近证明GCE可用于结合合适的探针进行超分辨率成像¹²。

细胞中蛋白质标记的两个最佳选择是双环[6.1.0]壬炔赖氨酸（BCN）和反式环辛烯赖氨酸（TCO;如图1所示），它们可以使用tRNA/合成酶对的变体（此处称为tRNA Pyl/^PylRS ^AF）进行遗传编码，其中Pyl代表吡咯赖氨酸，AF代表合理设计的双突变体（Y306A，Y384F），源自天然编码吡咯赖氨酸¹²的甲烷肉瘤^，²⁹，³⁰^，³¹.通过应变促进的逆电子需求Diels-Alder环加成（SPIEDAC）反应，这些氨基酸与四嗪偶联物发生化学选择性反应（图1）¹²，³⁰^，³¹。这种环加成反应非常快，并且与活细胞相容;如果适当的荧光团与四嗪部分¹²^，²⁶^，³²官能化，它们也可能是荧光的。本文提出了一种优化的方案，用于使用GCE监测传递到宿主细胞中的细菌效应子的动力学，然后使用dSTORM监测HeLa细胞中分泌蛋白的亚细胞定位。结果表明，通过GCE掺入ncAA，然后与含荧光四嗪的染料进行点击反应，代表了一种选择性标记，分泌蛋白可视化以及随后在宿主中进行亚细胞定位的通用方法。然而，这里详述的所有组件和程序都可以调整或替换，以便GCE系统可以适应其他生物学问题。

Protocol

1. 质粒构建将表达POI的基因克隆到表达大肠杆菌BL21（DE3）中表达POI的表达质粒（例如pET28a-sseJ 10TAG）。此步骤有助于确定突变体是否具有功能。为了可视化宿主细胞中分泌的沙门氏菌效应子，构建一个表达质粒（pWSK29-sseJ-HA），该质粒在其天然启动子的控制下表达靶POI SseJ，如先前的报告7，12，<sup…

Representative Results

该协议论文描述了一种基于GCE的方法，用于沙门氏菌分泌效应子的位点特异性荧光标记和可视化，如图1所示。含有ncAA的反式环辛烯生物正交基团（TCO）和荧光染料的化学结构如图1A所示。SseJ标记是通过GCE技术使用正交氨基酰基-tRNA合成酶/tRNA对在琥珀色终止密码子（见图1B）上遗传掺入生物?…

Discussion

本文描述的方法用于跟踪感染后由细菌T3SS注射到宿主细胞中的效应蛋白的精确位置。T3SS被 沙门氏菌、志贺氏菌和 耶尔森氏菌 等细胞内病原体用于将毒力成分转运到宿主中。超分辨率成像技术的发展使得以以前无法想象的分辨率¹²^，¹³^，²⁴可视化毒力因子成为可能。然而，某些标记限制排除了更深入的研究…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了德克萨斯州加尔维斯顿德克萨斯大学医学分院的启动资金的支持，以及L.J.K.的德克萨斯州之星奖。我们感谢Edward Lemke教授（德国海德堡欧洲分子生物学实验室）提供的质粒pEVOL-PylRS-AF。图1 中的图像是使用BioRender创建的。

Materials

10x Tris/Glycine SDS running buffer	BIO-RAD	1610732
Ammonium chloride	Fischer Scientific	A661-500
Ammonium sulphate	Fischer Scientific	BP212R-1
Ampicillin Sodium	Sigma-Aldrich	A0166	5 g
Antibiotic-Antimycotic (100x)	ThermiFischer Scientific	15240096
Arabinose	Sigma-Aldrich	A3256	500 g
Avanti J-26XP (High-Performance Centrifuge)	Beckman Coulter
Bacto-Agar	BD Diagnostics, Franklin Lakes, USA	214010
BDP-FL-tetrazine	Lumiprobe (USA)	2.14E+02
β-mercaptoethanol	Millipore	444203	250 mL
Bromophenol blue	Sigma-Aldrich	B8026
BSA	Sigma-Aldrich	A4503	500 g
Casein	Sigma-Aldrich	C8654
Catalase	Sigma-Aldrich	C9322
Chloramphenicol	Sigma-Aldrich	C1919
Click Amino Acid / trans-Cyclooct-2-en – L – Lysine (TCO*A)	SiChem GmbH	SC-8008	Size: 500 mg
DAPI (Hoechst33342)	Invitrogen	H3570
DeNovix DS-11+ Spectrophotometer	DeNovix
DMEM*	Corning	10-013-CV	* Used for maintaining HeLa Cell
DMEM**	Gibco	11965-092	**Used for bacterial infection in presence of ncAA, see section 5.4.
DMSO	Sigma-Aldrich	D8418	250 g
Donkey anti-rabbit Alexa fluoro555 secondary antibody	Invitrogen	A-31572
DPBS, 1x	Corning	21-031-CV
E. coli strain BL21 (DE3)	Novagen (Madison, WI)
EMCCD Camera	Andor	iXon Ultra 897-BV
Eppendorf Safe-Lock Tube 1.5 mL (PCR clean)	Eppendorf, Hamburg, Germany	30123.328
Fetal Bovine Serum (FBS)	Fischer	10082147
Fisherbrand Syringe Filters – Sterile (PVDF 0.22 µm)	Fischer Scientific	97203	Pack of 100
Gene Pulser Xcell Electroporator	BIO-RAD	1652660
Gentamycin	Sigma-Aldrich	G1272	10 mL
Gibco L-Glutamine (200 mM), 100x	Fischer Scientific	25-030-081
Glucose oxidase	Sigma-Aldrich	G7141-50KU
Glycerol	Fischer Scientific	BF229-4
HeLa cells	ATTC	CCL-2
HEPES Buffer	Corning	25-060-C1	100 mL
Hydrocloric acid	Fischer Scientific	A144-212
ImageJ			Image processing and analysis: http://rsbweb.nih.gov/ij
IntantBlue	Expedeon	ISB1L	Coomassie-based stain
Isopropyl β-D-1-thiogalactopyranoside (IPTG)	Sigma-Aldrich	I5502
Janelia Fluoro 646-tetrazine	Tocris Bioscience	7279
Kanamycin	Sigma-Aldrich	60615	5 g
LB Broth	BD Difco	244620	500 g
Lysozyme	Sigma-Aldrich	L6876
μManager (v. 1.4.2)			https://micro-manager.org/Download_Micro-Manager_Latest_Release
MES	Sigma-Aldrich	M3671	250 g
Micro pulser cuvette	BIO-RAD	165-2086	0.2 cm electrode gap, pkg. of 50
Nikon N-STORM	Nikon Instruments Inc.		https://www.microscope.healthcare.nikon.com/products/super-resolution-microscopes/n-storm-super-resolution
Nunc EasYFlask Cell Culture Flasks	ThermiFischer Scientific	156499
Omnipur Casamino Acid	Calbiochem	2240	500 g
Paraformaldehhyde (PFA)	Electron Microscopy Sciences	15710
PBS (10x)	Roche	11666789001
Penicillin-Streptomycin Solution (100x)	GenDEPOT	CA005-010	100 mL
pEVOL-PylRS-AF			For plasmid construction and map see following references 1. Angew Chem Int Ed Engl. 2011, 50(17), 3878-81. 2. Angew Chem Int Ed Engl., 2012, 51, 4166-70.
Plasmid Mini-prep Kit	Qiagen	27106
plasmid pWSK29-sseJ10TAG-HA			Ref.: elife. 2021, 10, e67789.
plasmid pWSK29-sseJ-HA			Ref.: elife. 2021, 10, e67789. Vector map of PWSK29: https://www.addgene.org/172972/
Pluronic F-127	Millipore	540025	Protein grade, 10% Solution
Potassium phosphate monobasic	Fischer Scientific	P285-500
Potassium sulphate	Acros Organic	424220250
Protease Inhibitor Cocktail Set I – Calbiochem	Sigma-Aldrich	539131	100x Solution
Rabbit anti-HA primary antibody	Sigma-Aldrich	H6908
S. enterica. serovar Typhimurium 14028s			Ref.: PLoS Biol. 2015, 13, e1002116.
Saponin	Sigma-Aldrich	47036-50G-F
Sodium dodecyl sulfate (SDS)	Sigma-Aldrich	L3771
Sodium hydroxide	Fischer Scientific	SS255-1
Sodium Pyruvate (100 mM), 100x	Corning	25-060-C1	100 mL
Sonic Dismembrator Model 100	Fischer Scientific	24932
STED microsocpe (Leica TCS SP8 STED 3X system)	Leica Microsystems, Wetzlar, Germany		https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp8-sted-one/
ThunderSTORM			https://zitmen.github.io/thunderstorm/
Trizma Base	Sigma-Aldrich	T1503
Trypsin-EDTA (1x), 0.25%	GenDEPOT	CA014-010
Tween-20	Sigma-Aldrich	P9416	100 mL
X-well tissue culture chamber slides	SARSTEDT	94.6190.802

Riferimenti

Marlovits, T. C., et al. Structural insights into the assembly of the type III secretion needle complex. Science. 306 (5698), 1040-1042 (2004).
Kubori, T., et al. Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science. 280 (5363), 602-605 (1998).
LaRock, D. L., Chaudhary, A., Miller, S. I. Salmonellae interactions with host processes. Nature Reviews Microbiology. 13 (4), 191-205 (2015).
Galan, J. E., Waksman, G. Protein-injection machines in bacteria. Cell. 172 (6), 1306-1318 (2018).
Feng, X., Oropeza, R., Kenney, L. J. Dual regulation by phospho-OmpR of ssrA/B gene expression in Salmonella pathogenicity island 2. Molecular Microbiology. 48 (4), 1131-1143 (2003).
Walthers, D., et al. Salmonella enterica response regulator SsrB relieves H-NS silencing by displacing H-NS bound in polymerization mode and directly activates transcription. Journal of Biological Chemistry. 286 (3), 1895-1902 (2011).
Chakraborty, S., Mizusaki, H., Kenney, L. J. A FRET-based DNA biosensor tracks OmpR-dependent acidification of Salmonella during macrophage infection. PLoS Biology. 13 (4), e1002116 (2015).
Liew, A. T. F., et al. Single cell, super-resolution imaging reveals an acid pH-dependent conformational switch in SsrB regulates SPI-2. eLife. 8, e45311 (2019).
Knuff, K., Finlay, B. B. What the SIF is happening-the role of intracellular Salmonella-induced filaments. Frontiers in Cellular and Infection Microbiology. 7, 335 (2017).
Drecktrah, D., et al. Dynamic behavior of Salmonella-induced membrane tubules in epithelial cells. Traffic. 9 (12), 2117-2129 (2008).
Garcia del-Portillo, F., Zwick, M. B., Leung, K. Y., Finlay, B. B. Salmonella induces the formation of filamentous structures containing lysosomal membrane glycoproteins in epithelial cells. Proceedings of the National Academy of Sciences. 90 (22), 10544-10548 (1993).
Singh, M. K., Zangoui, P., Yamanaka, Y., Kenney, L. J. Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors. elife. 10, e67789 (2021).
Singh, M. K., Kenney, L. J. Super-resolution imaging of bacterial pathogens and visualization of their secreted effectors. FEMS Microbiology Reviews. 45 (2), fuaa050 (2020).
Akeda, Y., Galan, J. E. Chaperone release and unfolding of substrates in type III secretion. Nature. 437 (7060), 911-915 (2005).
Su, W. W. Fluorescent proteins as tools to aid protein production. Microbial Cell Factories. 4 (1), 12 (2005).
Wang, S., Moffitt, J. R., Dempsey, G. T., Xie, X. S., Zhuang, X. Characterization and development of photoactivatable fluorescent proteins for single-molecule-based superresolution imaging. Proceedings of the National Academy of Sciences. 111 (23), 8452-8457 (2014).
Ghodke, H., Caldas, V. E. A., Punter, C. M., van Oijen, A. M., Robinson, A. Single-molecule specific mislocalization of red fluorescent proteins in live Escherichia coli. Biophysical Journal. 111 (1), 25-27 (2016).
Gahlmann, A., Moerner, W. E. Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging. Nature Reviews Microbiology. 12 (1), 9-22 (2014).
Keppler, A., et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nature Biotechnology. 21 (1), 86-89 (2003).
Gautier, A., et al. An engineered protein tag for multiprotein labeling in living cells. Chemical Biology. 15 (2), 128-136 (2008).
Young, A. M., Minson, M., McQuate, S. E., Palmer, A. E. Optimized fluorescence complementation platform for visualizing Salmonella effector proteins reveals distinctly different intracellular niches in different cell types. ACS Infectious Diseases. 3 (8), 575-584 (2017).
Martin, B. R., Giepmans, B. N., Adams, S. R., Tsien, R. Y. Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nature Biotechnology. 23 (10), 1308-1314 (2005).
Adams, S. R., et al. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: Synthesis and biological applications. Journal of the American Chemical Society. 124 (21), 6063-6076 (2002).
Gao, Y., Spahn, C., Heilemann, M., Kenney, L. J. The pearling transition provides evidence of force-driven endosomal tubulation during Salmonella infection. mBio. 9 (3), e01083-e01018 (2018).
Brumell, J. H., Goosney, D. L., Finlay, B. B. SifA, a type III secreted effector of Salmonella typhimurium, directs Salmonella-induced filament (Sif) formation along microtubules. Traffic. 3 (6), 407-415 (2002).
Lang, K., Chin, J. W. Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chemical Reviews. 114 (9), 4764-4806 (2014).
Davis, L., Chin, J. W. Designer proteins: applications of genetic code expansion in cell biology. Nature Reviews Molecular Cell Biology. 13 (3), 168-182 (2012).
Chin, J. W., et al. Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. Journal of the American Chemical Society. 124 (31), 9026-9027 (2002).
Kipper, K., et al. Application of noncanonical amino acids for protein labeling in a genomically recoded Escherichia coli. ACS Synthetic Biology. 6 (2), 233-255 (2017).
Uttamapinant, C., et al. Genetic code expansion enables live-cell and super-resolution imaging of site-specifically labeled cellular proteins. Journal of American Chemical Society. 137 (14), 4602-4605 (2015).
Plass, T., et al. Amino acids for Diels-Alder reactions in living cells. Angewandte Chemie International Edition. 51 (17), 4166-4170 (2012).
Lang, K., et al. Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions. Journal of the American Chemical Society. 134 (25), 10317-10320 (2012).
Xu, H., et al. Re-exploration of the codon context effect on amber codon-guided incorporation of noncanonical amino acids in Escherichia coli by the blue-white screening assay. ChemBioChem. 17 (13), 1250-1256 (2016).
Nakamura, Y., Gojobori, T., Ikemura, T. Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucleic Acids Research. 28 (1), 292 (2000).
Spahn, C., et al. Sequential super-resolution imaging of bacterial regulatory proteins: the nucleoid and the cell membrane in single, fixed E. coli cells. Methods in Molecular Biology. 1624, 269-289 (2017).
Grimm, J., English, B., Chen, J., et al. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nature Methods. 12 (3), 244-250 (2015).
Xu, J., Ma, H., Liu, Y. Stochastic Optical Reconstruction Microscopy (STORM). Current Protocols in Cytometry. 81 (1), 12-46 (2017).
Meineke, B., Heimgärtner, J., Eirich, J., Landreh, M., Elsässer, S. J. Site-specific incorporation of two ncAAs for two-color bioorthogonal labeling and crosslinking of proteins on live mammalian cells. Cell Reports. 31 (12), 107811 (2020).
Bessa-Neto, D., et al. Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids unveils masked epitopes in live neurons. Nature Communications. 12 (1), 6715 (2021).
Beliu, G., et al. Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy. Communication Biology. 2, 261 (2019).
Arsić, A., Hagemann, C., Stajković, N., Schubert, T., Nikić-Spiegel, I. Minimal genetically encoded tags for fluorescent protein labeling in living neurons. Nature Communications. 13 (1), 314 (2022).

Play Video

PDF

DOI

DOWNLOAD MATERIALS LIST

Citazione di questo articolo

Singh, M. K., Kenney, L. J. Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion. J. Vis. Exp. (192), e64382, doi:10.3791/64382 (2023).