基于一种Ⅵ型分泌系统重排热点家族效应物的异源蛋白工程化装载与结构解析

doi:10.3969/j.issn.1674-8115.2026.07.010

摘要/Abstract

摘要：

目的·以达卡气单胞菌（Aeromonas dhakensis）Ⅵ型分泌系统（type Ⅵ secretion system，T6SS）的重排热点（rearrangement hotspot，RHS）家族效应物Ⅵ型分泌系统效应物I（type Ⅵ secretion system effector I，TSEI）为模型，系统评估其桶状结构对异源蛋白的装载能力，从结构角度探究其作为工程化跨细胞蛋白递送模块的可行性。方法·将达卡气单胞菌T6SS相关蛋白缬氨酸-甘氨酸重复蛋白（valine-glycine repeat protein G，VGRG）、Ⅵ型分泌系统效应物伴侣蛋白I（type Ⅵ secretion system effector chaperon I，TECI），以及经异源蛋白替换重组改造的效应物TSEI对应基因，分别构建至适用于大肠埃希菌表达的载体中。采用化学感受态转化方式，使各组分蛋白在大肠埃希菌中共表达，并通过调控诱导条件，获得稳定表达的复合物。细胞破碎后，利用标签介导的亲和层析对2种目标复合物进行初步纯化，之后结合凝胶过滤层析进一步分离复合物，以提升样品的均一性与完整性。引入甘油密度梯度离心步骤，去除聚集体，富集完整装配的复合物。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis，SDS-PAGE）及蛋白质印迹法（Western blotting）对各组分进行鉴定，确定复合物的组成与最佳状态。使用冷冻电子显微镜（电镜）对获得的高纯度重组样品进行数据采集，借助单颗粒分析流程，对电镜图像依次进行颗粒挑选、二维分类与三维重构，得到重组复合物的三维结构模型。利用UCSF Chimera等可视化软件，将已知或预测的蛋白结构模型与电子密度图拟合，推测各组分在复合物中的相对空间排布，为分析TSEI改造体在T6SS相关复合物中的装配状态提供结构依据。结果·通过大肠埃希菌共表达，以及多步亲和纯化、凝胶过滤层析与甘油密度梯度离心，经SDS-PAGE与Western blotting分析证实，分别获得了2种组成明确的重组效应模块复合物，这表明RHS桶结构对异源蛋白具有良好的装载容忍性。冷冻电镜密度图显示，樱桃荧光蛋白（monomeric cherry fluorescent protein，MCHERRY）替换后复合物整体构象稳定，RHS桶腔内可见额外密度，提示成功装载；而CRISPR相关核酸内切酶Φ（CRISPR-associated endonuclease Φ，CASΦ）替换则很可能导致桶状骨架稳定性下降，完整封装结构形成受限。上述结果表明，RHS桶在一定范围内具备容纳异源蛋白的能力，且其结构适应边界得以揭示，这为T6SS工程化蛋白递送平台的构建提供了思路与结构依据。结论·基于重组表达结果并结合冷冻电镜结构，系统验证了达卡气单胞菌T6SS效应物TSEI的RHS桶递送异源蛋白的可行性，为基于T6SS的定向蛋白运输与功能改造提供了实验依据。

关键词: Ⅵ型分泌系统, 重排热点家族蛋白, 异源蛋白递送, 冷冻电镜技术, 蛋白工程化改造

Abstract:

Objective ·To systematically evaluate the loading capacity of the barrel-shaped structure of the rearrangement hotspot (RHS) effector type Ⅵ secretion system effector I (TSEI) from Aeromonas dhakensis (A. dhakensis) within the type Ⅵ secretion system (T6SS) for heterologous proteins and explore its feasibility as an engineered intercellular protein delivery module from a structural perspective. Methods ·The genes encoding the A. dhakensis T6SS-related proteins valine-glycine repeat protein G (VGRG), type Ⅵ secretion system effector chaperone I (TECI), and the engineered TSEI effector generated by heterologous protein replacement were individually cloned into vectors suitable for expression in Escherichia coli (E. coli).The component proteins were co-expressed in E. coli via chemical competent transformation. Stable expression of the complexes was achieved by regulating the induction conditions. Following cell lysis, the two target complexes were initially purified separately using tag-mediated affinity chromatography, and then were further separated via size-exclusion chromatography to improve sample homogeneity and integrity. A glycerol density gradient centrifugation step was introduced to remove aggregates and enrich fully assembled complexes. The composition and optimal state of the complexes were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. High-purity recombinant samples were subjected to cryo-electron microscopy (cryo-EM) data collection. Single-particle analysis, including particle picking, two-dimensional classification, and three-dimensional reconstruction, was carried out to obtain 3D structural models. UCSF Chimera and other visualization software were used to fit known or predicted protein structures into the electron density maps, enabling inference of the relative spatial arrangement of components and providing structural evidence for the assembly state of the engineered TSEI within T6SS-related complexes. Results ·Through co-expression in E. coli and sequential purification using affinity chromatography, size-exclusion chromatography, and glycerol density gradient centrifugation, two recombinant effector module complexes with well-defined compositions were successfully acquired, as confirmed by SDS-PAGE and Western blotting analyses. These results indicated that the RHS barrel structure exhibited good tolerance for heterologous protein loading. Cryo-EM density maps showed that, the overall conformation of the complex remained stable after replacement with monomeric cherry fluorescent protein (MCHERRY), with additional density observed inside the RHS barrel, suggesting successful loading. In contrast, replacement with CRISPR-associated endonuclease Φ (CASΦ) likely reduced the stability of the barrel scaffold and restricted the formation of a fully encapsulated structure. These findings demonstrated that the RHS barrel possesses the capacity to accommodate heterologous proteins within a certain range and revealed its structural adaptation boundaries, providing structural insights for the development of T6SS-based engineered protein delivery platforms. Conclusion ·Based on recombinant expression and cryo-EM structural analysis, this study systematically validated the feasibility of the RHS barrel of the A. dhakensis T6SS effector TSEI for delivering heterologous proteins, offering experimental support for T6SS-based targeted protein transport and functional engineering.

Key words: type Ⅵ secretion system, rearrangement hotspot (RHS) family protein, heterologous protein delivery, cryo-electron microscopy, protein engineering

中图分类号:

Q518.2

潘岳, 黄晶. 基于一种Ⅵ型分泌系统重排热点家族效应物的异源蛋白工程化装载与结构解析[J]. 上海交通大学学报（医学版）, 2026, 46(7): 916-927.

Pan Yue, Huang Jing. Engineering and structural analysis of heterologous protein loading based on an effector of the rearrangement hotspot family in the type Ⅵ secretion system[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026, 46(7): 916-927.

图/表 7

表1 冷冻电镜数据收集与处理

Tab 1 Cryo-electron microscopy data collection and processing

Data collection and processing	TSEI^MCHERRY-TECI-VGRG	TSEI^CASΦ-TECI-VGRG
Microscope	Titan Krios	Titan Krios
Detector	K3	K3
Voltage/kV	300	300
Electron exposure/(e^-∙Å^-2)	50	50
Defocus range/µm	-0.8‒ -3.0	-0.8‒ -3.0
Pixel size/Å	1.10	1.10
Symmetry imposed	C1	C1
Initial particle images/n	859 839	2 350 745
Final particle images/n	305 766	437 649
Map resolution/Å(FSC threshold 0.143)	4.23	8.80

图1 T6SS效应物TSEI的结构域组成与异源重组基因设计Note： A. Schematic diagram of the domain architecture of the self-cleaving RHS effector TSEI. B. WebLogo showing conserved residues in the C-terminal region of RHS, derived from sequence alignment of 48 representative RHS homologs across diverse bacterial species. The dashed line indicates the autocleavage site. C. Schematic representation of the native toxic domain ofTSEI. D. Design of recombinant TSEI constructs with MCHERRY or CASΦ replacement (created using BioRender). N-TER—N terminal; C-TER—C terminal.

Fig 1 Domain organization of the T6SS effector TSEI and the design of heterologous recombinant constructs

图2 TSEIMCHERRY-TECI-VGRG复合物蛋白纯化Note： A. Gel filtration profile of the TSEIMCHERRY-TECI-VGRGcomplex resolved using a Superose 6 Increase 10/300 GL column. B. SDS-PAGE analysis corresponding to the eluted fractions in “A”. C. Western blotting analysis of the peak fractions from “A”, confirmingthatTECI andTSEIMCHERRY were detected using an anti-HIS antibody, while VGRG was detected using an anti-FLAG antibody. D. Coomassie blue-stained SDS-PAGE of fractionated TSEIMCHERRY-TECI-VGRGcomplexes following GraFix cross-linking.

Fig 2 Purification of the TSEIMCHERRY-TECI-VGRG complex

图3 TSEIMCHERRY-TECI-VGRG复合物的冷冻电镜分析Note： A. Representative cryo-electron microscopy micrograph image of the TSEIMCHERRY-TECI-VGRG complex (×81 000). B. Representative two-dimensional (2D) class averages of cryo-electron microscopy particles from the TSEIMCHERRY-TECI-VGRG complex. C. Angular distribution of particle projections from the TSEIMCHERRY-TECI-VGRG complex reconstruction. D. Gold-standard Fourier shell correlation (FSC) curve of the final cryo-electron microscopy reconstruction map of the TSEIMCHERRY-TECI-VGRG complex.

Fig 3 Cryo-electron microscopy analysis of the TSEIMCHERRY-TECI-VGRG complex

图4 TSEIMCHERRY-TECI-VGRG复合物的电子密度图与结构分析Note： A. Surface representation of the cryo-electron micrograph density map of the TSEIMCHERRY-TECI-VGRG complex, presented in six orthogonal views. B. Docking of AlphaFold-predicted structures of TSEIRHS and TECI into the cryo-electron density map of the TSEIMCHERRY-TECI-VGRG complex, shown in three representative views. C. X-ray crystal structure of MCHERRY (PDB ID: 6YLM).

Fig 4 Electron density and structural analysis of theTSEIMCHERRY-TECI-VGRG complex

图5 TSEICASΦ-TECI-VGRG复合物蛋白纯化Note： A. Gel filtration analysis of the TSEICASΦ-TECI-VGRG complex on a Superose 6 Increase 10/300 GL column. B. Corresponding SDS-PAGE analysis of the fractions from A. C. Western blotting analysis of the peak fractions from A, with TECI and TSEICASΦconfirmed by an anti-HIS antibody and VGRG confirmed by an anti-FLAG antibody. D. Coomassie-stained SDS-PAGE of fractionated TSEICASΦ-TECI-VGRG complexes after GraFix cross-linking.

Fig 5 Purification of the TSEICASΦ-TECI-VGRG complex

图6 TSEICASΦ-TECI-VGRG复合物的冷冻电镜分析与电子密度图Note： A. Representative cryo-electron microscopy micrograph of the TSEICASΦ-TECI-VGRGcomplex (×81 000). B. Representative two-dimensional (2D) class averages of cryo-electron microscopy particles derived from theTSEICASΦ-TECI-VGRG complex. C. Angular distribution of particle projections from the TSEICASΦ-TECI-VGRG complex reconstruction. D. Gold-standard Fourier shell correlation (FSC) curve of the final cryo-electron micrograph reconstruction map of the TSEICASΦ-TECI-VGRGcomplex. E. Surface representation of the cryo-electron micrograph density map of the TSEICASΦ-TECI-VGRG complex, presented in four representative views.

Fig 6 Cryo-electron microscopy analysis and electron density map of the TSEICASΦ-TECI-VGRGcomplex

参考文献 39

[1]	Galán J E, Waksman G. Protein-injection machines in bacteria[J]. Cell, 2018, 172(6): 1306-1318.
[2]	Bleves S, Galán J E, Llosa M. Bacterial injection machines: evolutionary diverse but functionally convergent[J]. Cell Microbiol, 2020, 22(5): e13157.
[3]	Lynch J P, González-Prieto C, Reeves A Z, et al. Engineered Escherichia coli for the in situ secretion of therapeutic nanobodies in the gut[J]. Cell Host Microbe, 2023, 31(4): 634-649.e8.
[4]	Green C A, Kamble N S, Court E K, et al. Engineering the flagellar type Ⅲ secretion system: improving capacity for secretion of recombinant protein[J]. Microb Cell Fact, 2019, 18(1): 10.
[5]	Burdette L A, Leach S A, Kennedy N, et al. Characterization and engineering of the type 3 secretion system needle monomer from Salmonella through the construction and screening of a comprehensive mutagenesis library[J]. mSphere, 2024, 9(8): e0036724.
[6]	Carleton H A, Lara-Tejero M, Liu X, et al. Engineering the type Ⅲ secretion system in non-replicating bacterial minicells for antigen delivery[J]. Nat Commun, 2013, 4: 1590.
[7]	Vergunst A C, Schrammeijer B, den Dulk-Ras A, et al. VirB/D4-dependent protein translocation from Agrobacterium into plant cells[J]. Science, 2000, 290(5493): 979-982.
[8]	Schrammeijer B, den Dulk-Ras A, Vergunst A C, et al. Analysis of Vir protein translocation from Agrobacterium tumefaciens using Saccharomyces cerevisiae as a model: evidence for transport of a novel effector protein VirE3[J]. Nucleic Acids Res, 2003, 31(3): 860-868.
[9]	Akeda Y, Galán J E. Chaperone release and unfolding of substrates in type Ⅲ secretion[J]. Nature, 2005, 437(7060): 911-915.
[10]	Lettl C, Haas R, Fischer W. Kinetics of CagA type Ⅳ secretion by Helicobacter pylori and the requirement for substrate unfolding[J]. Mol Microbiol, 2021, 116(3): 794-807.
[11]	Guzmán-Herrador D L, Fernández-Gómez A, Llosa M. Recruitment of heterologous substrates by bacterial secretion systems for transkingdom translocation[J]. Front Cell Infect Microbiol, 2023, 13: 1146000.
[12]	Pukatzki S, Ma A T, Revel A T, et al. Type Ⅵ secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin[J]. Proc Natl Acad Sci U S A, 2007, 104(39): 15508-15513.
[13]	Leiman P G, Basler M, Ramagopal U A, et al. Type Ⅵ secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin[J]. Proc Natl Acad Sci U S A, 2009, 106(11): 4154-4159.
[14]	Zheng J, Ho B, Mekalanos J J. Genetic analysis of anti-amoebae and anti-bacterial activities of the type Ⅵ secretion system in Vibrio cholerae[J]. PLoS One, 2011, 6(8): e23876.
[15]	Kapitein N, Mogk A. Deadly syringes: type Ⅵ secretion system activities in pathogenicity and interbacterial competition[J]. Curr Opin Microbiol, 2013, 16(1): 52-58.
[16]	Mougous J D, Cuff M E, Raunser S, et al. A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus[J]. Science, 2006, 312(5779): 1526-1530.
[17]	Ma A T, McAuley S, Pukatzki S, et al. Translocation of a Vibrio cholerae type Ⅵ secretion effector requires bacterial endocytosis by host cells[J]. Cell Host Microbe, 2009, 5(3): 234-243.
[18]	Unterweger D, Kostiuk B, Ötjengerdes R, et al. Chimeric adaptor proteins translocate diverse type Ⅵ secretion system effectors in Vibrio cholerae[J]. EMBO J, 2015, 34(16): 2198-2210.
[19]	Alcoforado Diniz J, Coulthurst S J. Intraspecies competition in Serratia marcescens is mediated by type Ⅵ-secreted Rhs effectors and a conserved effector-associated accessory protein[J]. J Bacteriol, 2015, 197(14): 2350-2360.
[20]	Loquet A, Sgourakis N G, Gupta R, et al. Atomic model of the type Ⅲ secretion system needle[J]. Nature, 2012, 486(7402): 276-279.
[21]	Nazarov S, Schneider J P, Brackmann M, et al. Cryo-EM reconstruction of Type Ⅵ secretion system baseplate and sheath distal end[J]. EMBO J, 2018, 37(4): e97103.
[22]	Jiang F, Waterfield N R, Yang J, et al. A Pseudomonas aeruginosa type Ⅵ secretion phospholipase D effector targets both prokaryotic and eukaryotic cells[J]. Cell Host Microbe, 2014, 15(5): 600-610.
[23]	Hersch S J, Lam L, Dong T G. Engineered type six secretion systems deliver active exogenous effectors and Cre recombinase[J]. mBio, 2021, 12(4): e0111521.
[24]	Liang X, Moore R, Wilton M, et al. Identification of divergent type Ⅵ secretion effectors using a conserved chaperone domain[J]. Proc Natl Acad Sci U S A, 2015, 112(29): 9106-9111.
[25]	Pei T T, Li H, Liang X, et al. Intramolecular chaperone-mediated secretion of an RHS effector toxin by a type Ⅵ secretion system[J]. Nat Commun, 2020, 11(1): 1865.
[26]	Lin R J, Capage M, Hill C W. A repetitive DNA sequence, RHS, responsible for duplications within the Escherichia coli K-12 chromosome[J]. J Mol Biol, 1984, 177(1): 1-18.
[27]	Hill C W, Sandt C H, Vlazny D A. Rhs elements of Escherichia coli: a family of genetic composites each encoding a large mosaic protein[J]. Mol Microbiol, 1994, 12(6): 865-871.
[28]	Jackson A P, Thomas G H, Parkhill J, et al. Evolutionary diversification of an ancient gene family (RHS) through C-terminal displacement[J]. BMC Genomics, 2009, 10: 584.
[29]	Koskiniemi S, Lamoureux J G, Nikolakakis K C, et al. RHS proteins from diverse bacteria mediate intercellular competition[J]. Proc Natl Acad Sci U S A, 2013, 110(17): 7032-7037.
[30]	Busby J N, Panjikar S, Landsberg M J, et al. The BC component of ABC toxins is an RHS-repeat-containing protein encapsulation device[J]. Nature, 2013, 501(7468): 547-550.
[31]	Jackson V A, Meijer D H, Carrasquero M, et al. Structures of teneurin adhesion receptors reveal an ancient fold for cell-cell interaction[J]. Nat Commun, 2018, 9(1): 1079.
[32]	Jurėnas D, Rosa L T, Rey M, et al. Mounting, structure and autocleavage of a type Ⅵ secretion-associated RHS polymorphic toxin[J]. Nat Commun, 2021, 12(1): 6998.
[33]	Jones C, Hachani A, Manoli E, et al. An rhs gene linked to the second type Ⅵ secretion cluster is a feature of the Pseudomonas aeruginosa strain PA14[J]. J Bacteriol, 2014, 196(4): 800-810.
[34]	Hayes B K, Harper M, Venugopal H, et al. Structure of a Rhs effector clade domain provides mechanistic insights into type Ⅵ secretion system toxin delivery[J]. Nat Commun, 2024, 15(1): 8709.
[35]	Pausch P, Al-Shayeb B, Bisom-Rapp E, et al. CRISPR-CasΦ from huge phages is a hypercompact genome editor[J]. Science, 2020, 369(6501): 333-337.
[36]	Scheres S H W. RELION: implementation of a Bayesian approach to cryo-EM structure determination[J]. J Struct Biol, 2012, 180(3): 519-530.
[37]	Pettersen E F, Goddard T D, Huang C C, et al. UCSF Chimera: a visualization system for exploratory research and analysis[J]. J Comput Chem, 2004, 25(13): 1605-1612.
[38]	Ahmad S, Tsang K K, Sachar K, et al. Structural basis for effector transmembrane domain recognition by type Ⅵ secretion system chaperones[J]. eLife, 2020, 9: e62816.
[39]	Colautti J, Tan H, Bullen N P, et al. A widespread accessory protein family diversifies the effector repertoire of the type Ⅵ secretion system spike[J]. Nat Commun, 2024, 15(1): 10108.