Engineering and structural analysis of heterologous protein loading based on an effector of the rearrangement hotspot family in the type Ⅵ secretion system

doi:10.3969/j.issn.1674-8115.2026.07.010

Abstract

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

CLC Number:

Q518.2

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.

Figures/Tables 7

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

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

Fig 2 Purification of the TSEIMCHERRY-TECI-VGRG complex

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

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

Fig 5 Purification of the TSEICASΦ-TECI-VGRG complex

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

TrendMD

References 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.