Abstract
Bacterial flagellin is a potent immunomodulatory agent. Previously, we successfully obtained flagellin from Escherichia coli Nissle 1917 (FliCEcN) and constructed two mutants with varying degrees of deletion in its highly variable regions (HVRs). We found that there was a difference in immune stimulation levels between the two mutants, with the mutant lacking the D2–D3 domain pair of FliCEcN having a better adjuvant effect. Therefore, this study further analyzed the structural characteristics of the aforementioned FliCEcN and its two mutants and measured their levels of Caco-2 cell stimulation to explore the impact of different domains in the HVRs of FliCEcN on its structure and immune efficacy. This study utilized AlphaFold2, SERS (Surface-enhanced Raman spectroscopy), and CD (circular dichroism) techniques to analyze the structural characteristics of FliCEcN and its mutants, FliCΔ174-506 and FliCΔ274-406, and tested their immune effects by stimulating Caco-2 cells in vitro. The results indicate that the D2 and D3 domains of FliCEcN have more complex interactions compared to the D1-D2 domain pair., and these domains also play a role in molecular docking with TLR5 (Toll-like receptor 5). Furthermore, FliCΔ274-406 has more missing side chain and characteristic amino acid peaks than FliCΔ174-506. The FliCEcN group was found to stimulate higher levels of IL-10 (interleukin 10) secretion, while the FliCΔ174-506 and FliCΔ274-406 groups had higher levels of IL-6 (interleukin 6) and TNF-α (tumor necrosis factor-α) secretion. In summary, the deletion of different domains in the HVRs of FliCEcN affects its structural characteristics, its interaction with TLR5, and the secretion of immune factors by Caco-2 cells.
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Data availability
The datasets generated in the present study are available from the authors upon request.
References
Acharya D, Sullivan MJ, Duell BL, Goh KGK, Katupitiya L, Gosling D, Chamoun MN, Kakkanat A, Chattopadhyay D, Crowley M, Crossman DK, Schembri MA, Ulett GC (2019) Rapid bladder interleukin-10 synthesis in response to uropathogenic escherichia coli is part of a defense strategy triggered by the major bacterial flagellar filament FliC and contingent on TLR5. mSphere. 4(6):e00545-19. https://doi.org/10.1128/mSphere.00545-19
Barkhordari M, Bagheri M, Irian S, Khani MH, Ebrahimi MM, Zahmatkesh A, Shahsavandi S (2021) Comparison of flagellin and an oil-emulsion adjuvant in inactivated Newcastle disease vaccine in stimulation of immunogenic parameters. Comp Immunol Microbiol Infect Dis 75:101622. https://doi.org/10.1016/j.biomaterials.2022.121542
Berndt A, Wilhelm A, Jugert C, Pieper J, Sachse K, Methner U (2007) Chicken cecum immune response to Salmonella enterica serovars of different levels of invasiveness. Infect Immun 75(12):5993–6007. https://doi.org/10.1128/IAI.00695-07
Biedma ME, Cayet D, Tabareau J, Rossi AH, Ivičak-Kocjan K, Moreno G, Errea A, Soulard D, Parisi G, Jerala R (2019) Recombinant flagellins with deletions in domains D1, D2, and D3: characterization as novel immunoadjuvants. Vaccine 37:652–663. https://doi.org/10.1016/j.vaccine.2018.12.009
Bruxelle JF, Mizrahi A, Hoÿs S, Collignon A, Janoir C, Péchiné S (2017) Clostridium difficile flagellin FliC: Evaluation as adjuvant and use in a mucosal vaccine against Clostridium difficile. PLoS One 12:e0187212. https://doi.org/10.1371/journal.pone.0187212
Chen CH, Chen CC, Wang WB, Lionel V, Liu CC, Huang LM, Wu SC (2022) Intranasal immunization with Zika virus envelope domain III-flagellin fusion protein elicits systemic and mucosal immune responses and protection against subcutaneous and intravaginal virus challenges. Pharmaceutics 14:1014. https://doi.org/10.3390/pharmaceutics14051014
Côté-Cyr M, Gauthier L, Zottig X, Bourgault S, Archambault D (2021) Recombinant Bacillus subtilis flagellin Hag is a potent immunostimulant with reduced proinflammatory properties compared to Salmonella enterica serovar Typhimurium FljB. Vaccine 40:11–17. https://doi.org/10.1016/j.vaccine.2021.11.049
Couper KN, Blount DG, Riley EM (2008) IL-10: the master regulator of immunity to infection. J Immunol 180(9):5771–5777. https://doi.org/10.4049/jimmunol.180.9.5771
Deng YJ (2021) Study on the flagellin of probiotic nissle 1917 as a new type of immune adjuvant. Master’s Thesis, Guizhou University, Guiyang, China (In Chinese)
Fasina YO, Holt PS, Moran ET, Moore RW, Conner DE, McKee SR (2008) Intestinal cytokine response of commercial source broiler chicks to Salmonella typhimurium infection. Poult Sci 87(7):1335–1346. https://doi.org/10.3382/ps.2007-00526
Goulet A, Cambillau C (2022) Present impact of alphafold2 revolution on structural biology, and an illustration with the structure prediction of the bacteriophage J-1 host adhesion device. Front Mol Biosci 9:907452. https://doi.org/10.3389/fmolb.2022.907452
Hinkula J, Nyström S, Devito C, Bråve A, Applequist SE (2019) Long-lasting mucosal and systemic immunity against influenza a virus is significantly prolonged and protective by nasal whole influenza immunization with mucosal adjuvant N3 and DNA-plasmid expressing flagellin in aging in- and outbred mice. Vaccines 7:64. https://doi.org/10.3390/vaccines7030064
Il Kim M, Lee C, Park J, Jeon BY, Hong M (2018) Crystal structure of Bacillus cereus flagellin and structure-guided fusion-protein designs. Sci Rep 8:5814. https://doi.org/10.1038/s41598-018-24254-w
Ivison SM, Himmel ME, Hardenberg G, Wark PA, Kifayet A, Levings MK, Steiner TS (2010) TLR5 is not required for flagellin-mediated exacerbation of DSS colitis. Inflamm Bowel Dis 16:401–409. https://doi.org/10.1002/ibd.21097
Jiang W, Shi L, Cai L, Wang X, Li J, Li H, Liang J, Gu Q, Ji G, Li J (2021) A two-adjuvant multiantigen candidate vaccine induces superior protective immune responses against SARS-CoV-2 challenge. Cell Rep 37:110112. https://doi.org/10.1016/j.celrep.2021.110112
Khim K, Bang YJ, Puth S, Choi Y, Lee YS, Jeong K, Lee SE, Rhee JH (2021) Deimmunization of flagellin for repeated administration as a vaccine adjuvant. NPJ Vaccin 6:116. https://doi.org/10.1038/s41541-021-00379-4
Kulandaisamy A, Srivastava A, Kumar P, Nagarajan R, Priya SB, Gromiha MM (2018) Identification and analysis of key residues in protein-RNA complexes. IEEE/ACM Trans Comput Biol Bioinform 15:1436–1444. https://doi.org/10.1109/TCBB.2018.2834387
Lee S, Son JH, Lee LP (2014) Rapid detection of methicillin-resistant staphylococcus aureus using bubble-free microfluidic PCR. Biophys J 106:417a
Liu F, Yang J, Zhang Y, Zhou D, Chen Y, Gai W, Shi W, Li Q, Tien P, Yan H (2010) Recombinant flagellins with partial deletions of the hypervariable domain lose antigenicity but not mucosal adjuvancy. Biochem Biophys Res Commun 392:582–587. https://doi.org/10.1016/j.bbrc.2010.01.077
López-Yglesias AH, Lu CC, Zhao X, Chou T, VandenBos T, Strong RK, Smith KD (2009) FliC’s hypervariable D3 domain is required for robust anti-flagellin primary antibody responses. Immunohorizons 3:422–432. https://doi.org/10.4049/immunohorizons
Nempont C, Cayet D, Rumbo M, Bompard C, Villeret V, Sirard JC (2008) Deletion of flagellin’s hypervariable region abrogates antibody-mediated neutralization and systemic activation of TLR5-dependent immunity. J Immunol 181:2036–2043. https://doi.org/10.4049/jimmunol.181.3.2036
Nene T, Yadav M, Yadav HS (2022) Plant catalase in silico characterization and phylogenetic analysis with structural modeling. J Genet Eng Biotechnol 20:125. https://doi.org/10.1186/s43141-022-00404-6
Pulendran B, Arunachalam PS, O’Hagan DT (2021) Emerging concepts in the science of vaccine adjuvants. Nat Rev Drug Discov 20:454–475. https://doi.org/10.1038/s41573-021-00163-y
Puth S, Verma V, Hong SH, Tan W, Lee SE, Rhee JH (2022) An all-in-one adjuvanted therapeutic cancer vaccine targeting dendritic cell cytosol induces long-lived tumor suppression through NLRC4 inflammasome activation. Biomaterials 286:121542
Rhee JH, Khim K, Puth S, Choi Y, Lee SE (2023) Deimmunization of flagellin adjuvant for clinical application. Curr Opin Virol 60:101330. https://doi.org/10.1016/j.coviro.2023.101330
Salazar-Gonzalez RM, McSorley SJ (2005) Salmonella flagellin, a microbial target of the innate and adaptive immune system. Immunol Lett 101:117–122. https://doi.org/10.1016/j.imlet.2005.05.004
Sawyer N, Arora PS (2018) Hydrogen bond surrogate stabilization of β-hairpins. ACS Chem Biol 13:2027–2032. https://doi.org/10.1021/acschembio.8b00641
Shouval DS, Biswas A, Goettel JA, McCann K, Conaway E, Redhu NS, Mascanfroni ID, Al Adham Z, Lavoie S, Ibourk M (2014) Interleukin-10 receptor signaling in innate immune cells regulates mucosal immune tolerance and anti-inflammatory macrophage function. Immunity 40:706–719. https://doi.org/10.1016/j.immuni.2014.03.011
Song WS, Jeon YJ, Namgung B, Hong M, Yoon SI (2017) A conserved TLR5 binding and activation hot spot on flagellin. Sci Rep 7:40878. https://doi.org/10.1038/srep40878
Sreerama N, Woody RW (2004) Computation and analysis of protein circular dichroism spectra. Methods Enzymol 383:318–351. https://doi.org/10.1016/S0076-6879(04)83013-1
Steimle A, Menz S, Bender A, Ball B, Weber ANR, Hagemann T, Lange A, Maerz JK, Parusel R, Michaelis L (2019) Flagellin hypervariable region determines symbiotic properties of commensal Escherichia coli strains. PLoS Biol 17:e3000334. https://doi.org/10.1371/journal.pbio.3000334
Wangkahart E, Secombes CJ, Wang T (2019) Studies on the use of flagellin as an immunostimulant and vaccine adjuvant in fish aquaculture. Front Immunol 9:3054. https://doi.org/10.3389/fimmu.2018.03054
Yan S, Wang S, Qiu J, Li M, Li D, Xu D, Li D, Liu Q (2021) Raman spectroscopy combined with machine learning for rapid detection of food-borne pathogens at the single-cell level. Talanta 226:122195. https://doi.org/10.1016/j.talanta.2021.122195
Yang Y, Ou B, Xia P, Zhou M, Li L, Zhu G (2016) The flagellin hypervariable region is a potential flagella display domain in probiotic Escherichia coli strain Nissle 1917. Arch Microbiol 198:603–610. https://doi.org/10.1007/s00203-016-1219-3
Yuki K, Mitsui Y, Shibamura-Fujiogi M, Hou L, Odegard KC, Soriano SG, Priebe GP, Koutsogiannaki S (2021) Anesthetics isoflurane and sevoflurane attenuate flagellin-mediated inflammation in the lung. Biochem Biophys Res Commun 557:254–260. https://doi.org/10.1016/j.bbrc.2021.04.045
Acknowledgements
The authors would like to thank the College for Veterinary Medicine, Guizhou University, China, for supporting and helping them to complete this research work in a timely manner. At the same time, we thank all the contributors who provided financial support for this project.
Funding
The Support Plan Project of Science and Technology of Guizhou Province in 2021 (Qian Ke He Support [2021] General 164), the Science and Technology Project of Guizhou Province (QKHJC-ZK[2023] YB105), the Plan of Science and Technology of Guizhou Province (Qian Ke He LH Zi [2017] No.7268), and the project of Introducing Talents in Guizhou University (GDRJHZ [2016]77).
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Conceptualization, Y.Y. and M.W.; data curation, S.L. and G.W.; funding acquisition, Y.Y., M.W. and G.W.; methodology, S.L. and Y.Y.; project administration, Y.Y.; supervision, Y.Y., M.W. and G.W.; validation, S.L.; writing—original draft, S.L.; writing—review and editing, S.L. and Y.Y. All authors have read and agreed to the published version of the manuscript.
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Li, S., Wen, M., Wen, G. et al. Structure and biological activity in vitro of Flagellin and its mutants from Escherichia coli Nissle 1917. Arch Microbiol 206, 221 (2024). https://doi.org/10.1007/s00203-024-03907-7
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DOI: https://doi.org/10.1007/s00203-024-03907-7