{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T07:09:18Z","timestamp":1791270558433,"version":"4.1.0"},"reference-count":43,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2016,1,12]],"date-time":"2016-01-12T00:00:00Z","timestamp":1452556800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2016,1,12]],"date-time":"2016-01-12T00:00:00Z","timestamp":1452556800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>We report the development of a well-defined flagellin-based nanoparticle stimulator and also provide a new mechanism of action model explaining how flagellin-triggered innate immunity has evolved to favor localized rather than potentially debilitating systemic immune stimulation. Cell-free protein synthesis (CFPS) was used to facilitate mutational analysis and precisely orientated display of flagellin on Hepatitis B core (HBc) protein virus-like particles (VLPs). The need for product stability and an understanding of mechanism of action motivated investigations indicating that the D0 domain of flagellin is sensitive to amino acid sequence independent hydrolysis \u2013 apparently due to the need for structural flexibility during natural flagellin polymerization. When D0-stabilized flagellin was attached to HBc VLPs with the D0 domain facing outward, flagellin\u2019s tendency to polymerize caused the VLPs to precipitate. However, attaching the D0 domain to the VLP surface produced a stable nanoparticle adjuvant. Surprisingly, attaching only 2 flagellins per VLP provided the same 1\u2009pM potency as did VLPs with about 33 attached flagellins suggesting that the TLR5 receptor is highly effective in delivering its intracellular signal. These observations suggest that flagellin\u2019s protease sensitivity, tendency to aggregate and very high affinity for TLR5 receptors limit its systemic distribution to favor localized immune stimulation.<\/jats:p>","DOI":"10.1038\/srep18379","type":"journal-article","created":{"date-parts":[[2016,1,12]],"date-time":"2016-01-12T10:04:31Z","timestamp":1452593071000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":54,"title":["Functional properties of flagellin as a stimulator of innate immunity"],"prefix":"10.1038","volume":"6","author":[{"given":"Yuan","family":"Lu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James R.","family":"Swartz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2016,1,12]]},"reference":[{"key":"BFsrep18379_CR1","doi-asserted-by":"crossref","first-page":"1099","DOI":"10.1038\/35074106","volume":"410","author":"F Hayashi","year":"2001","unstructured":"Hayashi, F. et al. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410, 1099\u20131103 (2001).","journal-title":"Nature"},{"key":"BFsrep18379_CR2","first-page":"169","volume":"6","author":"YL Qi","year":"2015","unstructured":"Qi, Y. L. et al. Incorporation of membrane-anchored flagellin or Escherichia coli heat-labile enterotoxin B subunit enhances the innmunogenicity of rabies virus-like particles in mice and dogs. Front. Microbiol. 6, 169 (2015).","journal-title":"Front. Microbiol."},{"key":"BFsrep18379_CR3","doi-asserted-by":"crossref","first-page":"6255","DOI":"10.4049\/jimmunol.1001855","volume":"186","author":"JT Bates","year":"2011","unstructured":"Bates, J. T., Graff, A. H., Phipps, J. P., Grayson, J. M. & Mizel, S. B. Enhanced antigen processing of flagellin fusion proteins promotes the antigen-specific CD8(+) T cell response independently of TLR5 and MyD88. J. Immunol. 186, 6255\u20136262 (2011).","journal-title":"J. Immunol."},{"key":"BFsrep18379_CR4","doi-asserted-by":"crossref","first-page":"4897","DOI":"10.1016\/j.vaccine.2011.05.001","volume":"29","author":"DN Taylor","year":"2011","unstructured":"Taylor, D. N. et al. Induction of a potent immune response in the elderly using the TLR-5 agonist, flagellin, with a recombinant hemagglutinin influenza-flagellin fusion vaccine (VAX125, STF2.HA1 SI). Vaccine 29, 4897\u20134902 (2011).","journal-title":"Vaccine"},{"key":"BFsrep18379_CR5","doi-asserted-by":"crossref","first-page":"2073","DOI":"10.1002\/bit.24903","volume":"110","author":"Y Lu","year":"2013","unstructured":"Lu, Y., Welsh, J. P., Chan, W. & Swartz, J. R. Escherichia coli-based cell free production of flagellin and ordered flagellin display on virus-like particles. Biotechnol. Bioeng. 110, 2073\u20132085 (2013).","journal-title":"Biotechnol. Bioeng."},{"key":"BFsrep18379_CR6","doi-asserted-by":"crossref","first-page":"1247","DOI":"10.1038\/ni1011","volume":"4","author":"KD Smith","year":"2003","unstructured":"Smith, K. D. et al. Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nat. Immunol. 4, 1247\u20131253 (2003).","journal-title":"Nat. Immunol."},{"key":"BFsrep18379_CR7","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1590\/S1517-83822008000100011","volume":"39","author":"CJM Braga","year":"2008","unstructured":"Braga, C. J. M. et al. Cytotoxic T cell adjuvant effects of three Salmonella enterica flagellins. Braz. J. Microbiol. 39, 44\u201349 (2008).","journal-title":"Braz. J. Microbiol."},{"key":"BFsrep18379_CR8","doi-asserted-by":"crossref","first-page":"958","DOI":"10.1128\/JB.188.3.958-967.2006","volume":"188","author":"S Yamamoto","year":"2006","unstructured":"Yamamoto, S. & Kutsukake, K. Flja-mediated posttranscriptional control of phase 1 flagellin expression in flagellar phase variation of Salmonella enterica serovar typhimurium. J. Bacteriol. 188, 958\u2013967 (2006).","journal-title":"J. Bacteriol."},{"key":"BFsrep18379_CR9","doi-asserted-by":"crossref","first-page":"1047","DOI":"10.1002\/bit.22517","volume":"104","author":"WC Yang","year":"2009","unstructured":"Yang, W. C. et al. Cell-free production of transducible transcription factors for nuclear reprogramming. Biotechnol. Bioeng. 104, 1047\u20131058 (2009).","journal-title":"Biotechnol. Bioeng."},{"key":"BFsrep18379_CR10","doi-asserted-by":"crossref","first-page":"2962","DOI":"10.1002\/bit.24581","volume":"109","author":"JP Welsh","year":"2012","unstructured":"Welsh, J. P., Lu, Y., He, X. S., Greenberg, H. B. & Swartz, J. R. Cell-free production of trimeric influenza hemagglutinin head domain proteins as vaccine antigens. Biotechnol. Bioeng. 109, 2962\u20132969 (2012).","journal-title":"Biotechnol. Bioeng."},{"key":"BFsrep18379_CR11","doi-asserted-by":"crossref","first-page":"1689","DOI":"10.1021\/bp034350b","volume":"20","author":"JH Yang","year":"2004","unstructured":"Yang, J. H., Kanter, G., Voloshin, A., Levy, R. & Swartz, J. R. Expression of active murine granulocyte-macrophage colony-stimulating factor in an Escherichia coli cell-free system. Biotechnol. Prog. 20, 1689\u20131696 (2004).","journal-title":"Biotechnol. Prog."},{"key":"BFsrep18379_CR12","doi-asserted-by":"crossref","first-page":"503","DOI":"10.1002\/bit.20283","volume":"89","author":"JH Yang","year":"2005","unstructured":"Yang, J. H. et al. Rapid expression of vaccine proteins for B-cell lymphoma in a cell-free system. Biotechnol. Bioeng. 89, 503\u2013511 (2005).","journal-title":"Biotechnol. Bioeng."},{"key":"BFsrep18379_CR13","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1002\/aic.13701","volume":"58","author":"JR Swartz","year":"2012","unstructured":"Swartz, J. R. Transforming biochemical engineering with cell-free biology. AIChE J. 58, 5\u201313 (2012).","journal-title":"AIChE J."},{"key":"BFsrep18379_CR14","doi-asserted-by":"crossref","first-page":"9247","DOI":"10.1073\/pnas.0502040102","volume":"102","author":"E Andersen-Nissen","year":"2005","unstructured":"Andersen-Nissen, E. et al. Evasion of toll-like receptor 5 by flagellated bacteria. Proc. Natl. Acad. Sci. USA 102, 9247\u20139252 (2005).","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"BFsrep18379_CR15","doi-asserted-by":"crossref","first-page":"5525","DOI":"10.1128\/IAI.68.10.5525-5529.2000","volume":"68","author":"PF McDermott","year":"2000","unstructured":"McDermott, P. F., Ciacci-Woolwine, F., Snipes, J. A. & Mizel, S. B. High-affinity interaction between gram-negative flagellin and a cell surface polypeptide results in human monocyte activation. Infect. Immun. 68, 5525\u20135529 (2000).","journal-title":"Infect. Immun."},{"key":"BFsrep18379_CR16","doi-asserted-by":"crossref","first-page":"6217","DOI":"10.4049\/jimmunol.170.12.6217","volume":"170","author":"SB Mizel","year":"2003","unstructured":"Mizel, S. B., Honko, A. N., Moors, M. A., Smith, P. S. & West, A. P. Induction of macrophage nitric oxide production by gram-negative flagellin involves signalling via heteromeric toll-like receptor 5\/toll-like receptor 4 complexes. J. Immunol. 170, 6217\u20136223 (2003).","journal-title":"J. Immunol."},{"key":"BFsrep18379_CR17","doi-asserted-by":"crossref","first-page":"1147","DOI":"10.4049\/jimmunol.179.2.1147","volume":"179","author":"S Saha","year":"2007","unstructured":"Saha, S. et al. Blocking of the TLR5 activation domain hampers protective potential of flagellin DNA vaccine. J. Immunol. 179, 1147\u20131154 (2007).","journal-title":"J. Immunol."},{"key":"BFsrep18379_CR18","doi-asserted-by":"crossref","first-page":"4103","DOI":"10.1016\/j.vaccine.2009.07.058","volume":"28","author":"I Skountzou","year":"2010","unstructured":"Skountzou, I. et al. Salmonella flagellins are potent adjuvants for intranasally administered whole inactivated influenza vaccine. Vaccine 28, 4103\u20134112 (2010).","journal-title":"Vaccine"},{"key":"BFsrep18379_CR19","doi-asserted-by":"crossref","first-page":"698","DOI":"10.1038\/nnano.2014.169","volume":"9","author":"A Hernandez-Garcia","year":"2014","unstructured":"Hernandez-Garcia, A. et al. Design and self-assembly of simple coat proteins for artificial viruses. Nat. Nanotechnol. 9, 698\u2013702 (2014).","journal-title":"Nat. Nanotechnol."},{"key":"BFsrep18379_CR20","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1007\/s12033-012-9598-4","volume":"53","author":"A Zeltins","year":"2013","unstructured":"Zeltins, A. Construction and characterization of virus-like particles: a review. Mol. Biotechnol. 53, 92\u2013107 (2013).","journal-title":"Mol. Biotechnol."},{"key":"BFsrep18379_CR21","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1016\/j.it.2006.10.005","volume":"27","author":"ST Reddy","year":"2006","unstructured":"Reddy, S. T., Swartz, M. A. & Hubbell, J. A. Targeting dendritic cells with biomaterials: developing the next generation of vaccines. Trends Immunol. 27, 573\u2013579 (2006).","journal-title":"Trends Immunol."},{"key":"BFsrep18379_CR22","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1021\/bc100367u","volume":"22","author":"KG Patel","year":"2011","unstructured":"Patel, K. G. & Swartz, J. R. Surface functionalization of virus-like particles by direct conjugation using azide-alkyne click chemistry. Bioconjugate Chem. 22, 376\u2013387 (2011).","journal-title":"Bioconjugate Chem."},{"key":"BFsrep18379_CR23","doi-asserted-by":"crossref","first-page":"866","DOI":"10.1021\/bc700390r","volume":"19","author":"E Strable","year":"2008","unstructured":"Strable, E. et al. Unnatural amino acid incorporation into virus-like particles. Bioconjugate Chem. 19, 866\u2013875 (2008).","journal-title":"Bioconjugate Chem."},{"key":"BFsrep18379_CR24","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/S0378-1097(02)01208-9","volume":"219","author":"AJ Ozin","year":"2003","unstructured":"Ozin, A. J., Claret, L., Auvray, F. & Hughes, C. The FliS chaperone selectively binds the disordered flagellin C-terminal D0 domain central to polymerisation. FEMS Microbiol. Lett. 219, 219\u2013224 (2003).","journal-title":"FEMS Microbiol. Lett."},{"key":"BFsrep18379_CR25","doi-asserted-by":"crossref","first-page":"e50300","DOI":"10.1371\/journal.pone.0050300","volume":"7","author":"J Song","year":"2012","unstructured":"Song, J. et al. PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites. PLoS One 7, e50300 (2012).","journal-title":"PLoS One"},{"key":"BFsrep18379_CR26","doi-asserted-by":"crossref","first-page":"6659","DOI":"10.1073\/pnas.87.17.6659","volume":"87","author":"LB Evnin","year":"1990","unstructured":"Evnin, L. B., V\u00e1squez, J. R. & Craik, C. S. Substrate specificity of trypsin investigated by using a genetic selection. Proc. Natl. Acad. Sci. USA 87, 6659\u20136663 (1990).","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"BFsrep18379_CR27","doi-asserted-by":"crossref","first-page":"4501","DOI":"10.1021\/cr000033x","volume":"102","author":"L Hedstrom","year":"2002","unstructured":"Hedstrom, L. Serine protease mechanism and specificity. Chem. Rev. 102, 4501\u20134524 (2002).","journal-title":"Chem. Rev."},{"key":"BFsrep18379_CR28","doi-asserted-by":"crossref","first-page":"652","DOI":"10.1038\/292652a0","volume":"292","author":"KH Swamy","year":"1981","unstructured":"Swamy, K. H. & Goldberg, A. L. E. coli contains eight soluble proteolytic activities, one being ATP dependent. Nature 292, 652\u2013654 (1981).","journal-title":"Nature"},{"key":"BFsrep18379_CR29","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1021\/bi971732f","volume":"37","author":"SG Roudiak","year":"1998","unstructured":"Roudiak, S. G., Seth, A., Knipfer, N. & Shrader, T. E. The lon protease from Mycobacterium smegmatis: molecular cloning, sequence analysis, functional expression and enzymatic characterization. Biochemistry 37, 377\u2013386 (1998).","journal-title":"Biochemistry"},{"key":"BFsrep18379_CR30","doi-asserted-by":"crossref","first-page":"1098","DOI":"10.1002\/1521-3773(20020402)41:7<1098::AID-ANIE1098>3.0.CO;2-9","volume":"41","author":"S Walter","year":"2002","unstructured":"Walter, S. & Buchner, J. Molecular chaperones\u2013cellular machines for protein folding. Angew. Chem. Int. Ed. 41, 1098\u20131113 (2002).","journal-title":"Angew. Chem. Int. Ed."},{"key":"BFsrep18379_CR31","doi-asserted-by":"crossref","first-page":"324","DOI":"10.1038\/nature10317","volume":"475","author":"FU Hartl","year":"2011","unstructured":"Hartl, F. U., Bracher, A. & Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475, 324\u2013332 (2011).","journal-title":"Nature"},{"key":"BFsrep18379_CR32","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1038\/nsmb.1614","volume":"16","author":"G Kramer","year":"2009","unstructured":"Kramer, G., Boehringer, D., Ban, N. & Bukau, B. The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins. Nat. Struct. Mol. Biol. 16, 589\u2013597 (2009).","journal-title":"Nat. Struct. Mol. Biol."},{"key":"BFsrep18379_CR33","doi-asserted-by":"crossref","first-page":"574","DOI":"10.1038\/nsmb.1591","volume":"16","author":"FU Hartl","year":"2009","unstructured":"Hartl, F. U. & Hayer-Hartl, M. Converging concepts of protein folding in vitro and in vivo. Nat. Struct. Mol. Biol. 16, 574\u2013581 (2009).","journal-title":"Nat. Struct. Mol. Biol."},{"key":"BFsrep18379_CR34","doi-asserted-by":"crossref","first-page":"563","DOI":"10.1016\/j.bbrc.2009.09.006","volume":"389","author":"JP Welsh","year":"2009","unstructured":"Welsh, J. P., Patel, K. G., Manthiram, K. & Swartz, J. R. Multiply mutated Gaussia luciferases provide prolonged and intense bioluminescence. Biochem. Biophys. Res. Commun. 389, 563\u2013568 (2009).","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"BFsrep18379_CR35","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1007\/s11010-012-1260-8","volume":"365","author":"SS Narayanan","year":"2012","unstructured":"Narayanan, S. S. & Nampoothiri, K. M. Biochemical characterization of recombinant methionine aminopeptidases (MAPs) from Mycobacterium tuberculosis H37Rv. Mol. Cell. Biochem. 365, 191\u2013202 (2012).","journal-title":"Mol. Cell. Biochem."},{"key":"BFsrep18379_CR36","doi-asserted-by":"crossref","first-page":"751","DOI":"10.1128\/jb.169.2.751-757.1987","volume":"169","author":"A Benbassat","year":"1987","unstructured":"Benbassat, A. et al. Processing of the initiation methionine from proteins - properties of the Escherichia coli methionine aminopeptidase and its gene structure. J. Bacteriol. 169, 751\u2013757 (1987).","journal-title":"J. Bacteriol."},{"key":"BFsrep18379_CR37","doi-asserted-by":"crossref","first-page":"2336","DOI":"10.1074\/mcp.M600225-MCP200","volume":"5","author":"F Frottin","year":"2006","unstructured":"Frottin, F. et al. The proteomics of N-terminal methionine cleavage. Mol. Cell. Proteomics 5, 2336\u20132349 (2006).","journal-title":"Mol. Cell. Proteomics"},{"key":"BFsrep18379_CR38","doi-asserted-by":"crossref","first-page":"e7565","DOI":"10.1371\/journal.pone.0007565","volume":"4","author":"JM Kuchenreuther","year":"2009","unstructured":"Kuchenreuther, J. M., Stapleton, J. A. & Swartz, J. R. Tyrosine, cysteine and S-adenosyl methionine stimulate in vitro [FeFe] hydrogenase activation. PLoS One 4, e7565 (2009).","journal-title":"PLoS One"},{"key":"BFsrep18379_CR39","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1073\/pnas.1308701110","volume":"111","author":"Y Lu","year":"2014","unstructured":"Lu, Y., Welsh, J. P. & Swartz, J. R. Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines. Proc. Natl. Acad. Sci. USA 111, 125\u2013130 (2014).","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"BFsrep18379_CR40","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1002\/bit.21567","volume":"99","author":"AR Goerke","year":"2008","unstructured":"Goerke, A. R. & Swartz, J. R. Development of cell-free protein synthesis platforms for disulfide bonded proteins. Biotechnol. Bioeng. 99, 351\u2013367 (2008).","journal-title":"Biotechnol. Bioeng."},{"key":"BFsrep18379_CR41","doi-asserted-by":"crossref","first-page":"1146","DOI":"10.1021\/bp050052y","volume":"21","author":"KA Calhoun","year":"2005","unstructured":"Calhoun, K. A. & Swartz, J. R. An economical method for cell-free protein synthesis using glucose and nucleoside monophosphates. Biotechnol. Prog. 21, 1146\u20131153 (2005).","journal-title":"Biotechnol. Prog."},{"key":"BFsrep18379_CR42","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1038\/nmeth.2089","volume":"9","author":"CA Schneider","year":"2012","unstructured":"Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671\u2013675 (2012).","journal-title":"Nat. Methods"},{"key":"BFsrep18379_CR43","doi-asserted-by":"crossref","first-page":"12360","DOI":"10.1073\/pnas.1510533112","volume":"112","author":"Y Lu","year":"2015","unstructured":"Lu, Y., Chan, W., Ko, B. Y., VanLang, C. C. & Swartz, J. R. Assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery. Proc. Natl. Acad. Sci. USA 112, 12360\u201312365 (2015).","journal-title":"Proc. Natl. Acad. Sci. USA"}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/srep18379","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/srep18379.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/srep18379.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,1,4]],"date-time":"2023-01-04T23:15:34Z","timestamp":1672874134000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/srep18379"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2016,1,12]]},"references-count":43,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2016,5,10]]}},"alternative-id":["BFsrep18379"],"URL":"https:\/\/doi.org\/10.1038\/srep18379","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2016,1,12]]},"assertion":[{"value":"2 July 2015","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"5 October 2015","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"12 January 2016","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing financial interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"18379"}}