{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,1]],"date-time":"2026-04-01T20:23:09Z","timestamp":1775074989495,"version":"3.50.1"},"reference-count":54,"publisher":"American Society for Microbiology","issue":"2","license":[{"start":{"date-parts":[[2016,4,26]],"date-time":"2016-04-26T00:00:00Z","timestamp":1461628800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2016,5,4]],"date-time":"2016-05-04T00:00:00Z","timestamp":1462320000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"name":"National Key Basic Research Program of China","award":["2011CB504901"],"award-info":[{"award-number":["2011CB504901"]}]},{"name":"National Key Basic Research Program of China","award":["2013CB910804"],"award-info":[{"award-number":["2013CB910804"]}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["81125012"],"award-info":[{"award-number":["81125012"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["81373316"],"award-info":[{"award-number":["81373316"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["81471912"],"award-info":[{"award-number":["81471912"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mBio"],"published-print":{"date-parts":[[2016,5,4]]},"abstract":"<jats:title>ABSTRACT\u00a0<\/jats:title><jats:p>Conjugate vaccines are known to be one of the most effective and safest types of vaccines against bacterial pathogens. Previously, vaccine biosynthesis has been performed by using N-linked glycosylation systems. However, the structural specificity of these systems for sugar substrates has hindered their application. Here, we report a novel protein glycosylation system (O-linked glycosylation via<jats:named-content content-type=\"genus-species\">Neisseria meningitidis<\/jats:named-content>) that can transfer virtually any glycan to produce a conjugate vaccine. We successfully established this system in<jats:italic>Shigella<\/jats:italic>spp., avoiding the construction of an expression vector for polysaccharide synthesis. We further found that different protein substrates can be glycosylated using this system and that the O-linked glycosylation system can also effectively function in other Gram-negative bacteria, including some strains whose polysaccharide structure was not suitable for conjugation using the N-linked glycosylation system. The results from a series of animal experiments show that the conjugate vaccine produced by this O-linked glycosylation system offered a potentially protective antibody response. Furthermore, we elucidated and optimized the recognition motif, named MOOR, for the<jats:italic>O<\/jats:italic>-glycosyltransferase PglL. Finally, we demonstrated that the fusion of other peptides recognized by major histocompatibility complex class II around MOOR had no adverse effects on substrate glycosylation, suggesting that this optimized system will be useful for future vaccine development. Our results expand the glycoengineering toolbox and provide a simpler and more robust strategy for producing bioconjugate vaccines against a variety of pathogens.<\/jats:p><jats:p><jats:bold>IMPORTANCE<\/jats:bold>Recently, the rapid development of synthetic biology has allowed bioconjugate vaccines with N-linked protein glycosylation to become a reality. However, the difficulty of reestablishing the exogenous polysaccharide synthetic pathway in<jats:named-content content-type=\"genus-species\">Escherichia coli<\/jats:named-content>hinders their application. Here, we show that an O-linked protein glycosylation system from<jats:named-content content-type=\"genus-species\">Neisseria meningitidis<\/jats:named-content>, which has a lower structure specificity for sugar substrates, could be engineered directly in attenuated pathogens to produce effective conjugate vaccines. To facilitate the further design of next-generation bioconjugate vaccines, we optimized a novel short motif consisting of 8\u00a0amino acids that is sufficient for glycosylation. Our results expand the application potential of O-linked protein glycosylation and demonstrate a simpler and more robust strategy for producing bioconjugate vaccines against different pathogens. In the future, bacterial antigenic polysaccharides could be attached to major histocompatibility complex binding peptides to improve immunological memory or attached to protein subunit vaccine candidates to provide double immune stimulation.<\/jats:p>","DOI":"10.1128\/mbio.00443-16","type":"journal-article","created":{"date-parts":[[2016,4,27]],"date-time":"2016-04-27T01:43:09Z","timestamp":1461721389000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":86,"title":["Biosynthesis of Conjugate Vaccines Using an O-Linked Glycosylation System"],"prefix":"10.1128","volume":"7","author":[{"given":"Chao","family":"Pan","sequence":"first","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, Fengtai, Beijing, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peng","family":"Sun","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, Fengtai, Beijing, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bo","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, Fengtai, Beijing, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Haoyu","family":"Liang","sequence":"additional","affiliation":[{"name":"National Institutes for Food and Drug Control, Beijing, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhehui","family":"Peng","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, Fengtai, Beijing, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yan","family":"Dong","sequence":"additional","affiliation":[{"name":"National Institutes for Food and Drug Control, Beijing, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dongshu","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, Fengtai, Beijing, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiankai","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, Fengtai, Beijing, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bin","family":"Wang","sequence":"additional","affiliation":[{"name":"National Institutes for Food and Drug Control, Beijing, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ming","family":"Zeng","sequence":"additional","affiliation":[{"name":"National Institutes for Food and Drug Control, Beijing, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jun","family":"Wu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, Fengtai, Beijing, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9975-8978","authenticated-orcid":false,"given":"Li","family":"Zhu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, Fengtai, Beijing, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hengliang","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, Fengtai, Beijing, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1084\/jem.54.3.437"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/0952-7915(95)80109-X"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1038\/nm.2535"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1093\/bmb\/ldh021"},{"key":"e_1_3_2_6_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(07)60564-9"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.1086\/587897"},{"key":"e_1_3_2_8_2","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2958.1999.01415.x"},{"key":"e_1_3_2_9_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.298.5599.1790"},{"key":"e_1_3_2_10_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0500044102"},{"key":"e_1_3_2_11_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.1995.mmi_17061201.x"},{"key":"e_1_3_2_12_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.01318-07"},{"key":"e_1_3_2_13_2","doi-asserted-by":"publisher","DOI":"10.1093\/glycob\/cwq148"},{"key":"e_1_3_2_14_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2006.06.182"},{"key":"e_1_3_2_15_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M206114200"},{"key":"e_1_3_2_16_2","doi-asserted-by":"publisher","DOI":"10.1038\/sj.emboj.7601087"},{"key":"e_1_3_2_17_2","doi-asserted-by":"publisher","DOI":"10.1186\/1475-2859-9-61"},{"key":"e_1_3_2_18_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2015.06.102"},{"key":"e_1_3_2_19_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0509207103"},{"key":"e_1_3_2_20_2","doi-asserted-by":"publisher","DOI":"10.1021\/bi602633n"},{"key":"e_1_3_2_21_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.0020031"},{"key":"e_1_3_2_22_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1574-6968.2011.02394.x"},{"key":"e_1_3_2_23_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.03742-12"},{"key":"e_1_3_2_24_2","doi-asserted-by":"publisher","DOI":"10.1111\/1574-6976.12034"},{"key":"e_1_3_2_25_2","doi-asserted-by":"publisher","DOI":"10.1038\/378032a0"},{"key":"e_1_3_2_26_2","doi-asserted-by":"publisher","DOI":"10.1093\/glycob\/cwt087"},{"key":"e_1_3_2_27_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M807113200"},{"key":"e_1_3_2_28_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0809504106"},{"key":"e_1_3_2_29_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0062768"},{"key":"e_1_3_2_30_2","doi-asserted-by":"publisher","DOI":"10.1093\/glycob\/cwh151"},{"key":"e_1_3_2_31_2","doi-asserted-by":"publisher","DOI":"10.1111\/jphp.12321"},{"key":"e_1_3_2_32_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.120163297"},{"key":"e_1_3_2_33_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.177.4.1059-1068.1995"},{"key":"e_1_3_2_34_2","doi-asserted-by":"publisher","DOI":"10.1099\/00221287-146-1-1"},{"key":"e_1_3_2_35_2","first-page":"215","article-title":"Mucosal vaccines based on the use of cholera toxin B subunit as immunogen and antigen carrier","volume":"82","author":"Lebens M","year":"1994","unstructured":"Lebens M, Holmgren J. 1994. Mucosal vaccines based on the use of cholera toxin B subunit as immunogen and antigen carrier. Dev Biol Stand 82:215\u2013227.","journal-title":"Dev Biol Stand"},{"key":"e_1_3_2_36_2","doi-asserted-by":"publisher","DOI":"10.1139\/o86-004"},{"key":"e_1_3_2_37_2","doi-asserted-by":"publisher","DOI":"10.1128\/iai.64.7.2709-2715.1996"},{"key":"e_1_3_2_38_2","doi-asserted-by":"publisher","DOI":"10.1006\/jmbi.1995.0455"},{"key":"e_1_3_2_39_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2006.07.004"},{"key":"e_1_3_2_40_2","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.1300467"},{"key":"e_1_3_2_41_2","doi-asserted-by":"publisher","DOI":"10.1038\/300066a0"},{"key":"e_1_3_2_42_2","doi-asserted-by":"crossref","first-page":"717","DOI":"10.4049\/jimmunol.149.2.717","article-title":"Use of human universally antigenic tetanus toxin T cell epitopes as carriers for human vaccination","volume":"149","author":"Valmori D","year":"1992","unstructured":"Valmori D, Pessi A, Bianchi E, Corradin G. 1992. Use of human universally antigenic tetanus toxin T cell epitopes as carriers for human vaccination. J Immunol 149:717\u2013721.","journal-title":"J Immunol"},{"key":"e_1_3_2_43_2","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201300390"},{"key":"e_1_3_2_44_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2015.07.100"},{"key":"e_1_3_2_45_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2005.04.004"},{"key":"e_1_3_2_46_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.virusres.2004.02.030"},{"key":"e_1_3_2_47_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2008.09.038"},{"key":"e_1_3_2_48_2","doi-asserted-by":"publisher","DOI":"10.1086\/323648"},{"key":"e_1_3_2_49_2","doi-asserted-by":"publisher","DOI":"10.1099\/jmm.0.039438-0"},{"key":"e_1_3_2_50_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2009.06.013"},{"key":"e_1_3_2_51_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.biochem.75.103004.142545"},{"key":"e_1_3_2_52_2","doi-asserted-by":"publisher","DOI":"10.1098\/rsob.140227"},{"key":"e_1_3_2_53_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2006.11.032"},{"key":"e_1_3_2_54_2","doi-asserted-by":"publisher","DOI":"10.1093\/glycob\/7.8.1053-c"},{"key":"e_1_3_2_55_2","doi-asserted-by":"publisher","DOI":"10.1093\/glycob\/6.4.439"}],"container-title":["mBio"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/mBio.00443-16","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/mBio.00443-16","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,8,17]],"date-time":"2023-08-17T19:54:43Z","timestamp":1692302083000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/mBio.00443-16"}},"subtitle":[],"editor":[{"given":"Antonio","family":"Cassone","sequence":"additional","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2016,5,4]]},"references-count":54,"journal-issue":{"issue":"2","published-print":{"date-parts":[[2016,5,4]]}},"alternative-id":["10.1128\/mBio.00443-16"],"URL":"https:\/\/doi.org\/10.1128\/mbio.00443-16","relation":{},"ISSN":["2161-2129","2150-7511"],"issn-type":[{"value":"2161-2129","type":"print"},{"value":"2150-7511","type":"electronic"}],"subject":[],"published":{"date-parts":[[2016,5,4]]},"assertion":[{"value":"2016-03-11","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2016-03-23","order":1,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2016-04-26","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e00443-16"}}