{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T03:29:39Z","timestamp":1790220579703,"version":"4.1.0"},"reference-count":37,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2025,3,26]],"date-time":"2025-03-26T00:00:00Z","timestamp":1742947200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100006769","name":"Russian Science Foundation","doi-asserted-by":"publisher","award":["23-15-00132"],"award-info":[{"award-number":["23-15-00132"]}],"id":[{"id":"10.13039\/501100006769","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background\/Objectives:\u00a0Yersinia pestis is an important zoonotic pathogen responsible for the rare but deadly disease of people with bubonic, septic, or pneumonic forms of plague. The emergence of multidrug-resistant Y. pestis strains has attracted more and more researchers\u2019 attention to the search for molecular targets for antivirulence therapy, including anti-nutritional-virulence therapy. The glnALG operon plays a crucial role in regulating the nitrogen content within a bacterial cell. This operon codes for three genes: the structural gene glnA and the two regulatory genes glnL and glnG. In this study, we tested the effect of the deletion of glnA and glnALG on the pathogenic properties of Y. pestis. Methods: To assess the contribution of nitrogen metabolism to Y. pestis virulence, knockout mutants \u0394glnA and \u0394glnALG were constructed. The former was unable to synthesize glutamine, while the latter was not only defective in glutamine synthesis but also lacked the two-component sensor\u2013transcriptional activator pair GlnL and GlnG, which could partially compensate for the decrease in intracellular glutamine concentrations by transporting it from the host or by catabolic reactions. For vaccine studies, immunized mice and guinea pigs were injected s.c. with 200 LD100 of the wild-type Y. pestis strain. Results: A single knockout mutation in the glnA gene did not affect the virulence of Y. pestis in mice and guinea pigs. Knockout of the entire glnALG gene cluster was required for attenuation in these animals. The \u0394glnALG strain of Y. pestis did not cause death in mice (LD50 &gt; 105 CFU) and guinea pigs (LD50 &gt; 107 CFU) when administered subcutaneously and provided 100% protection of animals when subsequently infected with 200 LD100 of the Y. pestis virulent wild-type strain 231. Conclusions:\u00a0Y. pestis, defective in both the glutamine synthetase GlnA and the two-component sensor\u2013transcriptional activator pair GlnL-GlnG, completely lost virulence and provided potent protective immunity to mice and guinea pigs subsequently challenged with a wild-type Y. pestis strain, demonstrating the potential use of the glnALG operon as a new molecular target for developing a safe and efficient live plague vaccine.<\/jats:p>","DOI":"10.3390\/vaccines13040353","type":"journal-article","created":{"date-parts":[[2025,3,28]],"date-time":"2025-03-28T04:36:48Z","timestamp":1743136608000},"page":"353","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Protection Elicited by Glutamine Auxotroph of Yersinia pestis"],"prefix":"10.3390","volume":"13","author":[{"given":"Svetlana V.","family":"Dentovskaya","sequence":"first","affiliation":[{"name":"Laboratory for Plague Microbiology, Especially Dangerous Infections Department, State Research Center for Applied Microbiology and Biotechnology, 142279 Obolensk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7280-3660","authenticated-orcid":false,"given":"Anastasia S.","family":"Vagaiskaya","sequence":"additional","affiliation":[{"name":"Laboratory for Plague Microbiology, Especially Dangerous Infections Department, State Research Center for Applied Microbiology and Biotechnology, 142279 Obolensk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3946-1755","authenticated-orcid":false,"given":"Mikhail E.","family":"Platonov","sequence":"additional","affiliation":[{"name":"Laboratory for Plague Microbiology, Especially Dangerous Infections Department, State Research Center for Applied Microbiology and Biotechnology, 142279 Obolensk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9223-2105","authenticated-orcid":false,"given":"Alexandra S.","family":"Trunyakova","sequence":"additional","affiliation":[{"name":"Laboratory for Plague Microbiology, Especially Dangerous Infections Department, State Research Center for Applied Microbiology and Biotechnology, 142279 Obolensk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5472-8724","authenticated-orcid":false,"given":"Ekaterina A.","family":"Krasil\u2019nikova","sequence":"additional","affiliation":[{"name":"Laboratory for Plague Microbiology, Especially Dangerous Infections Department, State Research Center for Applied Microbiology and Biotechnology, 142279 Obolensk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0776-8392","authenticated-orcid":false,"given":"Elizaveta M.","family":"Mazurina","sequence":"additional","affiliation":[{"name":"Laboratory for Plague Microbiology, Especially Dangerous Infections Department, State Research Center for Applied Microbiology and Biotechnology, 142279 Obolensk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tat\u2019yana V.","family":"Gapel\u2019chenkova","sequence":"additional","affiliation":[{"name":"Laboratory for Plague Microbiology, Especially Dangerous Infections Department, State Research Center for Applied Microbiology and Biotechnology, 142279 Obolensk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8400-5276","authenticated-orcid":false,"given":"Nadezhda A.","family":"Lipatnikova","sequence":"additional","affiliation":[{"name":"Laboratory for Plague Microbiology, Especially Dangerous Infections Department, State Research Center for Applied Microbiology and Biotechnology, 142279 Obolensk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6985-6822","authenticated-orcid":false,"given":"Rima Z.","family":"Shaikhutdinova","sequence":"additional","affiliation":[{"name":"Laboratory for Plague Microbiology, Especially Dangerous Infections Department, State Research Center for Applied Microbiology and Biotechnology, 142279 Obolensk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sergei A.","family":"Ivanov","sequence":"additional","affiliation":[{"name":"Laboratory for Plague Microbiology, Especially Dangerous Infections Department, State Research Center for Applied Microbiology and Biotechnology, 142279 Obolensk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1959-1739","authenticated-orcid":false,"given":"Tat\u2019yana I.","family":"Kombarova","sequence":"additional","affiliation":[{"name":"Laboratory of Biomodels, State Research Center for Applied Microbiology and Biotechnology, 142279 Obolensk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3811-9691","authenticated-orcid":false,"given":"Florent","family":"Sebbane","sequence":"additional","affiliation":[{"name":"Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur Lille, U1019\u2014UMR 9017\u2014CIIL\u2014Center for Infection and Immunityof Lille, F-59000 Lille, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5499-7999","authenticated-orcid":false,"given":"Andrey P.","family":"Anisimov","sequence":"additional","affiliation":[{"name":"Laboratory for Plague Microbiology, Especially Dangerous Infections Department, State Research Center for Applied Microbiology and Biotechnology, 142279 Obolensk, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,26]]},"reference":[{"key":"ref_1","unstructured":"Reitzer, L.J., Neidhardt, F., Ingraham, J.L., Low, K.B., Magasanik, B., Schaechter, M., and Umbarger, H.E. (1987). Ammonia Assimilation and the Biosynthesis of Glutamine, Glutamate, Aspartate, Asparagine, L-alanine, and D-alanine. Escherichia coli and Salmonella Typhimurium: Cellular and Molecular Biology, ASM Press."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1006\/jmbi.1996.0342","article-title":"Salmonella Typhimurium Apparently Perceives External Nitrogen Limitation as Internal Glutamine Limitation","volume":"259","author":"Ikeda","year":"1996","journal-title":"J. Mol. Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"14674","DOI":"10.1073\/pnas.97.26.14674","article-title":"Nitrogen Regulatory Protein C-Controlled Genes of Escherichia coli: Scavenging as a Defense against Nitrogen Limitation","volume":"97","author":"Zimmer","year":"2000","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"260","DOI":"10.1007\/BF00430437","article-title":"Cloning and Complete Nucleotide Sequence of the Escherichia coli Glutamine Permease Operon (glnHPQ)","volume":"205","author":"Nohno","year":"1986","journal-title":"Molec. Gen. Genet."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1714","DOI":"10.1128\/AAC.05558-11","article-title":"Deciphering the Magainin Resistance Process of Escherichia coli Strains in Light of the Cytosolic Proteome","volume":"56","author":"Rodrigues","year":"2012","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"El Khoury, J.Y., Boucher, N., Bergeron, M.G., Leprohon, P., and Ouellette, M. (2017). Penicillin Induces Alterations in Glutamine Metabolism in Streptococcus pneumoniae. Sci. Rep., 7.","DOI":"10.1038\/s41598-017-15035-y"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1460","DOI":"10.1128\/jb.176.5.1460-1467.1994","article-title":"The femC Locus of Staphylococcus aureus Required for Methicillin Resistance Includes the Glutamine Synthetase Operon","volume":"176","author":"Gustafson","year":"1994","journal-title":"J. Bacteriol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Millanao, A.R., Mora, A.Y., Saavedra, C.P., Villagra, N.A., Mora, G.C., and Hidalgo, A.A. (2020). Inactivation of Glutamine Synthetase-Coding Gene glnA Increases Susceptibility to Quinolones Through Increasing Outer Membrane Protein F in Salmonella enterica Serovar Typhi. Front. Microbiol., 11.","DOI":"10.3389\/fmicb.2020.00428"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"456","DOI":"10.1128\/IAI.71.1.456-464.2003","article-title":"Inhibition of Mycobacterium tuberculosis Glutamine Synthetase as a Novel Antibiotic Strategy against Tuberculosis: Demonstration of Efficacy In Vivo","volume":"71","author":"Harth","year":"2003","journal-title":"Infect. Immun."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Crowther, G.J., Shanmugam, D., Carmona, S.J., Doyle, M.A., Hertz-Fowler, C., Berriman, M., Nwaka, S., Ralph, S.A., Roos, D.S., and Van Voorhis, W.C. (2010). Identification of Attractive Drug Targets in Neglected-Disease Pathogens Using an In Silico Approach. PLoS Negl. Trop. Dis., 4.","DOI":"10.1371\/journal.pntd.0000804"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1046\/j.1365-2958.1999.01333.x","article-title":"Regulation of Nitrogen Metabolism in Bacillus subtilis: Vive La Diff\u00e9rence!","volume":"32","author":"Fisher","year":"1999","journal-title":"Mol. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"587","DOI":"10.1128\/iai.65.2.587-596.1997","article-title":"Simultaneous Prevention of Glutamine Synthesis and High-Affinity Transport Attenuates Salmonella Typhimurium Virulence","volume":"65","author":"Klose","year":"1997","journal-title":"Infect. Immun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3669","DOI":"10.1099\/mic.0.043828-0","article-title":"Glutamine Synthetase Encoded by glnA-1 Is Necessary for Cell Wall Resistance and Pathogenicity of Mycobacterium bovis","volume":"156","author":"Chandra","year":"2010","journal-title":"Microbiology"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"e01838-17","DOI":"10.1128\/AEM.01838-17","article-title":"glnA Truncation in Salmonella Enterica Results in a Small Colony Variant Phenotype, Attenuated Host Cell Entry, and Reduced Expression of Flagellin and SPI-1-Associated Effector Genes","volume":"84","author":"Aurass","year":"2018","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Anisimov, A.P., Vagaiskaya, A.S., Trunyakova, A.S., and Dentovskaya, S.V. (2025). Live Plague Vaccine Development: Past, Present, and Future. Vaccines, 13.","DOI":"10.3390\/vaccines13010066"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1795","DOI":"10.2147\/DDDT.S98939","article-title":"The Arsenal of Pathogens and Antivirulence Therapeutic Strategies for Disarming Them","volume":"2016","author":"Hadjifrangiskou","year":"2016","journal-title":"DDDT"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Asha, I.J., Gupta, S.D., Hossain, M.M., Islam, M.N., Akter, N.N., Islam, M.M., Das, S.C., and Barman, D.N. (2024). In Silico Characterization of a Hypothetical Protein (PBJ89160.1) from Neisseria meningitidis Exhibits a New Insight on Nutritional Virulence and Molecular Docking to Uncover a Therapeutic Target. Evol. Bioinform. Online, 20.","DOI":"10.1177\/11769343241298307"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1367","DOI":"10.1093\/infdis\/jiu274","article-title":"Yersinia pestis Requires the 2-Component Regulatory System OmpR-EnvZ to Resist Innate Immunity during the Early and Late Stages of Plague","volume":"210","author":"Reboul","year":"2014","journal-title":"J. Infect. Dis."},{"key":"ref_19","first-page":"30","article-title":"Comparative Sequencing of Transcriptomes of Yersinia pestis subsp. microti bv. Ulegeica Different by Virulence for Guinea Pigs","volume":"2","author":"Solomentsev","year":"2017","journal-title":"Russ. J. Bacteriol."},{"key":"ref_20","unstructured":"Anisimov, A.P., Kislichkina, A.A., Kopylov PKh Krasilnikova, E.A., Bogun, A.G., and Dentovskaya, S.V. (2017, January 5). Search for Factors of Selective Virulence of Rhamnose-Positive Strains of Yersinia pestis. Proceedings of the 22nd International Scientific Conference \u201cCurrent Issues on Zoonotic Diseases\u201d, Ulaanbaatar, Mongolia. (In Russian)."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e01373-20","DOI":"10.1128\/mra.01373-20","article-title":"Whole-Genome Assembly of Yersinia pestis 231, the Russian Reference Strain for Testing Plague Vaccine Protection","volume":"10","author":"Kislichkina","year":"2021","journal-title":"Microbiol. Resour. Announc."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"6640","DOI":"10.1073\/pnas.120163297","article-title":"One-Step Inactivation of Chromosomal Genes in Escherichia coli K-12 Using PCR Products","volume":"97","author":"Datsenko","year":"2000","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/0378-1119(95)00193-A","article-title":"Gene Disruption in Escherichia coli: TcR and KmR Cassettes with the Option of Flp-Catalyzed Excision of the Antibiotic-Resistance Determinant","volume":"158","author":"Cherepanov","year":"1995","journal-title":"Gene"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4310","DOI":"10.1128\/iai.59.12.4310-4317.1991","article-title":"Construction of an eae Deletion Mutant of Enteropathogenic Escherichia coli by Using a Positive-Selection Suicide Vector","volume":"59","author":"Donnenberg","year":"1991","journal-title":"Infect. Immun."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Dentovskaya, S.V., Vagaiskaya, A.S., Platonov, M.E., Trunyakova, A.S., Kotov, S.A., Krasil\u2019nikova, E.A., Titareva, G.M., Mazurina, E.M., Gapel\u2019chenkova, T.V., and Shaikhutdinova, R.Z. (2021). Peptidoglycan-Free Bacterial Ghosts Confer Enhanced Protection against Yersinia pestis Infection. Vaccines, 10.","DOI":"10.20944\/preprints202111.0558.v1"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Trunyakova, A.S., Platonov, M.E., Ivanov, S.A., Kopylov, P.K., Dentovskaya, S.V., and Anisimov, A.P. (2024). A Recombinant Low-Endotoxic Yersinia pseudotuberculosis Strain\u2014over-Producer of Yersinia pestis F1 Antigen. Mol. Gen. Microbio. Viro., 42.","DOI":"10.17116\/molgen20244202110"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"524","DOI":"10.1134\/S0003683819040082","article-title":"Characteristics of the Chromatographic Cleaning and Protectiveness of the LcrV Isoform of Yersinia pestis","volume":"55","author":"Kopylov","year":"2019","journal-title":"Appl. Biochem. Microbiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"965","DOI":"10.1517\/14712598.4.6.965","article-title":"Yersinia pestis (Plague) Vaccines","volume":"4","author":"Titball","year":"2004","journal-title":"Expert Opin. Biol. Ther."},{"key":"ref_29","unstructured":"Finney, D. (1978). Statistical Method in Biological Assay, Charles Griffin. [3rd ed.]."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2757","DOI":"10.1093\/nar\/15.6.2757","article-title":"The Complete Nucleotide Sequence of the ginALG Operon of Escherichia coli K12","volume":"15","author":"Urdea","year":"1987","journal-title":"Nucl. Acids Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4356","DOI":"10.1128\/IAI.00242-09","article-title":"High-Throughput Identification of New Protective Antigens from a Yersinia pestis Live Vaccine by Enzyme-Linked Immunospot Assay","volume":"77","author":"Li","year":"2009","journal-title":"Infect. Immun."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"628","DOI":"10.1128\/MMBR.00025-13","article-title":"Nitrogen Assimilation in Escherichia coli: Putting Molecular Data into a Systems Perspective","volume":"77","author":"Westerhoff","year":"2013","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"882","DOI":"10.1111\/cmi.12138","article-title":"Microbial Quest for Food in Vivo: \u2018Nutritional Virulence\u2019 as an Emerging Paradigm","volume":"15","author":"Bumann","year":"2013","journal-title":"Cell Microbiol."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Eisenreich, W., Heesemann, J., Rudel, T., and Goebel, W. (2013). Metabolic Host Responses to Infection by Intracellular Bacterial Pathogens. Front. Cell. Infect. Microbiol., 3.","DOI":"10.3389\/fcimb.2013.00024"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"604","DOI":"10.1128\/mr.59.4.604-622.1995","article-title":"Nitrogen Control in Bacteria","volume":"59","author":"Merrick","year":"1995","journal-title":"Microbiol. Rev."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Henle, W., Kikuth, W., Meyer, K.F., Nauck, E.G., and Tomcsik, J. (1963). Virulence of Pasteurella pestis and immunity to plague. Ergebnisse der Mikrobiologie Immunit\u00e4tsforschung und Experimentellen Therapie, Springer.","DOI":"10.1007\/978-3-662-36742-1"},{"key":"ref_37","unstructured":"Anisimova, T.I., Sayapina, L.V., Sergeeva, G.M., Isupov, I.V., Beloborodov, R.A., Samoilova, L.V., Anisimov, A.P., Ledvanov, M.Y., Shvedun, G.P., and Zadumina, S.Y. (2002). Main Requirements for Vaccine Strains of the Plague Pathogen: Methodological Guidelines MU 3.3.1.1113-02, Federal Centre of State Epidemic Surveillance of Ministry of Health of Russian Federation."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/13\/4\/353\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:02:40Z","timestamp":1760029360000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/13\/4\/353"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,3,26]]},"references-count":37,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2025,4]]}},"alternative-id":["vaccines13040353"],"URL":"https:\/\/doi.org\/10.3390\/vaccines13040353","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,3,26]]}}}