{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T06:19:45Z","timestamp":1790662785398,"version":"4.1.0"},"reference-count":53,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2022,7,21]],"date-time":"2022-07-21T00:00:00Z","timestamp":1658361600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003246","name":"The Netherlands Organization for Scientific Research (NWO)","doi-asserted-by":"publisher","award":["14921"],"award-info":[{"award-number":["14921"]}],"id":[{"id":"10.13039\/501100003246","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The Gram-negative bacterium Bordetella pertussis is the causative agent of a respiratory infection known as whooping cough. Previously developed whole-cell pertussis vaccines were effective, but appeared to be too reactogenic mainly due to the presence of lipopolysaccharide (LPS, also known as endotoxin) in the outer membrane (OM). Here, we investigated the possibility of reducing endotoxicity by modulating the LPS levels. The promoter of the lpxC gene, which encodes the first committed enzyme in LPS biosynthesis, was replaced by an isopropyl \u03b2-D-1-thiogalactopyranoside (IPTG)-inducible promoter. The IPTG was essential for growth, even when the construct was moved into a strain that should allow for the replacement of LPS in the outer leaflet of the OM with phospholipids by defective phospholipid transporter Mla and OM phospholipase A. LpxC depletion in the absence of IPTG resulted in morphological changes of the cells and in overproduction of outer-membrane vesicles (OMVs). The reduced amounts of LPS in whole-cell preparations and in isolated OMVs of LpxC-depleted cells resulted in lower activation of Toll-like receptor 4 in HEK-Blue reporter cells. We suggest that, besides lipid A engineering, also a reduction in LPS synthesis is an attractive strategy for the production of either whole-cell- or OMV-based vaccines, with reduced reactogenicity for B. pertussis and other Gram-negative bacteria.<\/jats:p>","DOI":"10.3390\/ijms23148027","type":"journal-article","created":{"date-parts":[[2022,7,21]],"date-time":"2022-07-21T10:36:56Z","timestamp":1658399816000},"page":"8027","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Regulated Expression of lpxC Allows for Reduction of Endotoxicity in Bordetella pertussis"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8022-499X","authenticated-orcid":false,"given":"Jes\u00fas","family":"P\u00e9rez-Ortega","sequence":"first","affiliation":[{"name":"Section Molecular Microbiology, Department of Biology, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands"},{"name":"Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ria","family":"van Boxtel","sequence":"additional","affiliation":[{"name":"Section Molecular Microbiology, Department of Biology, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3089-5899","authenticated-orcid":false,"given":"Eline F.","family":"de Jonge","sequence":"additional","affiliation":[{"name":"Section Molecular Microbiology, Department of Biology, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands"},{"name":"Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7633-4945","authenticated-orcid":false,"given":"Jan","family":"Tommassen","sequence":"additional","affiliation":[{"name":"Section Molecular Microbiology, Department of Biology, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands"},{"name":"Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,7,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"a000414","DOI":"10.1101\/cshperspect.a000414","article-title":"The bacterial cell envelope","volume":"2","author":"Silhavy","year":"2010","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"635","DOI":"10.1146\/annurev.biochem.71.110601.135414","article-title":"Lipopolysaccharide endotoxins","volume":"71","author":"Raetz","year":"2002","journal-title":"Annu. Rev. Biochem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"449","DOI":"10.1038\/33046","article-title":"Meningitis bacterium is viable without endotoxin","volume":"392","author":"Steeghs","year":"1998","journal-title":"Nature"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"7569","DOI":"10.1128\/IAI.73.11.7569-7577.2005","article-title":"Moraxella catarrhalis bacterium without endotoxin, a potential vaccine candidate","volume":"73","author":"Peng","year":"2005","journal-title":"Infect. Immun."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"4971","DOI":"10.1128\/AAC.00834-10","article-title":"Colistin resistance in Acinetobacter baumannii is mediated by complete loss of lipopolysaccharide production","volume":"54","author":"Moffatt","year":"2010","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"326","DOI":"10.1128\/CMR.18.2.326-382.2005","article-title":"Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies","volume":"18","author":"Mattoo","year":"2005","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1344","DOI":"10.3389\/fimmu.2019.01344","article-title":"Pertussis prevention: Reasons for resurgence, and differences in the current acellular pertussis vaccines","volume":"10","author":"Esposito","year":"2019","journal-title":"Front. Immunol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1016\/j.coi.2019.03.006","article-title":"Pertussis vaccines and protective immunity","volume":"59","author":"Kapil","year":"2019","journal-title":"Curr. Opin. Immunol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"605","DOI":"10.1038\/nrmicro3525","article-title":"Outer-membrane vesicles from Gram-negative bacteria: Biogenesis and functions","volume":"13","author":"Schwechheimer","year":"2015","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3080","DOI":"10.1016\/j.vaccine.2007.01.022","article-title":"Safety review: Two outer membrane vesicle (OMV) vaccines against systemic Neisseria meningitidis serogroup B disease","volume":"25","author":"Aavitsland","year":"2007","journal-title":"Vaccine"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"174","DOI":"10.4161\/hv.18500","article-title":"Bexsero: A multicomponent vaccine for prevention of meningococcal disease","volume":"8","author":"Gorringe","year":"2012","journal-title":"Hum. Vaccin. Immunother."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"38240","DOI":"10.1038\/srep38240","article-title":"Bordetella pertussis outer membrane vesicle vaccine confers equal efficacy in mice with milder inflammatory responses compared to a whole-cell vaccine","volume":"6","author":"Raeven","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1038\/s41579-019-0201-x","article-title":"Pushing the envelope: LPS modifications and their consequences","volume":"17","author":"Simpson","year":"2019","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Kawahara, K. (2021). Variation, modification and engineering of lipid A in endotoxin of Gram-negative bacteria. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22052281"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"5574","DOI":"10.1128\/IAI.00834-06","article-title":"Expression of the lipopolysaccharide-modifying enzymes PagP and PagL modulates the endotoxic activity of Bordetella pertussis","volume":"74","author":"Geurtsen","year":"2006","journal-title":"Infect. Immun."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Arenas, J., Pupo, E., Phielix, C., David, D., Zariri, A., Zamyatina, A., Tommassen, J., and van der Ley, P. (2020). Shortening the lipid A acyl chains of Bordetella pertussis enables depletion of lipopolysaccharide endotoxic activity. Vaccines, 8.","DOI":"10.3390\/vaccines8040594"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"334","DOI":"10.1016\/j.tim.2020.09.008","article-title":"Border control: Regulating LPS biogenesis","volume":"29","author":"Guest","year":"2021","journal-title":"Trends Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"14829","DOI":"10.1074\/jbc.M113.539494","article-title":"Assembly of lipopolysaccharide in Escherichia coli requires the essential LapB heat shock protein","volume":"289","author":"Klein","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Biernacka, D., Gorzelak, P., Klein, G., and Raina, S. (2020). Regulation of the first committed step in lipopolysaccharide biosynthesis catalyzed by LpxC requires the essential protein LapC (YejM) and HslVU protease. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21239088"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e00939-20","DOI":"10.1128\/mBio.00939-20","article-title":"An essential membrane protein modulates the proteolysis of LpxC to control lipopolysaccharide synthesis in Escherichia coli","volume":"11","author":"Fivenson","year":"2020","journal-title":"mBio"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e00598-20","DOI":"10.1128\/mBio.00598-20","article-title":"YejM modulates activity of the YciM\/FtsH protease complex to prevent lethal accumulation of lipopolysaccharide","volume":"11","author":"Guest","year":"2020","journal-title":"mBio"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1038\/s41586-020-2597-x","article-title":"Structure of the essential inner membrane lipopolysaccharide\u2013PbgA complex","volume":"584","author":"Clairfeuille","year":"2020","journal-title":"Nature"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Klein, G., and Raina, S. (2019). Regulated assembly of LPS, its structural alterations and cellular response to LPS defects. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20020356"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1090","DOI":"10.1128\/JB.01043-10","article-title":"Control of lipopolysaccharide biosynthesis by FtsH-mediated proteolysis of LpxC is conserved in enterobacteria but not in all Gram-negative bacteria","volume":"193","author":"Langklotz","year":"2011","journal-title":"J. Bacteriol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"780","DOI":"10.1128\/JB.00943-13","article-title":"Ght protein of Neisseria meningitidis is involved in the regulation of lipopolysaccharide biosynthesis","volume":"196","author":"Putker","year":"2014","journal-title":"J. Bacteriol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e00379-18","DOI":"10.1128\/mBio.00379-18","article-title":"The Escherichia coli phospholipase PldA regulates outer membrane homeostasis via lipid signaling","volume":"9","author":"May","year":"2018","journal-title":"mBio"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"E1565","DOI":"10.1073\/pnas.1601375113","article-title":"Disruption of lipid homeostasis in the Gram-negative cell envelope activates a novel cell death pathway","volume":"113","author":"Sutterlin","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"de Jonge, E.F., Vogrinec, L., van Boxtel, R., and Tommassen, J. (2022). Inactivation of the Mla System and Outer-Membrane Phospholipase a Results in Disrupted Outer-Membrane Lipid Asymmetry and Hypervesiculation in Bordetella pertussis. [Ph.D. Thesis, Utrecht University].","DOI":"10.1016\/j.crmicr.2022.100172"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e00694-18","DOI":"10.1128\/mSphere.00694-18","article-title":"In vivo gene essentiality and metabolism in Bordetella pertussis","volume":"4","author":"Gonyar","year":"2019","journal-title":"mSphere"},{"key":"ref_30","first-page":"e000496","article-title":"Fundamental differences in physiology of Bordetella pertussis dependent on the two-component system Bvg revealed by gene essentiality studies","volume":"6","author":"Belcher","year":"2020","journal-title":"Microb. Genom."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e00420-19","DOI":"10.1128\/JB.00420-19","article-title":"Robust suppression of lipopolysaccharide deficiency in Acinetobacter baumannii by growth in minimal medium","volume":"201","author":"Nagy","year":"2019","journal-title":"J. Bacteriol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"E8518","DOI":"10.1073\/pnas.1806714115","article-title":"Phospholipid retention in the absence of asymmetry strengthens the outer membrane permeability barrier to last-resort antibiotics","volume":"115","author":"Powers","year":"2018","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"101433","DOI":"10.1016\/j.smim.2020.101433","article-title":"Outer membrane vesicle vaccines","volume":"50","author":"Micoli","year":"2020","journal-title":"Semin. Immunol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"36575","DOI":"10.1038\/srep36575","article-title":"Modulating endotoxin activity by combinatorial bioengineering of meningococcal lipopolysaccharide","volume":"6","author":"Zariri","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1452","DOI":"10.1080\/21505594.2021.1929037","article-title":"Reduction of endotoxicity in Bordetella bronchiseptica by lipid A engineering: Characterization of lpxL1 and pagP mutants","volume":"12","author":"Plisnier","year":"2021","journal-title":"Virulence"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1191","DOI":"10.1038\/nature07830","article-title":"The structural basis of lipopolysaccharide recognition by the TLR4\u2013MD-2 complex","volume":"458","author":"Park","year":"2009","journal-title":"Nature"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"4669","DOI":"10.4049\/jimmunol.175.7.4669","article-title":"Molecular basis of reduced potency of underacylated endotoxins","volume":"175","author":"Teghanemt","year":"2005","journal-title":"J. Immunol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"544","DOI":"10.1128\/CVI.00339-08","article-title":"Immunogenicity of a whole-cell pertussis vaccine with low lipopolysaccharide content in infants","volume":"16","author":"Zorzeto","year":"2009","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"339","DOI":"10.4161\/hv.22847","article-title":"An improved whole cell pertussis vaccine with reduced content of endotoxin","volume":"9","author":"Dias","year":"2013","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"701","DOI":"10.1002\/(SICI)1522-2683(19990101)20:4\/5<701::AID-ELPS701>3.0.CO;2-5","article-title":"Extraction of Escherichia coli proteins with organic solvents prior to two-dimensional electrophoresis","volume":"20","author":"Molloy","year":"1999","journal-title":"Electrophoresis"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"7980","DOI":"10.4049\/jimmunol.177.11.7980","article-title":"TLR4 mediates vaccine-induced protective cellular immunity to Bordetella pertussis: Role of IL-17-producing T cells","volume":"177","author":"Higgins","year":"2006","journal-title":"J. Immunol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"821","DOI":"10.1128\/CVI.00074-07","article-title":"Lipopolysaccharide analogs improve efficacy of acellular pertussis vaccine and reduce type I hypersensitivity in mice","volume":"14","author":"Geurtsen","year":"2007","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"10515","DOI":"10.1038\/ncomms10515","article-title":"A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria","volume":"7","author":"Roier","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1128\/jb.58.2.127-134.1949","article-title":"A simplified liquid culture medium for the growth of Hemophilus pertussis","volume":"58","author":"Verwey","year":"1949","journal-title":"J. Bacteriol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"3962","DOI":"10.1016\/S0021-9258(19)45127-2","article-title":"Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of the cytoplasmic and outer membrane","volume":"247","author":"Osborn","year":"1972","journal-title":"J. Biol. Chem."},{"key":"ref_46","first-page":"100009","article-title":"Heat shock enhances outer-membrane vesicle release in Bordetella spp.","volume":"2","author":"Balhuizen","year":"2021","journal-title":"Curr. Res. Microb. Sci."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1038\/227680a0","article-title":"Cleavage of structural proteins during the assembly of the head of bacteriophage T4","volume":"227","author":"Laemmli","year":"1970","journal-title":"Nature"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1007\/978-1-4939-2871-2_3","article-title":"Experimental methods for studying the BAM complex in Neisseria meningitidis","volume":"1329","author":"Bos","year":"2015","journal-title":"Methods Mol. Biol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/0003-2697(82)90673-X","article-title":"A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels","volume":"119","author":"Tsai","year":"1982","journal-title":"Anal. Biochem."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"2885","DOI":"10.1099\/00221287-147-11-2885","article-title":"Role of the polymorphic region 1 of the Bordetella pertussis protein pertactin in immunity","volume":"147","author":"King","year":"2001","journal-title":"Microbiology"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"4645","DOI":"10.1073\/pnas.87.12.4645","article-title":"Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants","volume":"87","author":"Grant","year":"1990","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"2222","DOI":"10.1128\/IAI.73.4.2222-2231.2005","article-title":"Function of neisserial outer membrane phospholipase A in autolysis and assessment of its vaccine potential","volume":"73","author":"Bos","year":"2005","journal-title":"Infect. Immun."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/0378-1119(83)90040-9","article-title":"Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis","volume":"26","author":"Norrander","year":"1983","journal-title":"Gene"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/14\/8027\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:55:14Z","timestamp":1760140514000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/14\/8027"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,7,21]]},"references-count":53,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2022,7]]}},"alternative-id":["ijms23148027"],"URL":"https:\/\/doi.org\/10.3390\/ijms23148027","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,7,21]]}}}