{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T17:16:03Z","timestamp":1788455763042,"version":"build-2803163510"},"reference-count":52,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,10,16]],"date-time":"2025-10-16T00:00:00Z","timestamp":1760572800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p><jats:italic>Pseudomonas aeruginosa<\/jats:italic> is a Gram-negative opportunistic pathogen responsible for severe respiratory tract infections. We previously developed a live attenuated auxotrophic vaccine candidate, PAO1 \u0394<jats:italic>murI<\/jats:italic>, which conferred protection in murine infection models but exhibited significant reactogenicity when administered intranasally. To reduce the toxicity of PAO1 \u0394<jats:italic>murI<\/jats:italic> without compromising its protective efficacy, we engineered strains with a modified lipid A structure, as lipid A is one of the main toxic components of whole-cell vaccines.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Two lipid A-modifying enzymes, LpxR and PagL were overproduced in PAO1 \u0394<jats:italic>murI<\/jats:italic> derivatives. The resulting lipopolysaccharide (LPS) was analyzed by MALDI-TOF mass spectrometry. <jats:italic>In vitro<\/jats:italic> assays with HEK293-Blue reporter cells expressing murine and human Toll-like receptor 4 (TLR4) were used to assess LPS-associated toxicity, while <jats:italic>in vivo<\/jats:italic> reactogenicity and protective efficacy were evaluated in a murine acute pneumonia model.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>LPS extracted from the wild-type strain showed heterogeneous lipid A structures with varying degrees of acylation, and a predominant penta-acylated species. Expression of LpxR led to enrichment in tetra-acylated species, while PagL expression reduced the heterogeneity observed in the wild type. Both mutant strains showed decreased TLR4 activation <jats:italic>in vitro<\/jats:italic> as compared to the wild type. In mice, lipid A-modified derivatives retained protective efficacy; however, no reduction in reactogenicity was observed.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>Lipid A modifications mediated by LpxR and PagL attenuated TLR4 signaling <jats:italic>in vitro<\/jats:italic> but were insufficient to reduce <jats:italic>in vivo<\/jats:italic> reactogenicity. Additional modifications or targeting of other toxic components may be required. This strategy may serve as an initial basis for optimizing live attenuated <jats:italic>P. aeruginosa<\/jats:italic> vaccines, although additional approaches will likely be necessary to achieve substantial improvements.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fcimb.2025.1664169","type":"journal-article","created":{"date-parts":[[2025,10,16]],"date-time":"2025-10-16T05:32:10Z","timestamp":1760592730000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":3,"title":["LpxR and PagL expression in live attenuated auxotrophic Pseudomonas aeruginosa vaccines modulates lipid A reactogenicity in vitro while preserving immunogenicity"],"prefix":"10.3389","volume":"15","author":[{"given":"Miriam","family":"Moscoso","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"V\u00edctor","family":"Fuentes-Valverde","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patricia","family":"Garc\u00eda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan A.","family":"Vallejo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jes\u00fas","family":"P\u00e9rez-Ortega","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rebeca","family":"Santamarina-Fern\u00e1ndez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana","family":"Candela","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marina","family":"Ovia\u00f1o","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jes\u00fas","family":"Arenas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Germ\u00e1n","family":"Bou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,10,16]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"594","DOI":"10.3390\/vaccines8040594","article-title":"Shortening the lipid A acyl chains of Bordetella pertussis enables depletion of lipopolysaccharide endotoxic activity","volume":"8","author":"Arenas","year":"2020","journal-title":"Vaccines (Basel)."},{"key":"B2","doi-asserted-by":"publisher","first-page":"107","DOI":"10.1684\/ecn.2019.0434","article-title":"IL-10 induces TGF-beta secretion, TGF-beta receptor II upregulation, and IgA secretion in B cells","volume":"30","author":"Bagheri","year":"2019","journal-title":"Eur. Cytokine Netw."},{"key":"B3","doi-asserted-by":"publisher","first-page":"6883","DOI":"10.4049\/jimmunol.0901466","article-title":"What role does the route of immunization play in the generation of protective immunity against mucosal pathogens","volume":"183","author":"Belyakov","year":"2009","journal-title":"J. Immunol."},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.01532-16","article-title":"Outer membrane vesicle production facilitates LPS remodeling and outer membrane maintenance in Salmonella during environmental transitions","volume":"7","author":"Bonnington","year":"2016","journal-title":"mBio."},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1008311","article-title":"A live auxotrophic vaccine confers mucosal immunity and protection against lethal pneumonia caused by Pseudomonas aeruginosa","volume":"16","author":"Cabral","year":"2020","journal-title":"PloS Pathog."},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms15480","article-title":"Design of live attenuated bacterial vaccines based on D-glutamate auxotrophy","volume":"8","author":"Cabral","year":"2017","journal-title":"Nat. Commun."},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0008439","article-title":"Pseudomonas aeruginosa exploits lipid A and muropeptides modification as a strategy to lower innate immunity during cystic fibrosis lung infection","volume":"4","author":"Cigana","year":"2009","journal-title":"PloS One"},{"key":"B8","doi-asserted-by":"publisher","first-page":"2221","DOI":"10.1016\/S0140-6736(22)02185-7","article-title":"Global mortality associated with 33 bacterial pathogens in 2019: a systematic analysis for the Global Burden of Disease Study 2019","volume":"400","year":"2022","journal-title":"Lancet"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1016\/j.ebiom.2020.102974","article-title":"Lung immunoglobulin A immunity dysregulation in cystic fibrosis","volume":"60","author":"Collin","year":"2020","journal-title":"EBioMedicine"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.00181-19","article-title":"Antimicrobial resistance in ESKAPE pathogens","volume":"33","author":"De Oliveira","year":"2020","journal-title":"Clin. Microbiol. Rev."},{"key":"B11","doi-asserted-by":"publisher","first-page":"6127","DOI":"10.1093\/nar\/16.13.6127","article-title":"High efficiency transformation of E. coli by high voltage electroporation","volume":"16","author":"Dower","year":"1988","journal-title":"Nucleic Acids Res."},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.00940-16","article-title":"LPS remodeling triggers formation of outer membrane vesicles in Salmonella","volume":"7","author":"Elhenawy","year":"2016","journal-title":"mBio"},{"key":"B13","doi-asserted-by":"publisher","first-page":"191","DOI":"10.1128\/JB.188.1.191-201.2006","article-title":"The Pseudomonas aeruginosa lipid A deacylase: selection for expression and loss within the cystic fibrosis airway","volume":"188","author":"Ernst","year":"2006","journal-title":"J. Bacteriol"},{"key":"B14","doi-asserted-by":"publisher","first-page":"1088","DOI":"10.1086\/521367","article-title":"Unique lipid a modifications in Pseudomonas aeruginosa isolated from the airways of patients with cystic fibrosis","volume":"196","author":"Ernst","year":"2007","journal-title":"J. Infect. Dis."},{"key":"B15","doi-asserted-by":"publisher","first-page":"1561","DOI":"10.1126\/science.286.5444.1561","article-title":"Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa","volume":"286","author":"Ernst","year":"1999","journal-title":"Science"},{"key":"B16","doi-asserted-by":"publisher","first-page":"1622","DOI":"10.3390\/vaccines10101622","article-title":"Double auxotrophy to improve the safety of a live anti-pseudomonas aeruginosa vaccine","volume":"10","author":"Fuentes-Valverde","year":"2022","journal-title":"Vaccines (Basel)."},{"key":"B17","doi-asserted-by":"publisher","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":"B18","doi-asserted-by":"publisher","first-page":"8248","DOI":"10.1074\/jbc.M414235200","article-title":"Dissemination of lipid A deacylases (pagL) among gram-negative bacteria: identification of active-site histidine and serine residues","volume":"280","author":"Geurtsen","year":"2005","journal-title":"J. Biol. Chem."},{"key":"B19","doi-asserted-by":"publisher","first-page":"1541","DOI":"10.1073\/pnas.0337542100","article-title":"PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin","volume":"100","author":"Gust","year":"2003","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"B20","doi-asserted-by":"publisher","first-page":"557","DOI":"10.1016\/s0022-2836(83)80284-8","article-title":"Studies on transformation of Escherichia coli with plasmids","volume":"166","author":"Hanahan","year":"1983","journal-title":"J. Mol. Biol."},{"key":"B21","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1258\/la.2010.010031","article-title":"A guide to defining and implementing protocols for the welfare assessment of laboratory animals: eleventh report of the BVAAWF\/FRAME\/RSPCA\/UFAW Joint Working Group on Refinement","volume":"45","author":"Hawkins","year":"2011","journal-title":"Lab. Anim."},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2024.1446626","article-title":"Unravelling the mechanisms causing murepavadin resistance in Pseudomonas aeruginosa: lipopolysaccharide alterations and its consequences","volume":"14","author":"Hern\u00e1ndez-Garc\u00eda","year":"2024","journal-title":"Front. Cell Infect. Microbiol."},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.1093\/femspd\/ftv053","article-title":"Site-specific activity of the acyltransferases HtrB1 and HtrB2 in Pseudomonas aeruginosa lipid A biosynthesis","volume":"73","author":"Hittle","year":"2015","journal-title":"Pathog. Dis."},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1186\/1756-0500-7-13","article-title":"Capsule deletion via a lambda-Red knockout system perturbs biofilm formation and fimbriae expression in Klebsiella pneumoniae MGH 78578","volume":"7","author":"Huang","year":"2014","journal-title":"BMC Res. Notes"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00347-20","article-title":"Identification of the Pseudomonas aeruginosa O17 and O15 O-specific antigen biosynthesis loci reveals an ABC transporter-dependent synthesis pathway and mechanisms of genetic diversity","volume":"202","author":"Huszczynski","year":"2020","journal-title":"J. Bacteriol."},{"key":"B26","doi-asserted-by":"publisher","first-page":"100940","DOI":"10.1016\/j.lanmic.2024.07.003","article-title":"WHO publishes updated list of bacterial priority pathogens","volume":"5","author":"Jesudason","year":"2024","journal-title":"Lancet Microbe"},{"key":"B27","doi-asserted-by":"publisher","first-page":"20044","DOI":"10.1074\/jbc.M401275200","article-title":"3-O-deacylation of lipid A by PagL, a PhoP\/PhoQ-regulated deacylase of Salmonella Typhimurium, modulates signaling through Toll-like receptor 4","volume":"279","author":"Kawasaki","year":"2004","journal-title":"J. Biol. Chem."},{"key":"B28","doi-asserted-by":"publisher","first-page":"343","DOI":"10.1016\/j.bbrc.2012.10.054","article-title":"Lipid A 3'-O-deacylation by Salmonella outer membrane enzyme LpxR modulates the ability of lipid A to stimulate Toll-like receptor 4","volume":"428","author":"Kawasaki","year":"2012","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"B29","doi-asserted-by":"publisher","first-page":"1100","DOI":"10.3390\/vaccines10071100","article-title":"Pseudomonas aeruginosa: recent advances in vaccine development","volume":"10","author":"Killough","year":"2022","journal-title":"Vaccines (Basel)"},{"key":"B30","doi-asserted-by":"publisher","first-page":"324","DOI":"10.1179\/096805106X118906","article-title":"Conserved and variable structural features in the lipopolysaccharide of Pseudomonas aeruginosa","volume":"12","author":"Knirel","year":"2006","journal-title":"J. Endotoxin Res."},{"key":"B31","doi-asserted-by":"publisher","first-page":"R90","DOI":"10.1186\/gb-2006-7-10-r90","article-title":"Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial","volume":"7","author":"Lee","year":"2006","journal-title":"Genome Biol."},{"key":"B32","doi-asserted-by":"publisher","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"B33","doi-asserted-by":"publisher","first-page":"736","DOI":"10.1111\/j.1365-2672.2006.03127.x","article-title":"Optimization of electroporation-mediated transformation: Staphylococcus carnosus as model organism","volume":"102","author":"Lofblom","year":"2007","journal-title":"J. Appl. Microbiol."},{"key":"B34","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2013.00003","article-title":"Recognition of lipid A variants by the TLR4-MD-2 receptor complex","volume":"3","author":"Maeshima","year":"2013","journal-title":"Front. Cell Infect. Microbiol."},{"key":"B35","doi-asserted-by":"publisher","first-page":"1628","DOI":"10.1126\/science.1138963","article-title":"The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4","volume":"316","author":"Mata-Haro","year":"2007","journal-title":"Science"},{"key":"B36","doi-asserted-by":"publisher","first-page":"852","DOI":"10.1038\/35101614","article-title":"Complete genome sequence of Salmonella enterica serovar Typhimurium LT2","volume":"413","author":"McClelland","year":"2001","journal-title":"Nature"},{"key":"B37","doi-asserted-by":"publisher","first-page":"431","DOI":"10.1136\/vr.116.16.431","article-title":"Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment","volume":"116","author":"Morton","year":"1985","journal-title":"Vet. Rec"},{"key":"B38","doi-asserted-by":"publisher","first-page":"575","DOI":"10.1128\/JB.186.2.575-579.2004","article-title":"PmrAB, a two-component regulatory system of Pseudomonas aeruginosa that modulates resistance to cationic antimicrobial peptides and addition of aminoarabinose to lipid A","volume":"186","author":"Moskowitz","year":"2004","journal-title":"J. Bacteriol"},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2019.00539","article-title":"Emerging strategies to combat ESKAPE pathogens in the era of antimicrobial resistance: A review","volume":"10","author":"Mulani","year":"2019","journal-title":"Front. Microbiol."},{"key":"B40","doi-asserted-by":"publisher","first-page":"1464","DOI":"10.1073\/pnas.1218080110","article-title":"Modulating the innate immune response by combinatorial engineering of endotoxin","volume":"110","author":"Needham","year":"2013","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"B41","doi-asserted-by":"publisher","first-page":"166","DOI":"10.1111\/mmi.13018","article-title":"Extracellular zinc induces phosphoethanolamine addition to Pseudomonas aeruginosa lipid A via the ColRS two-component system","volume":"97","author":"Nowicki","year":"2015","journal-title":"Mol. Microbiol."},{"key":"B42","doi-asserted-by":"publisher","first-page":"1191","DOI":"10.1038\/nature07830","article-title":"The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex","volume":"458","author":"Park","year":"2009","journal-title":"Nature"},{"key":"B43","doi-asserted-by":"publisher","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":"P\u00e9rez-Ortega","year":"2021","journal-title":"Virulence"},{"key":"B44","doi-asserted-by":"publisher","first-page":"21974","DOI":"10.1074\/jbc.M603527200","article-title":"An outer membrane enzyme encoded by Salmonella Typhimurium lpxR that removes the 3'-acyloxyacyl moiety of lipid A","volume":"281","author":"Reynolds","year":"2006","journal-title":"J. Biol. Chem."},{"key":"B45","doi-asserted-by":"publisher","first-page":"7071","DOI":"10.1073\/pnas.0509392103","article-title":"Crystal structure and catalytic mechanism of the LPS 3-O-deacylase PagL from Pseudomonas aeruginosa","volume":"103","author":"Rutten","year":"2006","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"B46","doi-asserted-by":"publisher","first-page":"2012","DOI":"10.3390\/ijms26052012","article-title":"Pseudomonas aeruginosa vaccine development: lessons, challenges, and future innovations","volume":"26","author":"Santamarina-Fern\u00e1ndez","year":"2025","journal-title":"Int. J. Mol. Sci."},{"key":"B47","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.00451-20","article-title":"Defining the Mechanistic Correlates of Protection Conferred by Whole-Cell Vaccination against Pseudomonas aeruginosa Acute Murine Pneumonia","volume":"89","author":"Sen-Kilic","year":"2021","journal-title":"Infect. Immun."},{"key":"B48","doi-asserted-by":"publisher","first-page":"275","DOI":"10.1111\/j.1472-765X.2008.02417.x","article-title":"A simple and fast method for determining colony forming units","volume":"47","author":"Sieuwerts","year":"2008","journal-title":"Lett. Appl. Microbiol."},{"key":"B49","doi-asserted-by":"publisher","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":"B50","doi-asserted-by":"publisher","first-page":"959","DOI":"10.1038\/35023079","article-title":"Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen","volume":"406","author":"Stover","year":"2000","journal-title":"Nature"},{"key":"B51","doi-asserted-by":"publisher","first-page":"158","DOI":"10.1111\/mmi.12451","article-title":"A divergent Pseudomonas aeruginosa palmitoyltransferase essential for cystic fibrosis-specific lipid A","volume":"91","author":"Thaipisuttikul","year":"2014","journal-title":"Mol. Microbiol."},{"key":"B52","doi-asserted-by":"publisher","first-page":"919","DOI":"10.1111\/mmi.13857","article-title":"Probing the sRNA regulatory landscape of P. aeruginosa: post-transcriptional control of determinants of pathogenicity and antibiotic susceptibility","volume":"106","author":"Zhang","year":"2017","journal-title":"Mol. Microbiol."}],"container-title":["Frontiers in Cellular and Infection Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2025.1664169\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,16]],"date-time":"2025-10-16T05:32:11Z","timestamp":1760592731000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2025.1664169\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,16]]},"references-count":52,"alternative-id":["10.3389\/fcimb.2025.1664169"],"URL":"https:\/\/doi.org\/10.3389\/fcimb.2025.1664169","relation":{},"ISSN":["2235-2988"],"issn-type":[{"value":"2235-2988","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,16]]},"article-number":"1664169"}}