000164173 001__ 164173
000164173 005__ 20251127172930.0
000164173 0247_ $$2doi$$a10.3389/fcimb.2025.1664169
000164173 0248_ $$2sideral$$a146395
000164173 037__ $$aART-2025-146395
000164173 041__ $$adeu
000164173 100__ $$aMoscoso, Miriam
000164173 245__ $$aLpxR and PagL expression in live attenuated auxotrophic Pseudomonas aeruginosa vaccines modulates lipid A reactogenicity in vitro while preserving immunogenicity
000164173 260__ $$c2025
000164173 5203_ $$aIntroduction: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen responsible for severe respiratory tract infections. We previously developed a live attenuated auxotrophic vaccine candidate, PAO1 ΔmurI, which conferred protection in murine infection models but exhibited significant reactogenicity when administered intranasally. To reduce the toxicity of PAO1 ΔmurI 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.
Methods: Two lipid A-modifying enzymes, LpxR and PagL were overproduced in PAO1 ΔmurI derivatives. The resulting lipopolysaccharide (LPS) was analyzed by MALDI-TOF mass spectrometry. In vitro assays with HEK293-Blue reporter cells expressing murine and human Toll-like receptor 4 (TLR4) were used to assess LPS-associated toxicity, while in vivo reactogenicity and protective efficacy were evaluated in a murine acute pneumonia model.
Results: 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 in vitro as compared to the wild type. In mice, lipid A-modified derivatives retained protective efficacy; however, no reduction in reactogenicity was observed.
Discussion: Lipid A modifications mediated by LpxR and PagL attenuated TLR4 signaling in vitro but were insufficient to reduce in vivo reactogenicity. Additional modifications or targeting of other toxic components may be required. This strategy may serve as an initial basis for optimizing live attenuated P. aeruginosa vaccines, although additional approaches will likely be necessary to achieve substantial improvements.
000164173 536__ $$9info:eu-repo/grantAgreement/ES/MICINN AEI PID2020-114617RB-100
000164173 540__ $$9info:eu-repo/semantics/closedAccess$$aby$$uhttps://creativecommons.org/licenses/by/4.0/deed.es
000164173 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion
000164173 700__ $$aFuentes-Valverde, Víctor
000164173 700__ $$aGarcía, Patricia
000164173 700__ $$aVallejo, Juan A.
000164173 700__ $$aPérez-Ortega, Jesús
000164173 700__ $$aSantamarina-Fernández, Rebeca
000164173 700__ $$aCandela, Ana
000164173 700__ $$aOviaño, Marina
000164173 700__ $$0(orcid)0000-0002-8134-0693$$aArenas, Jesús$$uUniversidad de Zaragoza
000164173 700__ $$aBou, Germán
000164173 7102_ $$11009$$2773$$aUniversidad de Zaragoza$$bDpto. Patología Animal$$cÁrea Sanidad Animal
000164173 773__ $$g15 (2025), [16 pp.]$$tFrontiers in Cellular and Infection Microbiology$$x2235-2988
000164173 8564_ $$s5291075$$uhttps://zaguan.unizar.es/record/164173/files/texto_completo.pdf$$yVersión publicada
000164173 8564_ $$s2318651$$uhttps://zaguan.unizar.es/record/164173/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada
000164173 909CO $$ooai:zaguan.unizar.es:164173$$particulos$$pdriver
000164173 951__ $$a2025-11-27-15:16:26
000164173 980__ $$aARTICLE