Deletion of pyrE disrupts pyrimidine de novo synthesis, attenuates virulence, and confers protective immunity as a live attenuated vaccine candidate against Brucella melitensis

Introduction Brucellosis, caused by Brucella spp., is a major zoonotic disease threatening public health and livestock production. Current live attenuated vaccines suffer from residual virulence and interference with serodiagnosis. Targeting essential metabolic pathways to generate auxotrophic mutants is a promising strategy for developing safer vaccines. The de novo pyrimidine synthesis pathway is critical for bacterial survival in nutrient-restricted intracellular environments. pyrE encodes orotate phosphoribosyltransferase, the key enzyme catalyzing the first committed step of pyrimidine synthesis. However, the role of this pathway in Brucella pathogenicity remains largely unexplored. Methods A pyrE deletion mutant (Δ pyrE ) and its complemented strain were constructed in B. melitensis M5. Growth characteristics, stress susceptibility, adhesion, invasion, intracellular survival, and BCV trafficking were assessed in vitro . Virulence, immunogenicity, and protective efficacy were evaluated in a mouse model with M5-90Δ26 as reference. Results Δ pyrE exhibited uracil-dependent growth defects and impaired adhesion, invasion, and intracellular survival, but retained normal stress resistance and BCV trafficking to escape lysosomal degradation. In mice, Δ pyrE was significantly attenuated, with reduced splenic colonization and hepatic pathology. Immunization with Δ pyrE induced a durable Th1-biased immune response (elevated IgG2a and IFN-γ) and conferred protection comparable to M5-90Δ26, with superior long-term efficacy. Conclusion This study demonstrates that pyrE is essential for pyrimidine synthesis and virulence in Brucella . The Δ pyrE strain represents a safe, immunogenic, and protective vaccine candidate with long-term potential, providing a new strategy for brucellosis control.