The reduced expression and decreased stability of RNA polymerase core proteins are implicated in the virulence attenuation of Brucella abortus strain A19.

Brucellosis is a severe zoonotic infectious disease. Although Brucella abortus vaccine strain A19 is vital for controlling bovine brucellosis, the molecular mechanisms underlying its virulence attenuation remain inadequately understood. This investigation integrated proteomics and bioinformatics with molecular simulation and experimental validation to explore molecular distinctions between A19 and the wild-type Brucella abortus strain DT21. Proteomic analysis identified 254 differentially expressed proteins (DEPs), predominantly enriched in metabolic networks, suggesting alterations in environmental adaptation and metabolic remodeling in A19. Protein-protein interaction (PPI) network analysis highlighted key transcription (RpoB/RpoC) and translation (tuf-2-2/fusA) components as central hubs. Quantitative RT-qPCR confirmed the downregulation of transcripts for the hub proteins RpoB/RpoC and associated virulence factors in A19. Notably, comparison of RpoC protein sequences revealed that RpoC from non-Brucella abortus strains possesses two specific amino acid substitutions (I139V/L565S) compared to those of Brucella abortus strains. The bacterial two-hybrid assay confirmed that the RpoC substitutions led to a stronger interaction with RpoB, and molecular simulations further predicted that these substitutions enhance complex stability and rigidity in non-Brucella abortus strains. This implies that structural stability differences in the RNA polymerase (RNAP) may contribute to virulence variations within the Brucella genus. Collectively, the decreased RpoB-RpoC stability and lower RNAP levels likely impair transcription in A19, contributing to its attenuated virulence. This study deepens our understanding of transcriptional regulation in Brucella virulence and provides a molecular basis for the rational design of new Brucella vaccines.