Objective To conduct comparative genomic analysis on a smooth Brucella suis strain (2008005-S), isolated from animal source, and its derived rough mutant (2008005-R), investigate the molecular characteristics of the spontaneously derived 2008005-R under long-term nutrient-limited culture conditions, identify key genetic variations and their protein expression responses, evaluate its potential as a rough-type antigen, and provide theoretical support for the the screening of rough-type vaccine strains.

Methods The phenotypes profiles of the paired strains were characterized by using serological agglutination and phage lysis assays. Whole-genome sequencing and comparative genomic analysis were conducted for both strains, followed by detailed comparative genomic analysis to screen high-impact mutations. Further proteomic analysis was performed on the rough mutant, and a rough-type antigen prepared from this strain was used in rose bengal and tube agglutination tests with clinical patient sera.

Results Phenotypic testing confirmed that 2008005-R exhibited typical rough type characteristics. Comparative genomic analysis revealed three functional mutations: a frameshift mutation in lgtB (p.Ala219fs), a key gene involved in LPS core oligosaccharide synthesis, a frameshift mutation in omp31 (p.Ala32fs), which encodes an outer membrane protein.; and a missense mutation in zraS (p.Leu244Pro), encoding a sensor histidine kinase. Proteomics analysis revealed significant upregulation of the Omp31 homologous protein (A0A0E1X3W5) and histidine kinase homologous proriein (A0AB36PYL8), suggesting the presence of a compensatory response mechanism. Antigenicity experiments demonstrated that the rough-type antigen prepared from this rough strain reacted well with sera from clinical patients, and the phenotype remained stable after multiple passages and freeze-thaw cycles.

Conclusion Frameshift mutations in lgtB and omp31 moght be closely associated with the phenotypic of this rough-type Brucella mutant. These findings provide critical candidate genes for elucidating the molecular basis of phenotypic conversion of this strain. Proteomics revealed a possible compensatory mechanism in the rough-type conversion of this strain, involving the similar active upregulation of sequence andfunctional complementation of homologous proteins. Moreover, the strain exhibits stable rough type antigenic properties.