Comparative in silico characterization of virulence factors in major Brucella species reveals potential targets for vaccine and drug target discovery

Background and Aim: Brucellosis is a globally significant zoonotic disease that causes reproductive failure in animals and chronic infection in humans, resulting in substantial economic and public health burdens. The pathogenicity of Brucella species is largely mediated by virulence determinants such as the type IV secretion system and lipopolysaccharide biosynthetic machinery. Despite extensive studies on individual components, a comparative and integrative understanding of conserved virulence factors across major Brucella species remains limited. This study aimed to perform a comprehensive in silico characterization of key virulence proteins across representative Brucella species to identify potential targets for vaccine and antimicrobial development. Materials and Methods: Nine virulence-associated proteins (VirB3, VirB5, VirB7, WbkA, WbkB, WbkC, FabZ, gmd, and lpxC) from five major Brucella species were analyzed using bioinformatics approaches. Analyses included physicochemical characterization, subcellular localization prediction, conserved domain and motif identification, multiple sequence alignment, phylogenetic analysis, and protein–protein interaction network construction. Publicly available databases and tools such as NCBI, ProtParam, DeepLoc, MEME, MEGA11, and STRING were utilized. Results: Subcellular localization analysis revealed that VirB5 is extracellular and VirB7 is outer membrane-associated, whereas most other proteins were cytoplasmic or membrane-associated. Conserved motif analysis identified three shared motifs, particularly in VirB5 and WbkB, indicating functional conservation across species. Phylogenetic and sequence alignment analyses demonstrated high conservation of virulence proteins among the selected Brucella species. Protein-protein interaction networks highlighted VirB3, VirB5, VirB7, WbkC, FabZ, gmd, and lpxC as key interaction hubs. lpxC showed strong connectivity with lipid A biosynthesis proteins, suggesting its central functional role. Conclusion: This integrative in silico analysis identified conserved virulence proteins with potential translational relevance. VirB5 and VirB7 emerged as promising candidates for subunit vaccine development due to their extracellular or membrane localization and conserved motifs, while lpxC was identified as a potential antimicrobial target because of its central role in lipopolysaccharide biosynthesis. These findings provide a rational framework for future experimental validation and support the development of improved control strategies against brucellosis. Keywords: bioinformatics, Brucella, lipopolysaccharide, lpxC, protein–protein interaction, type IV secretion system, vaccine targets, virulence factors.