Gene in S2: Its Role in Lipopolysaccharide Biosynthesis and Bacterial Virulence in RAW264.7.

, the causative agent of brucellosis, poses a significant public health and animal husbandry threat. However, the role of the alanine racemase () gene, which encodes alanine racemase in , remains unclear. Here, we analyzed an deletion mutant and a complemented strain of S2. The knockout strain displayed an unaltered, smooth phenotype in acriflavine agglutination tests but lacked the core polysaccharide portion of lipopolysaccharide (LPS). Genes involved in the LPS synthesis were significantly upregulated in the deletion mutant. The deletion strain exhibited reduced intracellular viability in the macrophages, increased macrophage-mediated killing, and upregulation of the apoptosis markers. Bcl2, an anti-apoptotic protein, was downregulated, while the pro-apoptotic proteins, Bax, Caspase-9, and Caspase-3, were upregulated in the macrophages infected with the deletion strain. The infected macrophages showed increased mitochondrial membrane permeability, Cytochrome C release, and reactive oxygen species, activating the mitochondrial apoptosis pathway. These findings revealed that alanine racemase was dispensable in S2 but influenced the strain's rough features and triggered the mitochondrial apoptosis pathway during macrophage invasion. The deletion of the gene reduced the intracellular survival and virulence. This study enhances our understanding of the molecular mechanism underlying 's survival and virulence and, specifically, how gene affects host immune evasion by regulating bacterial LPS biosynthesis.