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

Abstract

Brucella suis, the causative agent of brucellosis, poses a significant public health and animal husbandry threat. However, the role of the alanine racemase (alr) gene, which encodes alanine racemase in Brucella, remains unclear. Here, we analyzed an alr deletion mutant and a complemented strain of Brucella suis 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 alr 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 B. suis S2 but influenced the strain’s rough features and triggered the mitochondrial apoptosis pathway during macrophage invasion. The deletion of the alr gene reduced the intracellular survival and virulence. This study enhances our understanding of the molecular mechanism underlying Brucella’s survival and virulence and, specifically, how alr gene affects host immune evasion by regulating bacterial LPS biosynthesis.

Regulation of the Gene for Alanine Racemase Modulates Amino Acid Metabolism with Consequent Alterations in Cell Wall Properties and Adhesive Capability in Brucella spp.

“…suis S2 wild-type strain with CVCC reference number CVCC70502 was obtained from the Shaanxi Provincial Institute for Veterinary Drug Control, Xi'an, Shaanxi, China. The ∆alr and C∆alr strains were constructed previously in our laboratory [34]. All Brucella strains were cultivated in CDM.…”