Disruption of Purine De Novo Synthesis Pathway Impairs Membrane Homeostasis, Intracellular Survival, and Virulence of Brucella melitensis

Guangyu Yang¹Mengsi Li¹Yang Li¹Simin Chen¹Jing Qu¹Jihong Li²Shaohui Wang¹Yanqing Bao¹Jingjing Qi¹Yafeng Dou^3*Mingxing Tian^1*

• ¹Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China
• ²Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
• ³Department of Pathology, The Second People's Hospital of China Three Gorges University, Yichang, China

Brucellosis is a significant zoonotic infectious disease caused by Brucella. As a facultative intracellular pathogen, the ability of Brucella to acquire essential nutrients within host cells is critical for its intracellular survival and pathogenicity. Previous studies have indicated that disruption of the de novo purine biosynthesis pathway significantly attenuates the virulence of Brucella, although the underlying mechanisms remain incompletely understood. In this study, using Brucella melitensis M5 as the parental strain, we constructed a deletion mutant and a complemented strain of the key purine biosynthesis gene purD (encoding phosphoribosylamine– glycine ligase) via homologous recombination. Growth curve analysis confirmed that the deletion of purD disrupted the de novo purine synthesis pathway. Further investigations revealed that the purine synthesis deficiency impaired bacterial tolerance to the anionic detergent sodium dodecyl sulfate, reduced membrane permeability, and altered lipid biosynthesis levels, as assessed by uptake assays using propidium iodide (PI), 1-N-phenylnaphthylamine (NPN), and Nile red (NR) staining. Cellular infection assays demonstrated that the purine biosynthesis defect significantly attenuated the ability of Brucella to invade host cells and survive intracellularly, with the intracellular survival defect primarily attributable to insufficient purine acquisition within host cells. In a mouse infection model, the purD mutant exhibited reduced colonization capacity in the spleen and liver, accompanied by diminished induction of splenomegaly and liver granuloma formation. In summary, this study elucidates the critical role of the de novo purine biosynthesis pathway in the pathogenesis of Brucella, providing important insights for the development of attenuated vaccines and novel antimicrobial agents targeting this pathway.