Disruption of Histidine Biosynthesis Impairs Outer Membrane Stability and Intracellular Survival of Brucella melitensis, Resulting in Attenuated Virulence

Background: Brucellosis is a global zoonosis caused by Brucella. Histidine biosynthesis is essential for bacterial growth, but its role in Brucella melitensis virulence remains unclear. HisD catalyzes the final two steps of histidine synthesis and is absent in mammals, making it a potential drug target. Results: We constructed a hisD deletion mutant (ΔhisD) and complemented strain (ChisD) via homologous recombination. ΔhisD failed to grow in medium without histidine supplementation. It showed reduced survival under polymyxin B and SDS stress, and impaired outer membrane integrity under polymyxin B challenge, though no defect was observed under non-stressed conditions. Intracellularly, ΔhisD replicated poorly in HeLa and RAW264.7 cells, and this defect was rescued by exogenous histidine. In a mouse model, ΔhisD exhibited lower bacterial loads in liver and spleen, reduced splenomegaly, and attenuated hepatic granuloma formation. Conclusions: Histidine biosynthesis deficiency attenuates Brucella virulence by restricting nutritional acquisition and conditionally compromising outer membrane stability. HisD is a promising target for anti-brucellosis drug development, and ΔhisD holds potential as a live attenuated vaccine candidate.