Comprehensive genomic identification of essential genes required for Brucella melitensis intracellular survival during macrophage infection

Brucella melitensis is a significant zoonotic pathogen transmissible via contact, digestive, and respiratory routes, posing severe threats to livestock economies and human health. While its virulence genes critically regulate intracellular survival and replication, the molecular mechanisms underlying pathogenesis remain elusive. This study employed Transposon Directed Insertion-site Sequencing (TraDIS) technology to identify key genes essential for the survival of Brucella melitensis strain 16 M within RAW 264.7 macrophages, resulting in the discovery of 374 anti-phagocytic associated essential genes. Through GO and KEGG analyses, these genes were categorized into eight functional classes, representative genes from each category were selected to generate respective gene-deletion mutants, with bacterial virulence and phenotypes validated via intracellular survival assays, in vitro stress tests, carbon source utilization experiments, and RT-qPCR. All ten constructed mutants exhibited reduced macrophage survival capabilities. Deletion of membrane-related genes cydDC and BME_RS07715 attenuated stress resistance, membrane stability, and immune evasion, knockout of metabolic gene ptsP impaired carbon source utilization and intracellular proliferation, and disruption of regulatory gene BME_RS00125 diminished TCA cycle activity and fatty acid metabolism, compromising energy supply and structural integrity. In this study, some key genes of Brucella surviving in macrophages were verified, and preliminarily elucidated the function of these genes in regulating bacterial virulence, providing a theoretical basis for exploring their mechanism.