Abstract
Brucella species (Brucella spp.) are facultative intracellular zoonotic pathogens responsible for brucellosis, a disease causing substantial global public health and economic burdens. Rifampicin remains a first-line therapeutic agent, but the molecular mechanisms underlying rifampicin resistance in Brucella remain poorly defined, especially the contribution of ribosome-associated regulatory proteins. HflX is a conserved ribosome-binding GTPase involved in ribosomal quality control and antibiotic resistance, yet its role in rifampicin resistance has not been reported. Here, we constructed hflX deletion and complemented strains of Brucella abortus 2308 and characterized their phenotypes using antimicrobial susceptibility tests, growth and time-kill assays, electron microscopy, proteomics, and RT-qPCR. Deletion of hflX significantly increased bacterial susceptibility to rifampicin, impaired growth recovery, and intensified intracellular stress without disrupting cell envelope integrity. Mechanistically, hflX depletion led to coordinated downregulation of RNA polymerase (RNAP) core subunits (rpoA, rpoB, and rpoC) and σ factors (rpoD and rpoH) at both protein and mRNA levels. Our findings demonstrate that HflX mediates rifampicin resistance in Brucella by regulating RNAP-associated gene expression and metabolic adaptation, establishing a novel HflX-RNAP regulatory axis. This study expands the understanding of antibiotic resistance in intracellular pathogens and highlights HflX as a promising target for developing anti-resistance strategies against brucellosis.