iProXProteomicsYuefeng ChuBrucella Abortus 2308http://www.iprox.org/page/project.html?id=IPX0016270000chuyuefeng@caas.cnState Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural SciencesiProXBrucella species (<i>Brucella</i> 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 <i>Brucella</i> 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 <i>hflX</i> deletion and complemented strains of <i>Brucella</i> abortus 2308 and characterized their phenotypes using antimicrobial susceptibility tests, growth and time-kill assays, electron microscopy, proteomics, and RT-qPCR. Deletion of <i>hflX</i> significantly increased bacterial susceptibility to rifampicin, impaired growth recovery, and intensified intracellular stress without disrupting cell envelope integrity. Mechanistically, <i>hflX</i> depletion led to coordinated downregulation of RNA polymerase (RNAP) core subunits (<i>rpoA</i>, <i>rpoB</i>, and <i>rpoC</i>) and σ factors (<i>rpoD</i> and <i>rpoH</i>) at both protein and mRNA levels. Our findings demonstrate that HflX mediates rifampicin resistance in <i>Brucella</i> 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.HflX mediates rifampicin resistance in &lt;i&gt;Brucella&lt;/i&gt; by downregulating the expression of RNA polymerase-associated genes.Geng Hao H, Qi Mengzhu M, Su Mengru M, Zhi Feijie F, Chu Yuefeng YfalseHflX mediates rifampicin resistance in Brucella by downregulating the expression of RNA polymerase-associated genesTo further investigate molecular changes associated with HflX knock during rifampicin exposure, we performed label-free quantitative proteomic analysis. The WT and ΔhflX strains were cultured for three generations in the presence of 1/2 MIC rifampicin (1 μg/mL) and harvested during the logarithmic growth phase, with at least three biological replicates per group. DIA-based proteomic analysis quantified protein abundance and identified differentially expressed proteins (DEPs) (Figure 5A). Bioinformatic analyses were then conducted to evaluate enriched pathways and co-expression networks, aiming to elucidate the regulatory role of HflX in Brucella.Thu Mar 19 00:00:00 GMT 2026PXD07584635939142063517