ABSTRACT: Coxiella burnetii, the etiologic agent of Q fever, is an obligate intracellular pathogen that replicates within a specialized acidic compartment termed the Coxiella-containing vacuole (CCV). Current treatment options for chronic Q fever require prolonged antibiotic therapy and are limited by toxicity and reduced efficacy within the acidic intracellular niche. Short-chain fatty acids (SCFAs) are host- and microbiota-derived metabolites with documented immunomodulatory and antimicrobial properties; however, their effects on C. burnetii remain unknown. We investigated whether acetate, the most abundant SCFA in the human body, influences C. burnetii growth and CCV biology. C. burnetii axenic and intracellular growth was measured by colony-forming unit (CFU) assay. Acetate MIC and MBC were determined using standard broth microdilution coupled with CFU assays. Confocal microscopy followed by quantitative measurements was used to determine the CCV expansion and CCV luminal pH. Uninfected and C. burnetii-infected HeLa cells were processed and sent for RNA-seq to Plasmidsaurus®. Acetate significantly inhibited C. burnetii replication in axenic ACCM-2 medium, completely preventing bacterial growth at concentrations of 20 - 80 mM. Broth microdilution assays identified a minimum inhibitory concentration (MIC) of 1.25 mM and a minimum bactericidal concentration (MBC) of 5 mM, demonstrating marked bacterial sensitivity to acetate. In infected HeLa cells, 24 h acetate treatment reduced intracellular bacterial burden by approximately 50% and markedly impaired CCV expansion, decreasing vacuolar size by 61–67% relative to control cells. Further, acetate elevated CCV luminal pH from approximately 5.2 to 5.5–5.7, indicating reduced vacuolar acidity, a condition known to be unfavorable for C. burnetii replication. Transcriptomic analysis revealed extensive host-cell reprogramming following acetate treatment, characterized by altered expression of genes involved in lipid metabolism, membrane trafficking, stress responses, apoptosis, complement activation, and inflammatory signaling pathways. Acetate restricts C. burnetii through multiple mechanisms, including direct antibacterial activity, disruption of CCV maturation and acidification, and broad modulation of host-cell transcriptional programs. These findings identify acetate as a previously unrecognized inhibitor of C. burnetii and highlight metabolic perturbation of the intracellular niche as a potential vulnerability of this pathogen. More broadly, our results demonstrate that host-derived metabolites can profoundly influence obligate intracellular bacterial survival and pathogenesis.
