A host-directed adjuvant sensitizes intracellular bacterial persisters to antibiotics

Intracellular bacterial reservoirs contribute to antibiotic treatment failure by fostering metabolically dormant persister cells that are highly tolerant to killing. However, strategies to effectively target intracellular persister cells remain limited. Here, we developed a high-throughput screen to identify compounds that modulate the metabolic activity of intracellular Staphylococcus aureus. The identified compound, KL1, increases intracellular bacterial metabolic activity and sensitizes persister populations of S. aureus to antibiotics, without causing cytotoxicity or bacterial outgrowth. KL1 also exhibits adjuvant activity against intramacrophage Salmonella enterica Typhimurium and Mycobacterium tuberculosis, as well as in murine infection models of S. aureus and S. Typhimurium infection. Transcriptomic analysis and further mechanistic studies reveal that KL1 modulates host immune response genes and suppresses the production of reactive species in host macrophages, alleviating a key inducer of antibiotic tolerance. Our findings highlight the potential to target intracellular persisters by stimulating their metabolism. To interrogate the mechanism of action of the compound KL1, RAW264.7 cells infected with S. aureus strain JE2-Lux at an MOI of 20 were treated with and without 40 uM KL1 for 24 h in triplicate and harvested for RNA-seq. Only the host transcripts were analyzed. The data were processed based on the standard procedure by Azenta Life Sciences.