Abstract
The rise of Klebsiella pneumoniae resistant to last-resort antimicrobials poses an urgent threat to global health. The ramR-ramA regulatory system critically influences drug resistance by regulating the AcrAB-TolC efflux pump, which also plays a crucial role in the pathogenicity of K. pneumoniae. However, the mechanism of the ramR-ramA system on bacteria-host interaction remains unclear. To determine how specific mutations in ramR influence eravacycline (ERV) resistance and their impact on the immune activation capabilities of K. pneumoniae, thereby highlighting potential targets for therapeutic intervention, we performed genetic sequencing to identify mutations in ramR. Then, the CRISPR-Cas9 technology was employed to construct specific ramR mutations into K. pneumoniae, which were then subjected to phenotypic and functional assays in both in vitro and in vivo (mouse models, macrophage, and blood-killing experiment) settings. ramR L58P and F165L genetic alterations disrupt the binding affinity of RamR to the ramA promoter, thereby upregulating the efflux pump expression and increasing ERV minimum inhibitory concentration values up to 64-fold compared to the wild-type. Concurrently, these mutations modulate lipid A structure by increasing 2-hydroxy fatty acid chain abundance. In mouse models, ramR L58P and F165L mutants showed lower bacterial burden in organs (spleen, lung, and kidney) 6 h post-infection, and are fast cleared in 48 h. Furthermore, despite lower intracellular bacterial loads, ramR L58P and F165L mutants induce heightened pro-inflammatory cytokine responses in macrophages and elevate systemic cytokine levels (interleukin [IL]2, IL4, IL6, IL12, interferon-α, and interferon-γ) in human blood co-culture experiments. This study illuminates the critical role of ramR mutations in conferring ERV resistance and enhancing immune responses in K. pneumoniae. The dual impact of these mutations on both antimicrobial resistance and immune activation not only underscores the challenges in treating infections but also advocates for heightened surveillance and innovative strategies to counteract the emerging threat of antimicrobial-resistant K. pneumoniae.