ArcA modulates multidrug resistance and compound susceptibility in <i>Klebsiella pneumoniae</i> through ArcB-independent regulation of the SMR efflux pump kpnEF

<i>Klebsiella pneumoniae</i> has become a major clinical and public health threat due to the increasing prevalence of healthcare-associated infections caused by multidrug-resistant strains. In this study, we demonstrated that the deletion of <i>arcA</i> of ArcAB two-component system diminished the susceptibility of <i>K. pneumoniae</i> to antibiotics, osmotic agents, disinfectants, and structural compounds, which was independent of <i>arcB</i>. RNA-seq analysis revealed a marked upregulation of SMR efflux pump genes <i>kpnEF</i> in the Δ<i>arcA</i> strain compared to the wild-type strain, while the Δ<i>arcB</i> strain exhibited no significant changes. Notably, the deletion of <i>kpnEF</i> in both Δ<i>arcA</i> and wild-type strains abolished their differential susceptibility to antibiotics, osmotic agents, disinfectants, and structural compounds. The EMSA experiments showed that ArcA-P regulated the <i>kpnEF</i> transcriptional expression by directly binding to its promoter region. These findings indicated that ArcA could directly modulate the expression of the KpnEF efflux pump independently of its sensor kinase ArcB. Through phosphorylation level detection and gene knockout experiments, we found that ArcA phosphorylation in the Δ<i>arcB</i> strain was primarily mediated by the AckA-Pta pathway. This study expanded the function of the ArcAB system and provided a critical theoretical foundation for elucidating the mechanisms underlying bacterial antibiotic resistance in <i>K. pneumoniae</i>.IMPORTANCE<i>Klebsiella pneumoniae</i> is an important opportunistic bacterial pathogen, which can acquire a series of antimicrobial resistance (AMR) genes. The emergence of carbapenem-resistant <i>K. pneumoniae</i> (CRKP) posed significant challenges to public health. Polymyxins are often regarded as the last line of defense against CRKP infections. In this study, the deletion of <i>arcA</i>, the regulator of the two-component system ArcAB, increased resistance of <i>K. pneumoniae</i> to antibiotics and decreased susceptibility to osmotic agents, disinfectants, and structural compounds, which was independent of ArcB. Further experiments have shown that ArcA regulated the expression of the small multidrug resistance (SMR) pump KpnEF through direct binding. This process required ArcA phosphorylation, which was independent of ArcB but dependent on the AckA-Pta pathway. This study deepened the regulatory network of ArcAB and provided a new target for the treatment of <i>K. pneumoniae</i>.