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bioRxiv · 10.1101/2025.09.26.678718

Strain-dependent contribution of the AcrAB-TolC efflux pump to Klebsiella pneumoniae physiology

Bhogal, K.·Clough, B.·Emmerson, C.·Organ, A.·Chen, Y.·Buckner, M. M. C.·Alav, I.
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Abstract

Klebsiella pneumoniae is a prominent opportunistic pathogen increasingly associated with multidrug resistance and virulence. One of the main mechanisms of antimicrobial resistance in K. pneumoniae is active efflux, primarily mediated by the Resistance-Nodulation-Division (RND) family of pumps. AcrAB-TolC is the key RND efflux pump in K. pneumoniae, regulated by the transcriptional activator RamA and its repressor RamR. Although overexpression of AcrAB-TolC has been linked to drug resistance in various clinical strains, its physiological roles in K. pneumoniae remain insufficiently studied. In this study, we generated isogenic deletions of acrB and ramR in both the genetically tractable K. pneumoniae Ecl8 and the virulent ATCC 43816 strains. We examined the phenotype of the {Delta}acrB and {Delta}ramR mutants by assessing antimicrobial susceptibility, biofilm formation, growth under infection-related conditions, and both in vitro and in vivo infection models. Loss of acrB increased susceptibility to drugs, decreased biofilm formation, and reduced in vitro virulence in both Ecl8 and ATCC 43816. However, only in Ecl8 was the loss of AcrB found to diminish growth under infection-like conditions and decrease in vivo virulence in the Galleria mellonella infection model. In contrast, in ATCC 43816, it had no effect. Our findings suggest that AcrAB-TolC exhibits strain-specific physiological functions, highlighting its dual role in antimicrobial resistance and pathogenicity, and thereby broadening our understanding of efflux-mediated adaptations in K. pneumoniae. Exploring the broader functions of RND efflux pumps in K. pneumoniae can provide insights into the potential effects of targeting them with inhibitor molecules. ImportanceInfections caused by multidrug-resistant bacterial pathogens are among the most urgent global public health challenges. Specifically, Klebsiella pneumoniae is classified by the World Health Organisation as a critical priority pathogen for the development of new treatments. Resistance-Nodulation-Division (RND) efflux pumps significantly contribute to multidrug resistance and virulence, making them promising targets for drug development. In this study, we demonstrated that the RND efflux pump AcrAB-TolC in K. pneumoniae is essential for drug efflux, biofilm formation, and in vitro virulence. Notably, we identified strain-specific roles for AcrAB-TolC in supporting growth under infection-related conditions and virulence in the Galleria mellonella infection model. Our findings highlight that the function of RND efflux pumps can vary between strains within a species. This implies that targeting RND efflux pumps with inhibitors may yield different effects depending on the strain background.

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Bhogal, K., Clough, B., Emmerson, C., Organ, A., Chen, Y., Buckner, M. M. C., Alav, I.. 2025-09-26. Strain-dependent contribution of the AcrAB-TolC efflux pump to Klebsiella pneumoniae physiology. https://doi.org/10.1101/2025.09.26.678718

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