Biology subjects

Alav, I.

Publications and source records attributed to Alav, I..

5 recordsLinked to original sources

bioRxiv2025

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

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.

microbiology↗

bioRxiv2024

Natural products from food sources can alter the spread of AMR plasmids in Enterobacterales

Antimicrobial resistance (AMR) poses a significant threat to global public health. Notably, resistance to carbapenem and extended-spectrum {beta}-lactam antibiotics in Gram-negative bacteria is a major impediment for the treatment of infections. Genes responsible for resistance to these antibiotics are frequently carried on plasmids, which can transfer between bacteria. Therefore, exploring strategies to prevent this transfer and/or the prevalence of AMR plasmids is timely and pertinent. Here, we show that certain natural product extracts and associated pure compounds can reduce the transmission of AMR plasmids into new bacterial hosts. Using our established high-throughput fluorescence-based screen we found that the natural products were more active in reducing transmission of the IncK plasmid pCT in Escherichia coli ST131, compared to Klebsiella pneumoniae Ecl8 carrying the IncFII plasmid pKpQIL. Furthermore, we found that the natural product rottlerin was more active in K. pneumoniae than in E. coli. Importantly, rottlerin was also associated with a reduced number of transconjugant bacteria in a clinical K. pneumoniae isolate harbouring a blaNDM-1 plasmid. Together, these results demonstrate the potential of natural products as promising anti-plasmid agents.

microbiology↗

bioRxiv2023

Metal complexes and conjugation: Harnessing the power of cobalt complexes to curtail plasmid transfer

BackgroundAntimicrobial resistance genes (ARG), such as extended spectrum {beta}-lactamase (ESBL) and carbapenemase genes, are commonly carried on plasmids. Plasmids can transmit between bacteria, disseminate globally, and cause clinically important resistance. Therefore, targeting plasmids could reduce ARG prevalence, and restore the efficacy of existing antibiotics. Here, we assessed the effect of four previously characterised bis(N-picolinamido)cobalt(II) complexes on the conjugative transfer of plasmids in Escherichia coli and Klebsiella pneumoniae. MethodsLiquid broth and solid agar conjugation assays were used to measure complex activity on four plasmids in E. coli. Additionally, the effect of cobalt complexes was tested on the transmission of the fluorescently tagged extended spectrum {beta}-lactamase encoding pCTgfp plasmid in E. coli and carbapenemase encoding pKpQILgfp plasmid in K. pneumoniae, using flow cytometry. ResultsAntimicrobial susceptibility testing of cobalt complexes revealed no antibacterial activity. The cobalt complexes significantly reduced conjugative transfer of RP4, R6K, and R388 plasmids on solid agar in E. coli and pKpQILgfp transmission in K. pneumoniae. None affected conjugative transfer of pKM101 or transmission of fluorescently tagged pCT in E. coli. The cobalt complexes had no effect on plasmid persistence, suggesting that they target conjugation rather than plasmid prevalence. ConclusionsTo the best of our knowledge, this is the first study to report reduced transmission of clinically relevant plasmids with cobalt complexes. These cobalt complexes are not cytotoxic towards mammalian cells and are not antibacterial, therefore they could be optimised and employed as conjugation inhibitors to reduce prevalence of AMR and/or virulence genes in animals and humans. SignificanceAntimicrobial resistance is a growing problem that poses a significant threat to modern medicine. Some of the most problematic resistance genes are carried on genetic elements, called plasmids, that can spread between bacteria. While our understanding of the mechanisms and drivers of gene transfer amongst bacteria is increasing, we lack effective tools to slow down/control these processes. Here we demonstrate for the first time that novel cobalt-based compounds have anti-plasmid activity on a subset of E. coli plasmids, and are extremely potent in K. pneumoniae carrying a clinical carbapenem-resistance plasmid, without impacting plasmid maintenance. This finding forms the foundations of a potential strategy to control the transfer of genes within Gram-negative bacteria, which has implications for AMR and virulence.

microbiology↗

bioRxiv2021

The membrane-proximal domain of the periplasmic adapter protein plays a role in vetting substrates utilising channels 1 and 2 of RND efflux transporters

Active efflux by resistance-nodulation-division (RND) efflux pumps is a major contributor to antibiotic resistance in clinically relevant Gram-negative bacteria. Tripartite RND pumps, such as AcrAB-TolC of Salmonella enterica serovar Typhimurium, comprise of an inner membrane RND transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor. Previously, we elucidated binding sites within the PAP AcrA (termed binding boxes) that were important for AcrB-transporter recognition. Here, we have refined the binding box model by identifying the most critical residues involved in PAP-RND binding and show that the corresponding RND-binding residues in the closely related PAP AcrE are also important for AcrB interactions. In addition, our analysis identified a membrane-proximal domain (MPD)-residue in AcrA (K366), that when mutated, differentially affects transport of substrates utilising different AcrB efflux-channels, namely channels 1 and 2, supporting a potential role for the PAP in sensing the substrate-occupied state of the proximal binding pocket (PBP) of the transporter and substrate vetting. Our model predicts that there is a close interplay between the MPD of the PAP and the RND transporter in the productive export of substrates utilising the PBP. ImportanceAntibiotic resistance greatly threatens our ability to treat infectious diseases. In Gram-negative bacteria, overexpression of tripartite efflux pumps, such as AcrAB-TolC, contributes to multidrug resistance because they export many different classes of antibiotics. The AcrAB-TolC pump is made up of three components: the periplasmic adaptor protein (PAP) AcrA, the RND-transporter AcrB, and the outer-membrane factor TolC. Here, we identified critical residues of AcrA that are important for its function with AcrB in Salmonella enterica serovar Typhimurium. Also, we show that AcrA shares these critical residues with AcrE, a closely related PAP, explaining their interoperability with AcrB. Importantly, we identified a residue in the membrane-proximal domain of AcrA that when mutated affected how different substrates access AcrB and impacted downstream efflux via TolC channel. Understanding the role that PAPs play in the assembly and function of tripartite RND pumps can guide novel ways to inhibit their function to combat antibiotic resistance.

microbiology↗

bioRxiv2021

Interchangeability of Periplasmic Adaptor Proteins AcrA and AcrE in forming functional efflux pumps with AcrD in Salmonella Typhimurium

BackgroundRND efflux pumps are important mediators of antibiotic resistance. RND pumps including the principal multidrug-efflux pump AcrAB-TolC in Salmonella, are tripartite systems, with an inner membrane RND-transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor (OMF). We previously identified the residues required for binding between the PAP AcrA and the RND-transporter AcrB and have demonstrated that PAPs can function with non-cognate transporters. AcrE and AcrD/AcrF are homologues of AcrA and AcrB, respectively. Here, we show that AcrE can interact with AcrD, which does not possess its own PAP, and establish that the residues previously identified in AcrB-binding are also involved in AcrD-binding. MethodsThe acrD and acrE genes were expressed into a strain lacking acrABDEF ({Delta}3RND). PAP residues involved in promiscuous interactions were predicted based on previously defined PAP-RND interactions and corresponding mutations generated in acrA and acrE. Antimicrobial susceptibility of the mutant strains was determined. ResultsCo-expression of acrD and acrE significantly decreased susceptibility of the {Delta}3RND strain to AcrD substrates showing that AcrE can form a functional complex with AcrD. The substrate profile of Salmonella AcrD differed from that of E. coli AcrD. Mutations targeting the previously defined PAP-RND interaction sites in AcrA/AcrE impaired efflux of AcrD-dependent substrates. ConclusionsThese data indicate that AcrE forms an efflux-competent pump with AcrD and thus presents an alternative PAP for this pump. Mutagenesis of the conserved RND-binding sites validates the interchangeability of AcrA and AcrE, highlighting them as potential drug targets for efflux inhibition.

microbiology↗