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1. The Multidrug Efflux Regulator AcrR of Escherichia coli Responds to Exogenous and Endogenous Ligands To Regulate Efflux and Detoxification

   https://doi.org/10.1128/msphere.00474-22  

   Harmon, Dana E.; Ruiz, Cristian (December 2022, mSphere)

   Bradford, Patricia A. (Ed.)

   ABSTRACT The transcriptional repressor AcrR is the main regulator of the multidrug efflux pump AcrAB-TolC, which plays a major role in antibiotic resistance and cell physiology in Escherichia coli and other Enterobacteriaceae . However, it remains unknown which ligands control the function of AcrR. To address this gap in knowledge, this study tested whether exogenous and/or endogenous molecules identified as potential AcrR ligands regulate the activity of AcrR. Using electrophoretic mobility shift assays (EMSAs) with purified AcrR and the acrAB promoter and in vivo gene expression experiments, we found that AcrR responds to both exogenous molecules and cellular metabolites produced by E. coli . In total, we identified four functional ligands of AcrR, ethidium bromide (EtBr), an exogenous antimicrobial known to be effluxed by the AcrAB-TolC pump and previously shown to bind to AcrR, and three polyamines produced by E. coli , namely, putrescine, cadaverine, and spermidine. We found that EtBr and polyamines bind to AcrR both in vitro and in vivo , which prevents the binding of AcrR to the acrAB promoter and, ultimately, induces the expression of acrAB . Finally, we also found that AcrR contributes to mitigating the toxicity produced by excess polyamines by directly regulating the expression of AcrAB-TolC and two previously unknown AcrR targets, the MdtJI spermidine efflux pump and the putrescine degradation enzyme PuuA. Overall, these findings significantly expand our understanding of the function of AcrR by revealing that this regulator responds to different exogenous and endogenous ligands to regulate the expression of multiple genes involved in efflux and detoxification. IMPORTANCE Multidrug efflux pumps can remove antibiotics and other toxic molecules from cells and are major contributors to antibiotic resistance and bacterial physiology. Therefore, it is essential to better understand their function and regulation. AcrAB-TolC is the main multidrug efflux pump in the Enterobacteriaceae family, and AcrR is its major transcriptional regulator. However, little is known about which ligands control the function of AcrR or which other genes are controlled by this regulator. This study contributes to addressing these gaps in knowledge by showing that (i) the activity of AcrR is controlled by the antimicrobial ethidium bromide and by polyamines produced by E. coli , and (ii) AcrR directly regulates the expression of AcrAB-TolC and genes involved in detoxification and efflux of excess polyamines. These findings significantly advance our understanding of the biological role of AcrR by identifying four ligands that control its function and two novel targets of this regulator. 

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2. Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein

   https://doi.org/10.1038/s41467-023-39615-x  

   Russell Lewis, Benjamin; Uddin, Muhammad R.; Moniruzzaman, Mohammad; Kuo, Katie M.; Higgins, Anna J.; Shah, Laila M. N.; Sobott, Frank; Parks, Jerry M.; Hammerschmid, Dietmar; Gumbart, James C.; et al (July 2023, Nature Communications)

   Abstract Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system fromEscherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations. We define the structural dynamics of AcrA and find that an inhibitor can inflict long-range stabilisation across all four of its domains, whereas an interacting efflux substrate has minimal effect. Our results support a model where an inhibitor forms a molecular wedge within a cleft between the lipoyl and αβ barrel domains of AcrA, diminishing its conformational transmission of drug-evoked signals from AcrB to TolC. This work provides molecular insights into multidrug adaptor protein function which could be valuable for developing antimicrobial therapeutics. 

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3. Bacterial efflux pump modulators prevent bacterial growth in macrophages and under broth conditions that mimic the host environment

   https://doi.org/10.1128/mbio.02492-23  

   Allgood, Samual C; Su, Chih-Chia; Crooks, Amy L; Meyer, Christian T; Zhou, Bojun; Betterton, Meredith D; Barbachyn, Michael R; Yu, Edward W; Detweiler, Corrella S (December 2023, mBio)

   Hughes, Kelly T (Ed.)

   ABSTRACT New approaches for combating microbial infections are needed. One strategy for disrupting pathogenesis involves developing compounds that interfere with bacterial virulence. A critical molecular determinant of virulence for Gram-negative bacteria are efflux pumps of the resistance-nodulation-division family, which includes AcrAB-TolC. We previously identified small molecules that bind AcrB, inhibit AcrAB-TolC, and do not appear to damage membranes. These efflux pump modulators (EPMs) were discovered in an in-cell screening platform called SAFIRE (Screen for Anti-infectives using Fluorescence microscopy of IntracellulaR Enterobacteriaceae). SAFIRE identifies compounds that disrupt the growth of a Gram-negative human pathogen,Salmonella entericaserotype Typhimurium (S. Typhimurium), in macrophages. We used medicinal chemistry to iteratively design ~200 EPM35 analogs and test them for activity in SAFIRE, generating compounds with nanomolar potency. Analogs were demonstrated to bind AcrB in a substrate binding pocket by cryo-electron microscopy. Despite having amphipathic structures, the EPM analogs do not disrupt membrane voltage, as monitored by FtsZ localization to the cell septum. The EPM analogs had little effect on bacterial growth in standard Mueller Hinton Broth. However, under broth conditions that mimic the micro-environment of the macrophage phagosome,acrABis required for growth, the EPM analogs are bacteriostatic, and the EPM analogs increase the potency of antibiotics. These data suggest that under macrophage-like conditions, the EPM analogs prevent the export of a toxic bacterial metabolite(s) through AcrAB-TolC. Thus, compounds that bind AcrB could disrupt infection by specifically interfering with the export of bacterial toxic metabolites, host defense factors, and/or antibiotics. IMPORTANCEBacterial efflux pumps are critical for resistance to antibiotics and for virulence. We previously identified small molecules that inhibit efflux pumps (efflux pump modulators, EPMs) and prevent pathogen replication in host cells. Here, we used medicinal chemistry to increase the activity of the EPMs against pathogens in cells into the nanomolar range. We show by cryo-electron microscopy that these EPMs bind an efflux pump subunit. In broth culture, the EPMs increase the potency (activity), but not the efficacy (maximum effect), of antibiotics. We also found that bacterial exposure to the EPMs appear to enable the accumulation of a toxic metabolite that would otherwise be exported by efflux pumps. Thus, inhibitors of bacterial efflux pumps could interfere with infection not only by potentiating antibiotics, but also by allowing toxic waste products to accumulate within bacteria, providing an explanation for why efflux pumps are needed for virulence in the absence of antibiotics. 

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4. Escherichia coli resistance mechanism AcrAB-TolC efflux pump interactions with commonly used antibiotics: a molecular dynamics study

   https://doi.org/10.1038/s41598-024-52536-z  

   Smith, Brooke L; Fernando, Sandun; King, Maria D (December 2024, Scientific Reports)

   Abstract While antibiotic resistance poses a threat from both Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB), GNB pose a more imminent public health hazard globally. GNB are a threat to growing antibiotic resistance because of the complex makeup of the membrane. The AcrAB-TolC efflux pump is a known resistance mechanism ofEscherichia coli (E. coli)cells. This study utilized molecular dynamics modeling to visualize some of the changes occurring at a molecular level when airborne bacteria are exposed to stress and antibiotics. This study was conducted to build upon previous experimental research showing that there is an increase in antibiotic resistance and efflux pump activity when exposed to aerosolization. AcrB and AcrAB-TolC proteins were simulated under standard and increased pressure to compare the effect of aerosolization on the binding to the three different antibiotics (puromycin (PUY), ampicillin (AMP) and sulfamethoxazole-trimethoprim (SXT)) to the AcrB binding site. Analysis such as root-mean-square deviation of atomic positions and root-mean-square fluctuation, the opening of TolC, and the significant molecular mechanics with generalized Born and surface area solvation (MM-GBSA) scores associated with specific ligands were recorded. Resistance in experimental data indicated a relationship between the docking scores and some ligand–protein interactions. Results showed that there was more flexibility in the proteins within simulations conducted under standard pressure for the AcrB protein and the full tripartite complex AcrAB-TolC, showing that increased pressure causes more rigidity. MM-GBSA scores, used to calculate the free energy of ligand–protein binding, did not show a significant change, but interestingly, the strongest MM-GBSA scores were for ligands that moved to another binding pocket and did not result in resistance or opening of the efflux pump. However, the ligand moved from the binding site and did not cause the opening of TolC to increase significantly, whereas PUY and AMP were bound to the binding site for the duration of all simulations. AMP ligands under increased pressure showed the largest change in opening of the TolC efflux pump and aligns with experimental data showingE. colicells had the most resistance to AMP after aerosolization. These results, in addition to other real-time changes such as OM proteins and mutations of targets within the cell, could be used to delineate and mitigate antibiotic resistance mechanisms. 

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5. Insight into the AcrAB-TolC Complex Assembly Process Learned from Competition Studies

   https://doi.org/10.3390/antibiotics10070830  

   Rajapaksha, Prasangi; Ojo, Isoiza; Yang, Ling; Pandeya, Ankit; Abeywansha, Thilini; Wei, Yinan (July 2021, Antibiotics)

   null (Ed.)

   The RND family efflux pump AcrAB-TolC in E. coli and its homologs in other Gram-negative bacteria are major players in conferring multidrug resistance to the cells. While the structure of the pump complex has been elucidated with ever-increasing resolution through crystallography and Cryo-EM efforts, the dynamic assembly process remains poorly understood. Here, we tested the effect of overexpressing functionally defective pump components in wild type E. coli cells to probe the pump assembly process. Incorporation of a defective component is expected to reduce the efflux efficiency of the complex, leading to the so called “dominant negative” effect. Being one of the most intensively studied bacterial multidrug efflux pumps, many AcrA and AcrB mutations have been reported that disrupt efflux through different mechanisms. We examined five groups of AcrB and AcrA mutants, defective in different aspects of assembly and substrate efflux. We found that none of them demonstrated the expected dominant negative effect, even when expressed at concentrations many folds higher than their genomic counterpart. The assembly of the AcrAB-TolC complex appears to have a proof-read mechanism that effectively eliminated the formation of futile pump complex. 

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