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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. Sodium Malonate Inhibits the AcrAB-TolC Multidrug Efflux Pump of Escherichia coli and Increases Antibiotic Efficacy

   https://doi.org/10.3390/pathogens11121409  

   Cauilan, Allea; Ruiz, Cristian (December 2022, Pathogens)

   There is an urgent need to find novel treatments for combating multidrug-resistant bacteria. Multidrug efflux pumps that expel antibiotics out of cells are major contributors to this problem. Therefore, using efflux pump inhibitors (EPIs) is a promising strategy to increase antibiotic efficacy. However, there are no EPIs currently approved for clinical use especially because of their toxicity. This study investigates sodium malonate, a natural, non-hazardous, small molecule, for its use as a novel EPI of AcrAB-TolC, the main multidrug efflux pump of the Enterobacteriaceae family. Using ethidium bromide accumulation experiments, we found that 25 mM sodium malonate inhibited efflux by the AcrAB-TolC and other MDR pumps of Escherichia coli to a similar degree than 50 μΜ phenylalanine-arginine-β-naphthylamide, a well-known EPI. Using minimum inhibitory concentration assays and molecular docking to study AcrB-ligand interactions, we found that sodium malonate increased the efficacy of ethidium bromide and the antibiotics minocycline, chloramphenicol, and ciprofloxacin, possibly via binding to multiple AcrB locations, including the AcrB proximal binding pocket. In conclusion, sodium malonate is a newly discovered EPI that increases antibiotic efficacy. Our findings support the development of malonic acid/sodium malonate and its derivatives as promising EPIs for augmenting antibiotic efficacy when treating multidrug-resistant bacterial infections. 

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3. Fitness trade-offs of multidrug efflux pumps in Escherichia coli K-12 in acid or base, and with aromatic phytochemicals

   https://doi.org/10.1128/aem.02096-23  

   Liu, Yangyang; Van_Horn, Andrew M; Pham, Minh_T N; Dinh, Bao_Ngoc N; Chen, Rachel; Raphael, Slaybrina_D R; Paulino, Alejandro; Thaker, Kavya; Somadder, Aaryan; Frost, Dominick J; et al (February 2024, Applied and Environmental Microbiology)

   Zhou, Ning-Yi (Ed.)

   ABSTRACT Multidrug efflux pumps are the frontline defense mechanisms of Gram-negative bacteria, yet little is known of their relative fitness trade-offs under gut conditions such as low pH and the presence of antimicrobial food molecules. Low pH contributes to the proton-motive force (PMF) that drives most efflux pumps. We show how the PMF-dependent pumps AcrAB-TolC, MdtEF-TolC, and EmrAB-TolC undergo selection at low pH and in the presence of membrane-permeant phytochemicals. Competition assays were performed by flow cytometry of co-culturedEscherichia coliK-12 strains possessing or lacking a given pump complex. All three pumps showed negative selection under conditions that deplete PMF (pH 5.5 with carbonyl cyanide 3-chlorophenylhydrazone or at pH 8.0). At pH 5.5, selection against AcrAB-TolC was increased by aromatic acids, alcohols, and related phytochemicals such as methyl salicylate. The degree of fitness cost for AcrA was correlated with the phytochemical’s lipophilicity (logP). Methyl salicylate and salicylamide selected strongly against AcrA, without genetic induction of drug resistance regulons. MdtEF-TolC and EmrAB-TolC each had a fitness cost at pH 5.5, but salicylate or benzoate made the fitness contribution positive. Pump fitness effects were not explained by gene expression (measured by digital PCR). Between pH 5.5 and 8.0,acrAandemrAwere upregulated in the log phase, whereasmdtEexpression was upregulated in the transition-to-stationary phase and at pH 5.5 in the log phase. Methyl salicylate did not affect pump gene expression. Our results suggest that lipophilic non-acidic molecules select against a major efflux pump without inducing antibiotic resistance regulons.IMPORTANCEFor drugs that are administered orally, we need to understand how ingested phytochemicals modulate drug resistance in our gut microbiome. Bacteria maintain low-level resistance by proton-motive force (PMF)-driven pumps that efflux many different antibiotics and cell waste products. These pumps play a key role in bacterial defense by conferring resistance to antimicrobial agents at first exposure while providing time for a pathogen to evolve resistance to higher levels of the antibiotic exposed. Nevertheless, efflux pumps confer energetic costs due to gene expression and pump energy expense. The bacterial PMF includes the transmembrane pH difference (ΔpH), which may be depleted by permeant acids and membrane disruptors. Understanding the fitness costs of efflux pumps may enable us to develop resistance breakers, that is, molecules that work together with antibiotics to potentiate their effect. Non-acidic aromatic molecules have the advantage that they avoid the Mar-dependent induction of regulons conferring other forms of drug resistance. We show that different pumps have distinct selection criteria, and we identified non-acidic aromatic molecules as promising candidates for drug resistance breakers. 

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4. TCA and SSRI Antidepressants Exert Selection Pressure for Efflux-Dependent Antibiotic Resistance Mechanisms in Escherichia coli

   https://doi.org/10.1128/mbio.02191-22  

   Ou, Jin; Elizalde, Paul; Guo, Hao-Bo; Qin, Hong; Tobe, Brian T.D.; Choy, John S. (December 2022, mBio)

   Bonomo, Robert A. (Ed.)

   ABSTRACT Microbial diversity is reduced in the gut microbiota of animals and humans treated with selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs). The mechanisms driving the changes in microbial composition, while largely unknown, is critical to understand considering that the gut microbiota plays important roles in drug metabolism and brain function. Using Escherichia coli , we show that the SSRI fluoxetine and the TCA amitriptyline exert strong selection pressure for enhanced efflux activity of the AcrAB-TolC pump, a member of the resistance-nodulation-cell division (RND) superfamily of transporters. Sequencing spontaneous fluoxetine- and amitriptyline-resistant mutants revealed mutations in marR and lon, negative regulators of AcrAB-TolC expression. In line with the broad specificity of AcrAB-TolC pumps these mutants conferred resistance to several classes of antibiotics. We show that the converse also occurs, as spontaneous chloramphenicol-resistant mutants displayed cross-resistance to SSRIs and TCAs. Chemical-genomic screens identified deletions in marR and lon, confirming the results observed for the spontaneous resistant mutants. In addition, deletions in 35 genes with no known role in drug resistance were identified that conferred cross-resistance to antibiotics and several displayed enhanced efflux activities. These results indicate that combinations of specific antidepressants and antibiotics may have important effects when both are used simultaneously or successively as they can impose selection for common mechanisms of resistance. Our work suggests that selection for enhanced efflux activities is an important factor to consider in understanding the microbial diversity changes associated with antidepressant treatments. IMPORTANCE Antidepressants are prescribed broadly for psychiatric conditions to alter neuronal levels of synaptic neurotransmitters such as serotonin and norepinephrine. Two categories of antidepressants are selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs); both are among the most prescribed drugs in the United States. While it is well-established that antidepressants inhibit reuptake of neurotransmitters there is evidence that they also impact microbial diversity in the gastrointestinal tract. However, the mechanisms and therefore biological and clinical effects remain obscure. We demonstrate antidepressants may influence microbial diversity through strong selection for mutant bacteria with increased AcrAB-TolC activity, an efflux pump that removes antibiotics from cells. Furthermore, we identify a new group of genes that contribute to cross-resistance between antidepressants and antibiotics, several act by regulating efflux activity, underscoring overlapping mechanisms. Overall, this work provides new insights into bacterial responses to antidepressants important for understanding antidepressant treatment effects. 

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5. The multidrug efflux pump regulator AcrR directly represses motility in Escherichia coli

   https://doi.org/10.1128/msphere.00430-23  

   Maldonado, Jessica; Czarnecka, Barbara; Harmon, Dana E.; Ruiz, Cristian (October 2023, mSphere)

   Bradford, Patricia A. (Ed.)

   ABSTRACT Efflux and motility are two key biological functions in bacteria. Recent findings have shown that efflux impacts flagellum biosynthesis and motility inEscherichia coliand other bacteria. AcrR is known to be the major transcriptional repressor of AcrAB-TolC, the main multidrug efflux pump inE. coliand otherEnterobacteriaceae. However, the underlying molecular mechanisms of how efflux and motility are co-regulated remain poorly understood. Here, we have studied the role of AcrR in direct regulation of motility inE. coli. By combining bioinformatics, electrophoretic mobility shift assays (EMSAs), gene expression, and motility experiments, we have found that AcrR represses motility inE. coliby directly repressing transcription of theflhDCoperon, but not the other flagellum genes/operons tested.flhDCencodes the master regulator of flagellum biosynthesis and motility genes. We found that such regulation primarily occurs by direct binding of AcrR to theflhDCpromoter region containing the first of the two predicted AcrR-binding sites identified in this promoter. This is the first report of direct regulation by AcrR of genes unrelated to efflux or detoxification. Moreover, we report that overexpression of AcrR restores to parental levels the increased swimming motility previously observed inE. colistrains without a functional AcrAB-TolC pump, and that such effect by AcrR is prevented by the AcrR ligand and AcrAB-TolC substrate ethidium bromide. Based on these and prior findings, we provide a novel model in which AcrR senses efflux and then co-regulates efflux and motility inE. colito maintain homeostasis and escape hazards. IMPORTANCEEfflux and motility play a major role in bacterial growth, colonization, and survival. InEscherichia coli, the transcriptional repressor AcrR is known to directly repress efflux and was later found to also repress flagellum biosynthesis and motility by Kim et al. (J Microbiol Biotechnol 26:1824–1828, 2016, doi: 10.4014/jmb.1607.07058). However, it remained unknown whether AcrR represses flagellum biosynthesis and motility directly and through which target genes, or indirectly because of altering the amount of efflux. This study reveals that AcrR represses flagellum biosynthesis and motility by directly repressing the expression of theflhDCmaster regulator of flagellum biosynthesis and motility genes, but not the other flagellum genes tested. We also show that the antimicrobial, efflux pump substrate, and AcrR ligand ethidium bromide regulates motility via AcrR. Overall, these findings support a novel model of direct co-regulation of efflux and motility mediated by AcrR in response to stress inE. coli. 

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