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The multidrug efflux transporters MexB and MexY in Pseudomonas aeruginosa and AcrB in Escherichia coli contribute to these organisms' multidrug resistance. Efflux pump inhibitor (EPI) ABI-PP inhibits MexB and AcrB, but not MexY. We previously determined the structure of ABI-PP bound to the hydrophobic trap (the inhibitor-binding pit) of AcrB and MexB. The insensitivity of MexY to ABI-PP was attributed to a bulky tryptophan (Trp). AcrB(Phe178Trp) became uninhibited by ABI-PP, while MexY(Trp177Phe) resensitized MexY for ABI-PP. Interestingly, ABI-PP was able to inhibit MexB(Phe178Trp). Thus, it is not clear which bulky amino acid mutations are critical for inhibitor binding in MexB. Here, we investigated the pit of MexB in more detail, and elucidated which Trp mutation locations in the pit were hindering ABI-PP binding, but did not affect the function of the efflux pumps. Mutating positions 139, 277, 279, and 612 to tryptophan eliminated the inhibitory effect. However, the tryptophan mutation at position 571 did not cause any effect. These results show that the effectiveness of EPIs is greatly affected by mutations in different locations, and that binding of EPIs is partly attributed by spatial characteristics. These results should be taken into account for new inhibitor and drug discovery.

Multidrug resistance (MDR) in bacteria can be caused by the over-expression of multidrug efflux pumps belonging to the Resistance-Nodulation-Division (RND) superfamily of proteins. These intrinsic or acquired pumps can export a wide range of antibiotics. Recently, amino acid substitutions within these pumps have been observed in resistant clinical strains. Among others, two of these worrying gain-of-function mutations are R717L and R717Q in the proximal binding pocket of efflux pump AcrB (AcrB-Sa) found in azithromycin-resistant Salmonella enterica spp. We investigated the ramifications of these (and other) mutations in phylogenetically closely related AcrB from Escherichia coli (AcrB-Ec). We found that AcrB-Ec harboring Arg717 substitutions were significantly more effective in exporting all tested macrolides, with an up to 8-fold increase in the minimum inhibitory concentration (MIC) values (from 16 to 128 μg/mL for azithromycin). Interestingly, gain-of-function was also seen for fluoroquinolones (2-fold higher MICs), while there was a consistent loss-of-function for the export of novobiocin and β-lactam cloxacillin (2-fold lower MICs), pointing to a protein adaptation, which simultaneously partly compromised the efflux ability of other compounds with different molecular properties. Disk diffusion susceptibility testing corroborated the findings, as the R717Q and R717L mutant strains had significantly smaller inhibition zones for macrolides and fluoroquinolones and a larger inhibition zone for novobiocin, compared to the wild type. The spread and independent emergences of these potent efflux pump mutations highlight the necessity of control of, and adjustments to, treatments with antibiotics and the need for novel antibiotics and efflux pump inhibitors.ImportanceThe over-expression of multidrug efflux pumps is a major cause of multidrug-resistant bacteria. The emergence of gain-of-function mutations in these pumps is additionally worrying, as these render last-resort antibiotic treatment options ineffective. The Arg717-mutations in related Salmonella Typhi and Paratyphi A AcrB caused azithromycin-resistant strains, with resistance levels past breakpoints. This is worrying, as azithromycin is a last-resort antibiotic after fluoroquinolones to treat typhoid and paratyphoid fever. Our findings that the same substitutions in closely related Escherichia coli AcrB cause an increase in MICs for both fluoroquinolones and macrolides is worrying as it shows the effect of one amino acid substitution on a multitude of treatment options. Additionally, the finding that the substitutions negatively impact the export of other drugs, such as β-lactam cloxacillin, suggests that treatment with multiple antibiotics may mitigate resistance and improve treatment. Our findings also imply the urge to develop novel antibiotics and inhibitors.

Multidrug resistance in Gram-negative bacteria can arise by the over-expression of multidrug efflux pumps, which can extrude a wide range of antibiotics. Here we describe the ancestral Haemophilus influenzae efflux pump AcrB (AcrB-Hi). We performed a phylogenetic analysis of hundreds of RND-type transporters. We found that AcrB-Hi is a relatively ancient efflux pump, which nonetheless can export the same range of antibiotics as its evolved colleague from Escherichia coli. AcrB-Hi was not inhibited by the efflux pump inhibitor ABI-PP, and could export bile salts weakly. This points to an environmental adaptation of RND transporters. We also explain the sensitivity of H. influenzae cells to β-lactams and novobiocin by the outer membrane porin OmpP2. This porin counterbalances the AcrB-Hi efflux by leaking the drugs back into the cells. We hypothesise that multidrug recognition by RND-type pumps is not an evolutionarily acquired ability, and has been present since ancient promiscuous transporters.

Multidrug resistance in Gram-negative bacteria can arise by the over-expression of multidrug efflux pumps, which can extrude a wide range of antibiotics. Here we describe the ancestral Haemophilus influenzae efflux pump AcrB (AcrB-Hi). We performed a phylogenetic analysis of hundreds of RND-type transporters. We found that AcrB-Hi is a relatively ancient efflux pump, which nonetheless can export the same range of antibiotics as its evolved colleague from Escherichia coli. AcrB-Hi was not inhibited by the efflux pump inhibitor ABI-PP, and could export bile salts weakly. This points to an environmental adaptation of RND transporters. We also explain the sensitivity of H. influenzae cells to β-lactams and novobiocin by the outer membrane porin OmpP2. This porin counterbalances the AcrB-Hi efflux by leaking the drugs back into the cells. We hypothesise that multidrug recognition by RND-type pumps is not an evolutionarily acquired ability, and has been present since ancient promiscuous transporters., Zwama et al. demonstrate that the AcrB efflux pump from H. influenzae (AcrB-Hi) is phylogenetically far removed from AcrB in E. coli but show similar specificity. They also show that efflux is counteracted by leakage of some antibiotics back into the bacterial cell through the OmpP2 channel which can explain H. influenzae sensitivity to these compounds.

The over-expression of multidrug efflux pumps belonging to the Resistance-Nodulation-Division (RND) superfamily is one of the main causes of multidrug-resistance (MDR) in Gram-negative pathogenic bacteria. AcrB is the most thoroughly studied RND transporter and has functioned as a model for our understanding of efflux-mediated MDR. This multidrug-exporter can recognize and transport a wide range of structurally unrelated compounds (including antibiotics, dyes, bile salts and detergents), while it shows a strict inhibitor specificity. Here we discuss our current knowledge of AcrB, and include recent advances, regarding its structure, mechanism of drug transport, substrate recognition, different intramolecular entry pathways and the drug export driven by remote conformational coupling.

A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding.

Biologically active plant phenols (phytochemicals) are a cornerstone of traditional medicine. Phytochemicals have attracted increasing attention from Western medicine, and thousands of studies on their activity are published each year. Phytochemicals exert a broad range of pharmacological effects including being antioxidant, anti-inflammatory, anticarcinogenic and antimicrobial, yet their mechanism(s) of action are usually ill defined. Some better-studied phytochemicals modulate the function of a multitude of unrelated proteins, with few identified binding sites. Different phenolic compounds often affect the same proteins, many of which are membrane-associated. Additionally, in spite of large variations in chemical structure, plant phenols often have synergistic effects. In this context, it may be relevant that phytochemicals generally are hydrophobic/amphipathic and tend to adsorb to lipid bilayers. Phytochemicals therefore could exert some of their actions indirectly by perturbing membrane properties. To test the hypothesis that plant phenols exert their action on protein function by altering lipid bilayer properties, we chose five heavily studied phytochemicals: capsaicin (chili peppers), curcumin (turmeric), EGCG ((-)-epigallocatechin gallate, green tea), genistein (soybeans), and resveratrol (grapes). We measured their propensity to alter bilayer properties using gramicidin A channels, as probes for changes in bilayer material properties, and explored the phytochemicals’ effect on various membrane proteins: potassium channels, membrane-anchored metalloproteases, mechanosensitive channels of large conductance and voltage-gated sodium channels. All the tested compounds alter bilayer properties at concentrations consistent with their reported biological activity; they also altered the function of the membrane proteins tested, albeit at varying concentrations. We show that phytochemicals can alter protein function through a bilayer-mediated manner; therefore, any studies on phytochemicals should keep their promiscuity in mind and before claiming specific interactions evaluate the possibility of an indirect membrane mediated mechanism.