Proximal Binding Pocket Arg717 Substitutions in Escherichia coli AcrB Cause Clinically Relevant Divergencies in Resistance Profiles.

Recent mutations in RND efflux pumps in clinical strains have further increased multidrug resistance. We show that R717L and R717Q substitutions (found in azithromycin-resistant Salmonella enterica spp.) in the Escherichia coli efflux pump AcrB dramatically increase macrolide, as well as fluoroquinolone, resistance. On the other hand, cells became more susceptible to novobiocin and ß-lactam cloxacillin. We urge the control of, and adjustments to, treatments with antibiotics and the need for novel antibiotics and efflux pump inhibitors.

Spatial Characteristics of the Efflux Pump MexB Determine Inhibitor Binding.

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.

Ever-Adapting RND Efflux Pumps in Gram-Negative Multidrug-Resistant Pathogens: A Race against Time.

The rise in multidrug resistance (MDR) is one of the greatest threats to human health worldwide. MDR in bacterial pathogens is a major challenge in healthcare, as bacterial infections are becoming untreatable by commercially available antibiotics. One of the main causes of MDR is the over-expression of intrinsic and acquired multidrug efflux pumps, belonging to the resistance-nodulation-division (RND) superfamily, which can efflux a wide range of structurally different antibiotics. Besides over-expression, however, recent amino acid substitutions within the pumps themselves-causing an increased drug efflux efficiency-are causing additional worry. In this review, we take a closer look at clinically, environmentally and laboratory-evolved Gram-negative bacterial strains and their decreased drug sensitivity as a result of mutations directly in the RND-type pumps themselves (from Escherichia coli, Salmonella enterica, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Acinetobacter baumannii and Legionella pneumophila). We also focus on the evolution of the efflux pumps by comparing hundreds of efflux pumps to determine where conservation is concentrated and where differences in amino acids can shed light on the broad and even broadening drug recognition. Knowledge of conservation, as well as of novel gain-of-function efflux pump mutations, is essential for the development of novel antibiotics and efflux pump inhibitors.

Function and Inhibitory Mechanisms of Multidrug Efflux Pumps.

Multidrug efflux pumps are inner membrane transporters that export multiple antibiotics from the inside to the outside of bacterial cells, contributing to bacterial multidrug resistance (MDR). Postgenomic analysis has demonstrated that numerous multidrug efflux pumps exist in bacteria. Also, the co-crystal structural analysis of multidrug efflux pumps revealed the drug recognition and export mechanisms, and the inhibitory mechanisms of the pumps. A single multidrug efflux pump can export multiple antibiotics; hence, developing efflux pump inhibitors is crucial in overcoming infectious diseases caused by multidrug-resistant bacteria. This review article describes the role of multidrug efflux pumps in MDR, and their physiological functions and inhibitory mechanisms.

Identification of Genetic Variants via Bacterial Respiration Gas Analysis.

Indole is a signal molecule derived from the conversion of tryptophan, and it is present in bacterial respiratory gas. Besides influencing bacterial growth, indole exhibits effects on human health, including a positive effect on inflammation and protection against pathogens. However, a high fecal indole concentration (FIC) can suggest an unbalanced gut flora or the presence of certain pathogens. To analyze the indole produced by bacteria, its collection and detection is required. Traditional methods usually require centrifugation of liquid bacterial culture medium and subsequent extraction of indole from the medium or partial purification of indole from fecal samples (e.g., by distillation or extraction). In this study, we demonstrate the possibility of identifying gas contents directly from bacteria, and we distinguish the difference in species and their genetics without the need to centrifuge or extract. Using an absorbent sheet placed above a liquid culture, we were able to collect gas content directly from bacteria. Gas chromatography-mass spectrometry (GC-MS) was used for the analysis. The GC-MS results showed a clear peak attributed to indole for wild-type Escherichia coli cells (MG1655 and MC4100 strains), whereas the indole peak was absent in the chromatograms of cells where proteins, part of the indole production pathway from tryptophan (TnaA and TnaB), were not expressed (by using tnaAB-deleted cells). The indole observed was measured to be present in a low nmol-range. This method can distinguish whether the bacterial genome contains the tnaAB gene or not and can be used to collect gas compounds from bacterial cultures quickly and easily. This method is useful for other goals and future research, such as for measurements in restrooms, for food-handling facilities, and for various applications in medical settings.

Phylogenetic and functional characterisation of the Haemophilus influenzae multidrug efflux pump AcrB.

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 ß-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.

Molecular mechanisms of AcrB-mediated multidrug export.

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.

Multiple entry pathways within the efflux transporter AcrB contribute to multidrug recognition.

AcrB is the major multidrug exporter in Escherichia coli. Although several substrate-entrances have been identified, the specificity of these various transport paths remains unclear. Here we present evidence for a substrate channel (channel 3)  from the central cavity of the AcrB trimer, which is connected directly to the deep pocket without first passing the switch-loop and the proximal pocket . Planar aromatic cations, such as ethidium, prefer channel 3 to channels 1 and 2. The efflux through channel 3 increases by targeted mutations and is not in competition with the export of drugs such as minocycline and erythromycin through channels 1 and 2. A switch-loop mutant, in which the pathway from the proximal to the deep pocket is hindered, can export only channel 3-utilizing drugs. The usage of multiple entrances thus contributes to the recognition and transport of a wide range of drugs with different physicochemical properties.

Hoisting-Loop in Bacterial Multidrug Exporter AcrB Is a Highly Flexible Hinge That Enables the Large Motion of the Subdomains.

The overexpression of RND-type exporters is one of the main causes of multidrug resistance (MDR) in Gram-negative pathogens. In RND transporters, such as Escherichia coli's main efflux pump AcrB, drug efflux occurs in the porter domain, while protons flow through the transmembrane domain: remote conformational coupling. At the border of a transmembrane helix (TM8) and subdomain PC2, there is a loop which makes a hoisting movement by a random-coil-to-α-helix change, and opens and closes a drug channel entrance. This loop is supposed to play a key role in the allosteric conformational coupling between the transmembrane and porter domain. Here we show the results of a series of flexibility loop-mutants of AcrB. We determined the crystal structure of a three amino acid truncated loop mutant, which is still a functional transporter, and show that the short α-helix between Cß15 and the loop unwinds to a random coil in the access and binding monomers and in the extrusion monomer it makes a partially stretched coil-to-helix change. The loop has undergone compensatory conformational changes and still facilitates the opening and closing of the channel. In addition, more flexible mutated loops (proline mutated and significantly elongated) can still function during export. The flexibility in this region is however limited, as an even more truncated mutant (six amino acid deletion) becomes mostly inactive. We found that the hoisting-loop is a highly flexible hinge that enables the conformational energy transmission passively.

Phytochemicals perturb membranes and promiscuously alter protein function.

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.