Small Multidrug Resistance Family Research Articles

Transport of metformin metabolites by guanidinium exporters of the small multidrug resistance family.

Proteins from the small multidrug resistance (SMR) family are frequently associated with horizontally transferred multidrug resistance gene arrays found in bacteria from wastewater and the human-adjacent biosphere. Recent studies suggest that a subset of SMR transporters might participate in the metabolism of the common pharmaceutical metformin by bacterial consortia. Here, we show that both genomic and plasmid-associated transporters of the SMRGdx functional subtype export byproducts of microbial metformin metabolism, with particularly high export efficiency for guanylurea. We use solid-supported membrane electrophysiology to evaluate the transport kinetics for guanylurea and native substrate guanidinium by four representative SMRGdx homologs. Using an internal reference to normalize independent electrophysiology experiments, we show that transport rates are comparable for genomic and plasmid-associated SMRGdx homologs, and using a proteoliposome-based transport assay, we show that 2 proton:1 substrate transport stoichiometry is maintained. Additional characterization of guanidinium and guanylurea export properties focuses on the structurally characterized homolog, Gdx-Clo, for which we examined the pH dependence and thermodynamics of substrate binding and solved an x-ray crystal structure with guanylurea bound. Together, these experiments contribute in two main ways. By providing the first detailed kinetic examination of the structurally characterized SMRGdx homolog Gdx-Clo, they provide a functional framework that will inform future mechanistic studies of this model transport protein. Second, this study casts light on a potential role for SMRGdx transporters in microbial handling of metformin and its microbial metabolic byproducts, providing insight into how native transport physiologies are co-opted to contend with new selective pressures.

Microsecond Motion of the Bacterial Transporter EmrE in Lipid Bilayers.

The bacterial transporter EmrE is a homo-dimeric membrane protein that effluxes cationic polyaromatic substrates against the concentration gradient by coupling to proton transport. As the archetype of the small multidrug resistance family of transporters, EmrE structure and dynamics provide atomic insights into the mechanism of transport by this family of proteins. We recently determined high-resolution structures of EmrE in complex with a cationic substrate, tetra(4-fluorophenyl)phosphonium (F4-TPP+), using solid-state NMR spectroscopy and an S64V-EmrE mutant. The substrate-bound protein exhibits distinct structures at acidic and basic pH, reflecting changes upon binding or release of a proton from residue E14, respectively. To obtain insight into the protein dynamics that mediate substrate transport, here we measure 15N rotating-frame spin-lattice relaxation (R1ρ) rates of F4-TPP+-bound S64V-EmrE in lipid bilayers under magic-angle spinning (MAS). Using perdeuterated and back-exchanged protein and 1H-detected 15N spin-lock experiments under 55 kHz MAS, we measured 15N R1ρ rates site-specifically. Many residues show spin-lock field-dependent 15N R1ρ relaxation rates. This relaxation dispersion indicates the presence of backbone motions at a rate of about 6000 s-1 at 280 K for the protein at both acidic and basic pH. This motional rate is 3 orders of magnitude faster than the alternating access rate but is within the range estimated for substrate binding. We propose that these microsecond motions may allow EmrE to sample different conformations to facilitate substrate binding and release from the transport pore.

Necessary and sufficient molecular determinants of quaternary ammonium antiseptic export by small multidrug resistance transporters.

The small multidrug resistance (SMR) family of transporters are exceptional models for studying polyspecific drug transport. The family is functionally split between two major clusters: the selective Guanidinium exporters (Gdx) that selectively transport the metabolite, guanidinium, and the polyspecific quaternary ammonium compound (Qac) drug exporters that transport a range of man-made compounds with different structural properties. Members from each cluster are small, structurally similar, and only differ in what substrates they transport which makes them ideal models for investigating the molecular determinants required for multidrug transport. Using a combination of directed evolution, high throughput mutagenesis, and next generation sequencing, we identified six mutations that are necessary and sufficient to convert a selective Gdx into a polyselective quaternary ammonium transporter. Biochemical, biophysical, and structural characterization of this transporter will reveal the mechanistic basis for promiscuous transport in the SMR family.

Investigating the regulation and function of bacterial quaternary ammonium compound exporters.

Quaternary ammonium cations were introduced as commonly used disinfectant compounds in the 1930s. Today, they are often used in disinfectant solutions and wipes. Bacterial resistance to these antiseptic compounds is mediated by transporters from the small multidrug resistance (SMR) family of proteins. SMR transporters are bacterial proton-coupled transporters that export cationic substrates including quaternary ammonium compounds. Here we examine the genetic regulation of SMR expression. In addition, we use solid supported membrane (SSM) electrophysiology to observe transport of quaternary ammonium compounds by purified, reconstituted QacE proteins. Our findings allow us to functionally characterize and obtain insight into the evolution of these quaternary ammonium antiseptic transporters so that we can further investigate how to mitigate the spread of antiseptic resistance among bacterial populations.

Metadynamics study of benzyltrimethylammonium binding free energy to EmrE protein in all atom molecular dynamics simulations.

EmrE is a membrane protein found in bacteria such as E. coli that transports positively charged, aromatic toxins outside the cell. It is a parallel dimer and belongs to the small multidrug resistance (SMR) family. Although it is very small, 110 amino acids per monomer, its structure was not resolved at high resolution until recently. The toxin transport is associated with an alternating access movement of the protein monomers. The toxin binds to the protein while the two GLU14 residues of the EmrE are deprotonated in the high pH environment, and is released on the other side when a GLU14 is protonated in low pH. We use metadynamics to calculate the free energy of the binding path of benzyltrimethylammonium to EmrE in a model bacterial membrane. Two geometric reaction coordinates were chosen for the metadynamics runs, distance between center of mass of toxin and the binding site, the angle of the toxin plane with the z-axis. Our results indicate that the free energy of benzyltrimethylammonium binding to EmrE through a water-only path is about −40 kJ/mol. We have also used funnel metadynamics to observe the effect of lipid interaction with the toxin to the binding free energy.

The molecular basis for antiseptic resistance in bacteria.

By providing broad resistance to ubiquitous disinfectants and antiseptics and other environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations. A fundamental understanding of substrate selectivity by SMR transporters is needed to identify the types of selective pressures that contribute to this process. Here, I will describe the molecular basis for the recognition of diverse substrates by SMR proteins, revealed through a combination of x-ray crystallography, biochemical, and electrophysiological approaches.

Can heavy metal pollution induce soil bacterial community resistance to antibiotics in boreal forests?

Abstract The emergence of microbial antibiotic resistance is a central threat to global health, food security, and development. It has been shown that heavy metal pollution can give rise to microbial resistance to antibiotics, but how wide‐spread this phenomenon is remains an open question that urgently needs filling to enable appropriate environmental risk assessments. Here, we determined whether long‐term differences in heavy metal pollution in boreal forests had affected soil microbial communities such that they had increased microbial resistance to antibiotics. First, we assessed variation in metal concentrations in samples collected across a distance trajectory from the pollution source, and also the microbial rates and levels of bacterial community resistance to the heavy metal Cu and the antibiotics tetracycline and vancomycin in those samples. Second, we tested if the exposure to Cu or tetracycline could increase bacterial community resistance to Cu and to antibiotics in soils with high versus low background levels of metal contamination. Metal pollution had affected microbial community structures and suppressed decomposer functioning. Importantly, bacterial community Cu resistance increased with higher metal concentrations, which coincided with an induced bacterial community resistance to tetracycline, but not to vancomycin. Laboratory experiments revealed that bacterial community Cu resistance could be further induced in both the low and high end of the pollution gradient, but also that these short‐term inductions of community metal tolerance did not coincide with enhanced antibiotic resistance. This yielded a surprising negative correlation between long‐term and short‐term effects by metals on microbial metal and antibiotic resistances. One mechanism that could provide protection against both metal cations and tetracycline is the small multidrug resistance (SMR) family, which is an energy demanding physiological mechanism that may take time to confer protection. This may explain the different microbial responses to long‐term gradients and metal addition experiments. Policy implications. We show that metal pollution in boreal forests will promote soil bacterial antibiotic resistance, revealing a so far unrecognized reservoir of microbial antibiotic resistance. We recommend that environmental risk assessments for any activity giving rise to increased soil metal concentrations need to also consider the induction of microbial antibiotic resistance.

Functional Role of YnfA, an Efflux Transporter in Resistance to Antimicrobial Agents in Shigella flexneri.

ABSTRACTShigella flexneri has become a significant public health concern accounting for the majority of shigellosis cases worldwide. Even though a multitude of efforts is being made into the development of a vaccine to prevent infections, the absence of a licensed global vaccine compels us to enormously depend on antibiotics as the major treatment option. The extensive-unregulated use of antibiotics for treatment along with natural selection in bacteria has led to the rising of multidrug-resistance Shigella strains. Out of the various mechanisms employed by bacteria to gain resistance, efflux transporters are considered to be one of the principal contributors to antimicrobial resistance. The small multidrug-resistance family consists of unique small proteins that act as efflux pumps and are involved in extruding various antimicrobial compounds. The present study aims to demonstrate the role of an efflux transporter YnfA belonging to the SMR family and its functional involvement in promoting antimicrobial resistance in S. flexneri. Employing various genetic, computational, and biochemical techniques, we show how disrupting the YnfA transporter, renders the mutant Shigella strain more susceptible to some antimicrobial compounds tested in this study, and significantly affects the overall transport activity of the bacteria against ethidium bromide and acriflavine when compared with the wild-type Shigella strain. We also assessed how mutating some of the conserved amino acid residues of YnfA alters the resistance profile and efflux activity of the mutant YnfA transporter. This study provides a functional understanding of an uncharacterized SMR transporter YnfA of Shigella.

Crystal structures of bacterial small multidrug resistance transporter EmrE in complex with structurally diverse substrates.

Proteins from the bacterial small multidrug resistance (SMR) family are proton-coupled exporters of diverse antiseptics and antimicrobials, including polyaromatic cations and quaternary ammonium compounds. The transport mechanism of the Escherichia coli transporter, EmrE, has been studied extensively, but a lack of high-resolution structural information has impeded a structural description of its molecular mechanism. Here, we apply a novel approach, multipurpose crystallization chaperones, to solve several structures of EmrE, including a 2.9 Å structure at low pH without substrate. We report five additional structures in complex with structurally diverse transported substrates, including quaternary phosphonium, quaternary ammonium, and planar polyaromatic compounds. These structures show that binding site tryptophan and glutamate residues adopt different rotamers to conform to disparate structures without requiring major rearrangements of the backbone structure. Structural and functional comparison to Gdx-Clo, an SMR protein that transports a much narrower spectrum of substrates, suggests that in EmrE, a relatively sparse hydrogen bond network among binding site residues permits increased sidechain flexibility.

The structural basis of substrate promiscuity within the small multidrug resistance family

The Small Multidrug Resistance (SMR) family of membrane proteins is known for conferring resistance to a wide range of antibiotics and xenobiotic compounds in bacteria. Moreover, their small size and rare dual topology architecture have made them a suitable model system for studying membrane proteins’ mechanism and evolution. We recently characterized a new subtype within this family, comprised of guanidinium exporters or Gdx proteins. Gdx proteins facilitate the transport of chemical compounds with guanidinyl moieties and share an overlapping substrate specificity with drug exporter subtype (QAC).

The substrate specificity of the small multidrug resistance famly

Efflux proteins from the small multidrug resistance (SMR) family are associated with resistance to toxic compounds in both bacteria and archaea. They are the smallest known efflux proteins capable of broad-spectrum transport of toxic compounds, making them an ideal model to study molecular evolution of multidrug resistance in microbes. Two major functional subtypes exist in the family: the guanidinium exporters (Gdx), and the quaternary ammonium compound exporters (Qac). Gdx proteins are selective for guanidinium (gdm+), a byproduct of bacterial nitrogen metabolism, whereas the Qacs transport anthropogenic, cationic toxic compounds that are mostly bulky and polyaromatic.

Asymmetric protonation of glutamate residues drives a preferred transport pathway in EmrE

EmrE is an Escherichia coli multidrug efflux pump and member of the small multidrug resistance (SMR) family that transports drugs as a homodimer by harnessing energy from the proton motive force. SMR family transporters contain a conserved glutamate residue in transmembrane 1 (Glu14 in EmrE) that is required for binding protons and drugs. Yet the mechanism underlying proton-coupled transport by the two glutamate residues in the dimer remains unresolved. Here, we used NMR spectroscopy to determine acid dissociation constants (pKa ) for wild-type EmrE and heterodimers containing one or two Glu14 residues in the dimer. For wild-type EmrE, we measured chemical shifts of the carboxyl side chain of Glu14 using solid-state NMR in lipid bilayers and obtained unambiguous evidence on the existence of asymmetric protonation states. Subsequent measurements of pKa values for heterodimers with a single Glu14 residue showed no significant differences from heterodimers with two Glu14 residues, supporting a model where the two Glu14 residues have independent pKa values and are not electrostatically coupled. These insights support a transport pathway with well-defined protonation states in each monomer of the dimer, including a preferred cytoplasmic-facing state where Glu14 is deprotonated in monomer A and protonated in monomer B under pH conditions in the cytoplasm of E. coli Our findings also lead to a model, hop-free exchange, which proposes how exchangers with conformation-dependent pKa values reduce proton leakage. This model is relevant to the SMR family and transporters comprised of inverted repeat domains.

Overexpression of Efflux Pumps Mediate Pan Resistance of Klebsiella pneumoniae Sequence Type 11.

A clinically isolated pan-resistant Klebsiella pneumoniae strain (ST11), KPN142 was subjected to whole-genome sequencing. Genomic sequence of KPN142 showed that limited antibiotic resistances (β-lactams [blashv-11], sulfonamides [sul1 and dfrA22], bacitracin [bacA], tetracycline [tet34], aminoglycosides [ksgA, kdpE, aph(3)Ia, aac(3)III, and ant(3)Ia], and chloramphenicol [catA1]) were mediated by enzymes, and efflux pumps contributed most to pan resistance. Five types of multidrug resistance efflux pump families were identified, including the resistance nodulation division superfamily (AcrAB-TolC, AcrD, MdtABC, and KexD), the ATP-binding cascade superfamily (MacAB), the small multidrug resistance family (KpnEF), the multidrug and toxic compound extrusion family (KdeA), and the major facilitator superfamily (EmrAB). There was an AcrAB-TolC efflux pump system, and inhibitory regulatory gene acrR and ramR of system carried deletion mutation, which lead to overexpression of AcrAB-TolC efflux pump, and in turn plays key role in the pan resistance of KPN142. Moreover, we did not find mgrb, a suppressor in the expression of phoPQ, overexpression of which may confer the resistance of KPN142 to colistin B. In addition, K. pneumoniae KPN142 carries IS1, IS3, and IntI1, which means that KPN142 is able to transfer drug-resistance genes. Of note, we detected the overexpression of acrB, ramA, phoP, and phoQ by real-time quantitative reverse transcription-polymerase chain reaction, and carbonyl cyanide chlorophenylhydrazone was able to reverse the resistance patterns of K. pneumoniae KPN142. In conclusion, we consider that the overexpression of AcrAB-TolC efflux pump mediates the resistance to most common clinical antimicrobial agents, and the overexpression of phoPQ mediates the resistance to colistin B in K. pneumoniae KPN142.

Biophysical Characterization and Structural Studies of Small Multidrug Resistance Family of Transporters

The Small Multidrug Resistance (SMR) family of membrane proteins is known for conferring resistance to a wide range of antibiotics and xenobiotic compounds in bacteria. Moreover, their small size and rare dual topology architecture have made them a suitable model system for studying membrane protein mechanism and evolution. Despite many years of research on multidrug exporters in the SMR family, the family's main function has remained a mystery. We show for the first time, using phylogenetic studies and membrane transport assays, that the majority members of this family are not drug exporters.

Complexity in the Membrane: The Fluc family of Fluoride Channels and Small Multidrug Resistance Family of Transporters as Models for Understanding Membrane Protein Structural and Functional Evolution

Complexity in the Membrane: The Fluc family of Fluoride Channels and Small Multidrug Resistance Family of Transporters as Models for Understanding Membrane Protein Structural and Functional Evolution

The structural basis of promiscuity in small multidrug resistance transporters

By providing broad resistance to environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations. A fundamental understanding of substrate selectivity by SMR transporters is needed to identify the types of selective pressures that contribute to this process. Using solid-supported membrane electrophysiology, we find that promiscuous transport of hydrophobic substituted cations is a general feature of SMR transporters. To understand the molecular basis for promiscuity, we solved X-ray crystal structures of a SMR transporter Gdx-Clo in complex with substrates to a maximum resolution of 2.3 Å. These structures confirm the family’s extremely rare dual topology architecture and reveal a cleft between two helices that provides accommodation in the membrane for the hydrophobic substituents of transported drug-like cations.

Substrate Recognition of Guanidinium Exporters (Gdx) from the Small Multidrug Resistance (SMR) Family

The small multidrug resistance (SMR) proteins are a bacterial family known to confer both antimicrobial and antiseptic resistance against a wide array of unnatural drugs. A small protein cluster containing drug exporters exist in this family and are highly promiscuous in exporting polyaromatic, cationic substrates. Interestingly, the majority of the protein population within the SMRs are guanidinium exporters (Gdx), which are structurally and sequentially similar to the drug exporters. Despite the similarities, both drug exporters and Gdx differ in their functionality. Although Gdx can export its native substrate, guanidinium (Gdm+), it is also selective for mono-substituted Gdm+ analogues. Drug exporters, on the other hand, cannot transport Gdm+, but are highly promiscuous in exporting polyaromatic, cationic, unnatural substrates, including disubstituted Gdm+ analogues. At the lens of substrate specificity, it was very natural to observe an evolutionary pathway from Gdx to drug exporters, but how this event occurred remains a mystery. Here we explore how Gdx proteins evolved a more specialized function to becoming drug exporters. To elucidate this evolutionary process, we will provide a step-wise bridge by mutating key residues on a representative Eco Gdx protein from Escherichia coli,thereby potentially yielding mutants that behave more like EmrE, the most well-studied drug exporter. We will rely on both rational design and directed evolution to generate these mutants, which will then be tested for their transport functionality of cationic, polyaromatic substrates using solid surface membrane (SSM) electrophysiology.

Overlapping Substrate Specificities in the Small Multidrug Resistance (SMR) Family of Transporters

Overlapping Substrate Specificities in the Small Multidrug Resistance (SMR) Family of Transporters

Inducing conformational preference of the membrane protein transporter EmrE through conservative mutations.

Transporters from bacteria to humans contain inverted repeat domains thought to arise evolutionarily from the fusion of smaller membrane protein genes. Association between these domains forms the functional unit that enables transporters to adopt distinct conformations necessary for function. The small multidrug resistance (SMR) family provides an ideal system to explore the role of mutations in altering conformational preference since transporters from this family consist of antiparallel dimers that resemble the inverted repeats present in larger transporters. Here, we show using NMR spectroscopy how a single conservative mutation introduced into an SMR dimer is sufficient to change the resting conformation and function in bacteria. These results underscore the dynamic energy landscape for transporters and demonstrate how conservative mutations can influence structure and function.

Dynamic membrane topology in an unassembled membrane protein.

Helical membrane proteins are typically assumed to attain stable transmembrane topologies immediately upon co-translational membrane insertion. Here we show that unassembled monomers of the small multidrug resistance (SMR) family exist in a dynamic equilibrium where the N-terminal transmembrane helix flips in and out of the membrane, with rates that depend on dimerization and the polypeptide sequence. Thus, membrane topology can display rapid dynamics in vivo and can be regulated by post-translational assembly.