A Transporter Interactome Is Essential for the Acquisition of Antimicrobial Resistance to Antibiotics.

Awareness of the problem of antimicrobial resistance (AMR) has escalated and drug-resistant infections are named among the most urgent problems facing clinicians today. Our experiments here identify a transporter interactome and portray its essential function in acquisition of antimicrobial resistance. By exposing E. coli cells to consecutive increasing concentrations of the fluoroquinolone norfloxacin we generated in the laboratory highly resistant strains that carry multiple mutations, most of them identical to those identified in clinical isolates. With this experimental paradigm, we show that the MDTs function in a coordinated mode to provide an essential first-line defense mechanism, preventing the drug reaching lethal concentrations, until a number of stable efficient alterations occur that allow survival. Single-component efflux transporters remove the toxic compounds from the cytoplasm to the periplasmic space where TolC-dependent transporters expel them from the cell. We postulate a close interaction between the two types of transporters to prevent rapid leak of the hydrophobic substrates back into the cell. The findings change the prevalent concept that in Gram-negative bacteria a single multidrug transporter, AcrAB-TolC type, is responsible for the resistance. The concept of a functional interactome, the process of identification of its members, the elucidation of the nature of the interactions and its role in cell physiology will change the existing paradigms in the field. We anticipate that our work will have an impact on the present strategy searching for inhibitors of AcrAB-TolC as adjuvants of existing antibiotics and provide novel targets for this urgent undertaking.

Topology determination of untagged membrane proteins.

The topology of integral membrane proteins with a weak topological tendency can be influenced when fused to tags, such as these used for topological determination or protein purification. Here, we describe a technique for topology determination of an untagged membrane protein. This technique, optimized for bacterial cells, allows the visualization of the protein in the native environment and incorporates the substituted-cysteine accessibility method.

New substrates on the block: clinically relevant resistances for EmrE and homologues.

Transporters of the small multidrug resistance (SMR) family are small homo- or heterodimers that confer resistance to multiple toxic compounds by exchanging substrate with protons. Despite the wealth of biochemical information on EmrE, the most studied SMR member, a high-resolution three-dimensional structure is missing. To provide proteins that are more amenable to biophysical and structural studies, we identified and partially characterized SMR transporters from bacteria living under extreme conditions of temperature and radiation. Interestingly, these homologues as well as EmrE confer resistance to streptomycin and tobramycin, two aminoglycoside antibiotics widely used in clinics. These are hydrophilic and clinically important substrates of SMRs, and study of their mode of action should contribute to understanding the mechanism of transport and to combating the phenomenon of multidrug resistance. Furthermore, our study of one of the homologues, a putative heterodimer, supports the suggestion that in the SMR family, heterodimers can also function as homodimers.

Topologically random insertion of EmrE supports a pathway for evolution of inverted repeats in ion-coupled transporters.

Inverted repeats in ion-coupled transporters have evolved independently in many unrelated families. It has been suggested that this inverted symmetry is an essential element of the mechanism that allows for the conformational transitions in transporters. We show here that small multidrug transporters offer a model for the evolution of such repeats. This family includes both homodimers and closely related heterodimers. In the former, the topology determinants, evidently identical in each protomer, are weak, and we show that for EmrE, an homodimer from Escherichia coli, the insertion into the membrane is random, and dimers are functional whether they insert into the cytoplasmic membrane with the N- and C-terminal domains facing the inside or the outside of the cell. Also, mutants designed to insert with biased topology are functional regardless of the topology. In the case of EbrAB, a heterodimer homologue supposed to interact antiparallel, we show that one of the subunits, EbrB, can also function as a homodimer, most likely in a parallel mode. In addition, the EmrE homodimer can be forced to an antiparallel topology by fusion of an additional transmembrane segment. The simplicity of the mechanism of coupling ion and substrate transport and the few requirements for substrate recognition provide the robustness necessary to tolerate such a unique and unprecedented ambiguity in the interaction of the subunits and in the dimer topology relative to the membrane. The results suggest that the small multidrug transporters are at an evolutionary junction and provide a model for the evolution of structure of transport proteins.

Parallel topology of genetically fused EmrE homodimers.

EmrE is a small H+-coupled multidrug transporter in Escherichia coli. Claims have been made for an antiparallel topology of this homodimeric protein. However, our own biochemical studies performed with detergent-solubilized purified protein support a parallel topology of the protomers. We developed an alternative approach to constrain the relative topology of the protomers within the dimer so that their activity can be assayed also in vivo before biochemical handling. Tandem EmrE was built with two identical monomers genetically fused tail to head (C-terminus of the first to N-terminus of the second monomer) with hydrophilic linkers of varying length. All the constructs conferred resistance to ethidium by actively removing it from the cytoplasm. The purified proteins bound substrate and transported methyl viologen into proteoliposomes by a proton-dependent mechanism. A tandem where one of the essential glutamates was replaced with glutamine transported only monovalent substrates and displayed a modified stoichiometry. The results support a parallel topology of the protomers in the functional dimer. The implications regarding insertion and evolution of membrane proteins are discussed.

Identification of tyrosine residues critical for the function of an ion-coupled multidrug transporter.

Aromatic residues may play several roles in integral membrane proteins, including direct interaction with substrates. In this work, we studied the contribution of tyrosine residues to the activity of EmrE, a small multidrug transporter from Escherichia coli that extrudes various drugs across the plasma membrane in exchange with protons. Each of five tyrosine residues was replaced by site-directed mutagenesis. Two of these residues, Tyr-40 and Tyr-60, can be partially replaced with hydroxyamino acids, but in the case of Tyr-40, replacement with either Ser or Thr generates a protein with modified substrate specificity. Replacement of Tyr-4 with either Trp or Phe generates a functional transporter. A Cys replacement at this position generates an uncoupled protein; it binds substrate and protons and transports the substrate downhill but is impaired in uphill substrate transport in the presence of a proton gradient. The role of these residues is discussed in the context of the published structures of EmrE.

Characterization of bacterial drug antiporters homologous to mammalian neurotransmitter transporters.

Multidrug transporters are ubiquitous proteins, and, based on amino acid sequence similarities, they have been classified into several families. Here we characterize a cluster of archaeal and bacterial proteins from the major facilitator superfamily (MFS). One member of this family, the vesicular monoamine transporter (VMAT) was previously shown to remove both neurotransmitters and toxic compounds from the cytoplasm, thereby conferring resistance to their effects. A BLAST search of the available microbial genomes against the VMAT sequence yielded sequences of novel putative multidrug transporters. The new sequences along with VMAT form a distinct cluster within the dendrogram of the MFS, drug-proton antiporters. A comparison with other proteins in the family suggests the existence of a potential ion pair in the membrane domain. Three of these genes, from Mycobacterium smegmatis, Corynebacterium glutamicum, and Halobacterium salinarum, were cloned and functionally expressed in Escherichia coli. The proteins conferred resistance to fluoroquinolones and chloramphenicol (at concentrations two to four times greater than that of the control). Measurement of antibiotic accumulation in cells revealed proton motive force-dependent transport of those compounds.

Exploring the binding domain of EmrE, the smallest multidrug transporter.

EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds. Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu14) from both EmrE monomers. Previous studies implied that other residues in the vicinity of Glu14 are part of the binding domain. Alkylation of Cys replacements in the same transmembrane domain inhibits the activity of the protein and this inhibition is fully prevented by substrates of EmrE. To monitor directly the reaction we tested also the extent of modification using fluorescein-5-maleimide. While most residues are not accessible or only partially accessible, four, Y4C, I5C, L7C, and A10C, were modified at least 80%. Furthermore, preincubation with tetraphenylphosphonium reduces the reaction of two of these residues by up to 80%. To study other essential residues we generated functional hetero-oligomers and challenged them with various methane thiosulfonates. Taken together the findings imply the existence of a binding cavity accessible to alkylating reagents where at least three residues from TM1, Tyr40 from TM2, and Trp63 in TM3 are involved in substrate binding.

Substrate-induced tryptophan fluorescence changes in EmrE, the smallest ion-coupled multidrug transporter.

Tryptophan residues may play several roles in integral membrane proteins including direct interaction with substrates. In this work we studied the contribution of tryptophan residues to substrate binding in EmrE, a small multidrug transporter of Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons. Each of the four tryptophan residues was replaced by site-directed mutagenesis. The only single substitutions that affected the protein's activity were those in position 63. While cysteine and tyrosine replacements yielded a completely inactive protein, the replacement of Trp63 with phenylalanine brought about a protein that, although it could not confer any resistance against the toxicants tested, could bind substrate with an affinity 2 orders of magnitude lower than that of the wild-type protein. Double or multiple cysteine replacements at the other positions generate proteins that are inactive in vivo but regain their activity upon solubilization and reconstitution. The findings suggest a possible role of the tryptophan residues in folding and/or insertion. Substrate binding to the wild-type protein and to a mutant with a single tryptophan residue in position 63 induced a very substantial fluorescence quenching that is not observed in inactive mutants or chemically modified protein. The reaction is dependent on the concentration of the substrate and saturates at a concentration of 2.57 microM with the protein concentration of 5 microM supporting the contention that the functional unit is a dimer. These findings strongly suggest the existence of an interaction between Trp63 and substrate, and the nature of this interaction can now be studied in more detail with the tools developed in this work.

In vitro synthesis of fully functional EmrE, a multidrug transporter, and study of its oligomeric state.

EmrE is a small multidrug transporter from Escherichia coli that provides a unique model for the study of polytopic membrane proteins. Here, we show its synthesis in a cell-free system in a fully functional form. The detergent-solubilized protein binds substrates with high affinity and, when reconstituted into proteoliposomes, transports substrate in a Deltamicro(H)(+)-dependent fashion. Here, we used the cell-free system to study the oligomeric properties of EmrE. EmrE functions as an oligomer, but the size of the functional oligomer has not been established unequivocally. Coexpression of two plasmids in the cell-free system allowed demonstration of functional complementation and pull-down experiments confirmed that the basic functional unit is the dimer. An additional interaction between dimers has been detected by using crosslinking between unique Cys residues. This finding implies the existence of a dimer of dimers.

Killing of intraerythrocytic Plasmodium falciparum by lysosomotropic amino acid esters.

Esters of amino acids are known to penetrate into cells by simple diffusion. Subsequently, they are hydrolyzed by hydrolases to release the parent amino acid. Due to the abundance of hydrolases in phagolysosomes, amino acids accumulate, there because the rate of influx and hydrolysis exceed the rate of amino acid efflux through specific carriers. The osmotic effect of this accumulation results in the disruption of the organelles. This mechanism has been demonstrated to be responsible for the killing of Leishmania amastigotes by amino acid esters. In this investigation, it is shown that all esters tested, including alcohol esters, N-acetyl esters and the esters of some dipeptides, inhibit the growth of Plasmodium falciparum in culture. Inhibition is time-dependent and, in some cases, ring-stage parasites are more sensitive than trophozoites. Similar to the findings with Leishmania, alcohol esters of Glu, Leu, Met, Phe and Trp are more toxic to Plasmodium whereas Ala, Gly, His and Ile are much less noxious. Esters caused the release of acridine orange that selectively accumulates in the phagolysosome-like food vacuole of the parasite, attesting the ostensible destruction of this organelle by osmotic lysis. The toxicity of the N-acetyl esters is probably associated in part to their ability to inhibit cytosolic proteases. Since excess of amino acids can also inhibit proteolysis, the effect of free amino acids on parasite growth was also tested. Of the 19 odd amino acids tested, only three, namely Cys, His and Trp, were found to be toxic to the parasites at millimolar concentrations and the reasons for their possible specific toxicity are discussed.

An amino acid cluster around the essential Glu-14 is part of the substrate- and proton-binding domain of EmrE, a multidrug transporter from Escherichia coli.

EmrE is a small multidrug transporter (110 amino acids long) from Escherichia coli that extrudes various drugs in exchange with protons, thereby rendering bacteria resistant to these compounds. Glu-14 is the only charged membrane-embedded residue in EmrE and is evolutionarily highly conserved. This residue has an unusually high pK and is an essential part of the binding domain, shared by substrates and protons. The occupancy of the binding domain is mutually exclusive, and, as such, this provides the molecular basis for the coupling between substrate and proton fluxes. Systematic cysteine-scanning mutagenesis of the residues in the transmembrane segment (TM1), where Glu-14 is located, reveals an amino acid cluster on the same face of TM1 as Glu-14 that is part of the substrate- and proton-binding domain. Substitutions at most of these positions yielded either inactive mutants or mutants with modified affinity to substrates. Substitutions at the Ala-10 position, one helix turn away from Glu-14, yielded mutants with modified affinity to protons and thereby impaired in the coupling of substrate and proton fluxes. Taken as a whole, the results strongly support the concept of a common binding site for substrate and protons and stress the importance of one face of TM1 in substrate recognition, binding, and H(+)-coupled transport.

Crosslinking of membrane-embedded cysteines reveals contact points in the EmrE oligomer.

EmrE is a small multidrug transporter that extrudes various drugs in exchange with protons, thereby rendering Escherichia coli cells resistant to these compounds. In this study, relative helix packing in the EmrE oligomer solubilized in detergent was probed by intermonomer crosslinking analysis. Unique cysteine replacements in transmembrane domains were shown to react with organic mercurials but not with sulfhydryl reagents, such as maleimides and methanethiosulfonates. A new protocol was developed based on the use of HgCl(2), a compound known to react rapidly and selectively with sulfhydryl groups. The reaction can bridge vicinal pairs of cysteines and form an intermolecular mercury-linked dimer. To circumvent problems inherent to mercury chemistry, a second crosslinker, hexamethylene diisocyanate, was used. After the HgCl(2) treatment, excess reagent was removed and the oligomers were dissociated with a strong denaturant. Only those previously crosslinked reacted with hexamethylene diisocyanate. Thus, vicinal cysteine-substituted residues in the EmrE oligomer were identified. It was shown that transmembrane domain (TM)-1 and TM4 in one subunit are in contact with the corresponding TM1 and TM4, respectively, in the other subunit. In addition, TM1 is also in close proximity to TM4 of the neighboring subunit, suggesting possible arrangements in the binding and translocation domain of the EmrE oligomer. This method should be useful for other proteins with cysteine residues in a low-dielectric environment.