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A A Neyfakh

Publications and source records attributed to A A Neyfakh.

At least 19 recordsLinked to original sources
PubMed1998

Paradoxical enhancement of the activity of a bacterial multidrug transporter caused by substitutions of a conserved residue.

Substitution of threonine or serine for the evolutionary conserved intramembrane proline P347 of the Bacillus subtilis multidrug transporter Bmr significantly increases the toxin-effluxing activity of Bmr without affecting its abundance in the cell. In cocultivation experiments, we demonstrate that although the mutant T347 Bmr is advantageous to cells growing in the presence of a toxin, the wild-type P347 Bmr is advantageous under the conditions of nutritional limitation. This may explain why Bmr has evolved the way it did, that is, with proline at position 347. These observations provide a basis for speculating that the evolution of Bmr has been determined by its presently unidentified natural function rather than by its ability to expel diverse toxins from the cell.

Acriflavine↗
PubMed1997

Broad ligand specificity of the transcriptional regulator of the Bacillus subtilis multidrug transporter Bmr.

The expression of the Bacillus subtilus multidrug-efflux transporter Bmr can be induced by two of its structurally dissimilar substrates, rhodamine 6G and tetraphenylphosphonium, through their direct interaction with the transcriptional regulator BmrR (Ahmed et al., J. Biol. Chem. 269, 28506). Here, by screening a chemical library, we identified four additional ligands of BmrR inducing Bmr expression at micromolar concentrations. BmrR ligands, although sharing a positive charge and moderate hydrophobicity, are structurally very diverse. At the same time, not all hydrophobic positively charged compounds, including many structural analogs of the inducers, induce Bmr expression, thus suggesting that local chemical interactions and not merely physical properties of the ligands are important for their recognition by BmrR. These results confirm that this soluble protein, like the membrane transporter it regulates, has a uniquely broad substrate specificity.

Bacillus subtilis↗
PubMed1997

Efflux of the natural polyamine spermidine facilitated by the Bacillus subtilis multidrug transporter Blt.

Multidrug transporters pump structurally dissimilar toxic molecules out of cells. It is not known, however, if detoxification is the primary physiological function of these transporters. The chromosomal organization of the gene encoding the Bacillus subtilis multidrug transporter Blt suggests a specific function for this protein; it forms a single operon with another gene, bltD, whose protein product is identified here as a spermine/spermidine acetyltransferase, an enzyme catalyzing a key step in spermidine degradation. Overexpression of the Blt transporter in B. subtilis leads not only to the multidrug-resistance phenotype but also to the efflux of large amounts of spermidine into the medium; this efflux is supressed by an inhibitor of Blt, reserpine. Taken together, these results strongly suggest that the natural function of the Blt transporter is the efflux of spermidine, whereas multiple drugs may be recognized by Blt merely opportunistically.

Acetylation↗
PubMed1997

Preliminary structural studies on the multi-ligand-binding domain of the transcription activator, BmrR, from Bacillus subtilis.

In the bacterium Bacillus subtilis, the DNA-binding regulatory protein, BmrR, activates transcription from the multidrug transporter gene, bmr, after binding either rhodamine or tetraphenylphosphonium. These two compounds, which have no structural similarity, are also substrates for the bacterial multidrug transporter. BmrR belongs to the MerR family of transcription activators but differs from the other family members in its ability to bind unrelated small molecule activators. As an initial step in the elucidation of the mechanism by which BmrR recognizes rhodamine and tetraphenylphosphonium and activates transcription, we have crystallized the 144-amino acid-residue carboxy terminal dimerization/ligand-binding domain of the BmrR, named the BRC (BmrR C-terminus). Tetragonal crystals of ligand-free BRC take the space group P4(1)2(1)2, or its enantiomorph P4(3)2(1)2, with unit cell dimensions a = b = 76.3 A, c = 96.0 A, alpha = beta = gamma = 90 degrees. Diffraction is observed to at least 2.7 A resolution at room temperature. In addition, we determined the secondary structure content of ligand-free and rhodamine-bound BRC by circular dichroism. In the ligand-free form, BRC has considerable beta-sheet content (41%) and little alpha-helix structure (13%). After BRC binds rhodamine, its beta-sheet content increases to 47% while the alpha-helix structure decreases to 11%. The structure of BRC will provide insight not only into its multidrug recognition mechanism but could as well aid in the elucidation of the recognition and efflux mechanisms of Bmr and other bacterial multidrug transporters.

Bacillus subtilis↗
PubMed1997

Natural functions of bacterial multidrug transporters.

Bacteria express several multidrug transporters that recognize structurally dissimilar toxic molecules and expel them from cells. These transporters may have evolved to protect bacteria from diverse environmental toxins or to transport specific physiological compounds with the ability to expel drugs being only a fortuitous side effect.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Apparent involvement of a multidrug transporter in the fluoroquinolone resistance of Streptococcus pneumoniae.

A Streptococcus pneumoniae strain selected for resistance to ethidium bromide demonstrated enhanced energy-dependent efflux of this toxic dye. Both the ethidium resistance and the ethidium efflux could be inhibited by the plant alkaloid reserpine. The ethidium-selected cells demonstrated cross-resistance to the fluoroquinolones norfloxacin and ciprofloxacin; this resistance could also be completely reversed by reserpine. Furthermore, reserpine potentiated the susceptibility of wild-type S. pneumoniae to fluoroquinolones and ethidium. The most plausible explanation for these results is that S. pneumoniae, like some other gram-positive bacteria, expresses a reserpine-sensitive multidrug transporter, which may play an important role in both intrinsic and acquired resistances of this pathogen to fluoroquinolone therapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Mutations affecting substrate specificity of the Bacillus subtilis multidrug transporter Bmr.

The Bacillus subtilis multidrug transporter Bmr, a member of the major facilitator superfamily of transporters, causes the efflux of a number of structurally unrelated toxic compounds from cells. We have shown previously that the activity of Bmr can be inhibited by the plant alkaloid reserpine. Here we demonstrate that various substitutions of residues Phe143 and Phe306 of Bmr not only reduce its sensitivity to reserpine inhibition but also significantly change its substrate specificity. Cross-resistance profiles of bacteria expressing mutant forms of the transporter differ from each other and from the cross-resistance profile of cells expressing wild-type Bmr. This result strongly suggests that Bmr interacts with its transported drugs directly, with residues Phe143 and Phe306 likely to be involved in substrate recognition.

Bacillus subtilis↗
PubMed1996

The drug-binding activity of the multidrug-responding transcriptional regulator BmrR resides in its C-terminal domain.

Rhodamine and tetraphenylphosphonium, the substrates of the Bacillus subtilis multidrug efflux transporter Bmr, induce the expression of Bmr through direct interaction with its transcriptional activator BmrR. Here we show that the C-terminal domain of BmrR, expressed individually, binds both these compounds and therefore can be used as a model for molecular analysis of the phenomenon of multidrug recognition.

Bacillus subtilis↗
PubMed1995

Two highly similar multidrug transporters of Bacillus subtilis whose expression is differentially regulated.

The Bacillus subtilis genome encodes two multidrug efflux transporters sharing 51% sequence identity: Bmr, described previously, and Blt, described here. Overexpression of either transporter in B. subtilis leads to a similar increase in resistance to ethidium bromide, rhodamine and acridine dyes, tetraphenylphosphonium, doxorubicin, and fluoroquinolone antibiotics. However, Blt differs widely from Bmr in its expression pattern. Under standard cultivation conditions, B. subtilis expresses Bmr but Blt expression is undetectable. We have previously shown that Bmr expression is regulated by BmrR, a member of the family of MerR-like transcriptional activators. Here we show that blt transcription is regulated by another member of the same family, BltR. The DNA-binding domains of BmrR and BltR are related, but their putative inducer-binding domains are dissimilar, suggesting that Bmr and Blt are expressed in response to different inducers. Indeed, rhodamine, a substrate of Bmr and Blt and a known inducer of Bmr expression, does not induce Blt expression. Blt expression has been observed only in B. subtilis, carrying mutation acfA, which, as we show here, alters the sequence of the blt gene promoter. Unlike bmr, which is transcribed as a monocistronic mRNA, blt is cotranscribed with a downstream gene encoding a putative acetyltransferase. Overall, the differences in transcriptional control and operon organization between bmr and blt suggest that the transporters encoded by these genes have independent functions involving the transport of distinct physiological compounds.

Acetyltransferases↗
PubMed1994

A protein that activates expression of a multidrug efflux transporter upon binding the transporter substrates.

Multidrug transporters are membrane proteins which, by an unknown mechanism, recognize diverse toxic compounds and efflux them from cells. We found that two substrates of the Bacillus subtilis multidrug transporter Bmr, rhodamine 6G and tetraphenylphosphonium (TPP), enhance Bmr expression at the level of transcription. Gene knock-out experiments demonstrated that an open reading frame located immediately downstream of the bmr gene is required for this enhancement. The protein product of this open reading frame, BmrR, shows distinct sequence homology to several known bacterial transcription activator proteins, such as MerR and TipAL. Gel-mobility shift and DNase protection assays indicated that BmrR binds specifically, as a dimer, to the bmr gene promoter. Furthermore, the affinity of this binding was enhanced by rhodamine and TPP, thus suggesting that these structurally dissimilar molecules interact directly with BmrR. Indeed, we found that BmrR bound rhodamine 6G stoichiometrically, one rhodamine molecule/BmrR dimer, and that TPP competed with rhodamine for this binding. Our results indicate that the enhancement of Bmr expression by some of its substrates is due to the ability of the regulatory protein, BmrR, to bind structurally dissimilar compounds resulting in enhanced transcription of the transporter gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Mutants of the Bacillus subtilis multidrug transporter Bmr with altered sensitivity to the antihypertensive alkaloid reserpine.

The Bacillus subtilis multidrug transporter Bmr effluxes structurally diverse toxic compounds out of bacterial cells. Antihypertensive alkaloid reserpine reverses Bmr-mediated multidrug resistance by inhibiting drug transport. We have obtained a mutant of the bmr gene that provides a normal level of multidrug resistance, which can, however, only be reversed by very high concentrations of reserpine. Reduction of Bmr sensitivity to reserpine has been caused by the substitution of Leu for Val286 in the Bmr molecule. This mutation also led to a dramatic decrease of [3H]reserpine binding to membrane vesicles prepared from the Bmr-overexpressing bacteria. Leucine is larger than valine by one methylene group. Substitution of Val286 with a smaller residue, glycine, had an opposite effect. It led to increased sensitivity of Bmr to reserpine and increased affinity of reserpine binding to the membranes prepared from Bmr-overexpressing bacteria. Neither of the mutations significantly changed the sensitivity of Bmr to rescinnamine, a structural analog of reserpine. The results suggest that Val286 is involved in the formation of the reserpine-binding site of the Bmr molecule.

Amino Acid Sequence↗
PubMed1993

Fluoroquinolone resistance protein NorA of Staphylococcus aureus is a multidrug efflux transporter.

The gene of the Staphylococcus aureus fluoroquinolone efflux transporter protein NorA confers resistance to a number of structurally dissimilar drugs, not just to fluoroquinolones, when it is expressed in Bacillus subtilis. NorA provides B. subtilis with resistance to the same drugs and to a similar extent as the B. subtilis multidrug transporter protein Bmr does. NorA and Bmr share 44% sequence similarity. Both the NorA- and Bmr-conferred resistances can be completely reversed by reserpine.

Anti-Infective Agents↗
PubMed1992

The multidrug efflux transporter of Bacillus subtilis is a structural and functional homolog of the Staphylococcus NorA protein.

The Bacillus subtilis multidrug efflux transporter Bmr demonstrates 44% amino acid sequence identity with a product of the Staphylococcus aureus gene norA, which is responsible for clinically relevant resistance to fluoroquinolones. We show here that overexpression of bmr in B. subtilis provides strong resistance to fluoroquinolones that can be reversed by reserpine, an inhibitor of Bmr.

Amino Acid Sequence↗
PubMed1991

Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system.

Bacillus subtilis cells selected for their resistance to rhodamine 6G demonstrated a multidrug-resistance (MDR) phenotype resembling that of mammalian MDR cells. Like MDR in mammalian cells, MDR in bacteria was mediated by the efflux of the drugs from the cells. The bacterial multidrug efflux system transported similar drugs and was sensitive to similar inhibitors as the mammalian multidrug transporter, P-glycoprotein. The gene coding for the bacterial multidrug transporter, like the P-glycoprotein gene in mammalian MDR cells, was amplified in the resistant bacteria. On the other hand, the bacterial multidrug transporter showed no sequence similarity to P-glycoprotein but exhibited an obvious homology to tetracycline efflux pumps and carbohydrate-ion symporters. These results show that the transport of structurally unrelated molecules can be mediated by members of different families of membrane transporters.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug selection.

Multidrug-resistant (MDR) cells demonstrate the increased activity of the membrane transport system performing efflux of diverse lipophylic drugs and fluorescent dyes from the cells. In order to detect MDR cells we have developed a simple test consisting of three steps: staining of the cells with fluorescent dye rhodamine 123, incubation in the dye-free medium and, finally, detection by fluorescence microscopy of the cells that have lost accumulated dye. The experiments with B-lymphoma cell lines with different degrees of MDR have shown that the cell fluorescence after the poststaining incubation is indeed inversely proportional to the degree of resistance. Application of this testing procedure to normal human or mouse leukocytes revealed the presence of the cells rapidly losing the dye in these populations. Cell fractionation experiments have shown that there are T-lymphocytes (most T-killers/suppressors and a part of T-helpers) that demonstrate rapid efflux of rhodamine 123. This characteristic was detected also in T-killer clones and cell line and in some T-lymphomas. The inhibitors of the MDR transport system, reserpine and verapamil, blocked the efflux of the dye from these cells. Rhodamine-losing T-lymphoma contained large amounts of the mRNA coding P-glycoprotein, the MDR efflux pump, and demonstrated increased resistance to rhodamine 123, gramicidin D, colchicine, and vincristine, the drugs belonging to the cross-resistance group for the MDR cells. The role of the increased activity of the MDR membrane transport system in T-lymphocytes is discussed.

Animals↗
PubMed1988

Use of fluorescent dyes as molecular probes for the study of multidrug resistance.

Fluorescence microscopy has shown that 18 different fluorescent dyes, staining various intracellular structures in transformed hamster fibroblasts (DM-15), did not stain or stained weakly multidrug-resistant cells selected from DM-15 by colchicine. Reduced staining by fluorescent dyes was characteristic also of five other tested multidrug-resistant cell lines of hamster and mouse origin, selected by actinomycin D, colcemid, rubomycin, and ruboxyl. The intensity of staining of two revertant cell lines was similar to that of parental sensitive cells. All tested inhibitors of multidrug resistance, including weak detergent, metabolic inhibitors, calcium channel blockers, calmodulin inhibitors, and reserpine, restored normal staining of multidrug-resistant cells. The dyes accumulated in resistant cells in presence of these inhibitors left the cells several minutes after the removal of the inhibitor from the incubation medium. Sensitive cells retained the dyes for several hours. The efflux of the dyes from resistant cells is an active process since it occurred even in the presence of the dyes in the incubation medium. The efflux could be blocked by all tested inhibitors of multidrug resistance and it is possibly a basic mechanism of the reduced staining of resistant cells. These data support the idea that multidrug resistance is based on active nonspecific efflux of the drugs and indicate that the simple procedure of cell staining can be used for the detection of resistant cells and further study of the phenomenon of multidrug resistance.

Animals↗
PubMed1988

The membrane transport system responsible for multidrug resistance is operating in nonresistant cells.

Cultured hamster fibroblasts of the DM-15 cell line stained by rhodamine 123 gradually release the dye when placed in dye-free medium. Here we demonstrate that reserpine, verapamil, and trifluoperazine are capable of blocking this release. We also show that reserpine can inhibit the efflux of another dye, phosphine 3R, from DM-15 cells and the release of rhodamine 123 from mouse embryo fibroblasts, four mouse cell lines, and MDCK cells. The three substances that block the release of the dyes are potent inhibitors of the membrane transport system implicated in the phenomenon of multidrug resistance (MDR). By using this system MDR cells can pump many structurally unrelated drugs and dyes, including rhodamine 123 and phosphine 3R, from the cytoplasm to the outer medium. It appears from our results that the membrane transport system responsible for MDR operates slowly in nonresistant cells and can play a role in normal cell physiology.

Animals↗