Efflux-mediated drug resistance in Gram-positive bacteria.

Gram-positive bacteria express numerous membrane transporters that promote the efflux of various drugs, including many antibiotics, from the cell to the outer medium. Drug transporters can be specific to a particular drug, or can have broad specificity, as in so-called multidrug transporters. This broad specificity can be a consequence of the hydrophobic nature of transported molecules, as suggested by recent structural studies of soluble multidrug-binding proteins. Although the functions of drug transporters may involve both the protection of bacteria from outside toxins and the transport of natural metabolites, their clinical importance lies largely in providing Gram-positive pathogens with resistance to macrolides, tetracyclines and fluoroquinolones. A number of agents, discovered in recent years, that inhibit drug transporters can potentially be used to overcome efflux-associated antibiotic resistance.

Recognition of multiple drugs by a single protein: a trivial solution of an old paradox.

Multidrug-efflux transporters recognize scores of structurally dissimilar toxic compounds and expel them from cells. The broad chemical specificity of these transporters challenges some of the basic dogmas of biochemistry and remains unexplained. To understand, at least in principle, how a protein can recognize multiple compounds, we analysed the transcriptional regulator of the Bacillus subtilis multidrug transporter Bmr. This regulator, BmrR, binds multiple dissimilar hydrophobic cations and, by activating the expression of the Bmr transporter, causes their expulsion from the cell. Crystallographic analysis of the complexes of the inducer-binding domain of BmrR with some of its inducers revealed that ligands cause disordering of the surface alpha-helix and penetrate the hydrophobic core of the protein, where they form multiple van der Waals and stacking interactions with hydrophobic amino acids and an electrostatic bond with the buried glutamic residue. Mutational analysis of the binding site suggests that each ligand forms a unique set of atomic contacts with the protein: each tested mutation exerted disparate effects on the binding of different ligands. The example of BmrR demonstrates that a protein can bind multiple compounds with micromolar affinities by using only electrostatic and hydrophobic interactions. Its ligand specificity can be broadened by the flexibility of the binding site. It therefore seems that the commonly expressed fascination with the broad specificity of multidrug transporters is misdirected and originates from an almost exclusive familiarity with the more sophisticated processes of specific molecular recognition that predominate among existing proteins.

Mechanism of ligand recognition by BmrR, the multidrug-responding transcriptional regulator: mutational analysis of the ligand-binding site.

The Bacillus subtilis transcriptional regulator BmrR recognizes dissimilar hydrophobic cations and, in response, activates the expression of a multidrug transporter which expels them out of the cell. The structure of the inducer-binding domain of BmrR, both free and in complex with one of the inducers, tetraphenylphosphonium (TPP), revealed an unusual internal binding site, covered by an amphipathic alpha-helix. Upon unfolding of this helix, the TPP molecule penetrates into the core of the protein, where it contacts six hydrophobic residues and forms an electrostatic bond with a buried glutamate, E134 [Zheleznova et al. (1999) Cell 96, 353-362]. Here, a structure-based mutational analysis was used to understand how BmrR interacts with a wide variety of ligands. We determined the effects of alanine substitutions of each of the seven residues interacting with TPP, and mutations within the amphipathic alpha-helix, on the binding affinities of six different BmrR inducers. The E134A substitution abolished the binding of all but one inducer. Mutations of the hydrophobic residues contacting the ligand, and of the alpha-helix, had more moderate effects, often with the affinity for some inducers increasing and others decreasing as a result of the same substitution. These results indicate that each inducer forms a unique set of contacts within the binding site. The flexible geometry of this site and the lack of involvement of hydrogen bonds in ligand binding are the likely reasons for the extremely broad inducer specificity of BmrR. The similarly broad substrate specificity of multidrug transporters can be governed by the same structural principles.

Multiple novel inhibitors of the NorA multidrug transporter of Staphylococcus aureus.

The multidrug transporter NorA contributes to the resistance of Staphylococcus aureus to fluoroquinolone antibiotics by promoting their active extrusion from the cell. Previous studies with the alkaloid reserpine, the first identified inhibitor of NorA, indicate that the combination of a chemical NorA inhibitor with a fluoroquinolone could improve the efficacy of this class of antibiotics. Since reserpine is toxic to humans at the concentrations required to inhibit NorA, we sought to identify new inhibitors of NorA that may be used in a clinical setting. Screening of a chemical library yielded a number of structurally diverse inhibitors of NorA that were more potent than reserpine. The new inhibitors act in a synergistic manner with the most widely used fluoroquinolone, ciprofloxacin, by substantially increasing its activity against both NorA-overexpressing and wild-type S. aureus isolates. Furthermore, the inhibitors dramatically suppress the emergence of ciprofloxacin-resistant S. aureus upon in vitro selection with this drug. Some of these new inhibitors, or their derivatives, may prove useful for augmentation of the antibacterial activities of fluoroquinolones in the clinical setting.

Inhibition of the emergence of ciprofloxacin resistance in Streptococcus pneumoniae by the multidrug efflux inhibitor reserpine.

Recent evidence supports the contribution of a multidrug efflux mechanism to fluoroquinolone resistance in Streptococcus pneumoniae. In this paper I show that reserpine, an inhibitor of multidrug transporters in gram-positive bacteria, dramatically suppresses the in vitro emergence of ciprofloxacin-resistant variants of S. pneumoniae, suggesting that the combination of a fluoroquinolone with an inhibitor of multidrug transport may help preserve the efficacy of this class of antibiotics.

Structural basis of multidrug recognition by BmrR, a transcription activator of a multidrug transporter.

Multidrug-efflux transporters demonstrate an unusual ability to recognize multiple structurally dissimilar toxins. A comparable ability to bind diverse hydrophobic cationic drugs is characteristic of the Bacillus subtilis transcription regulator BmrR, which upon drug binding activates expression of the multidrug transporter Bmr. Crystal structures of the multidrug-binding domain of BmrR (2.7 A resolution) and of its complex with the drug tetraphenylphosphonium (2.8 A resolution) revealed a drug-induced unfolding and relocation of an alpha helix, which exposes an internal drug-binding pocket. Tetraphenylphosphonium binding is mediated by stacking and van der Waals contacts with multiple hydrophobic residues of the pocket and by an electrostatic interaction between the positively charged drug and a buried glutamate residue, which is the key to cation selectivity. Similar binding principles may be used by other multidrug-binding proteins.

IL-4 inhibits P-glycoprotein in normal and malignant NK cells.

Patients presenting with a natural killer (NK) cell leukemia generally have a poor prognosis. NK cell tumors are generally resistant to numerous chemotherapeutic drugs and even combination chemotherapy usually results in only short term remissions. The drug resistance of NK cell leukemias may be at least partially explained by their expression of the multidrug resistant transporter, P-glycoprotein (Pgp). In this study, we demonstrate that the expression and function of Pgp activity on NK cells (leukemic and normal) can be reversed with IL-4.

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.

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.

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.

Differential sensitivity of resting and IL-2 activated NK cells to R-verapamil.

Natural killer (NK) cells are the first lymphoid population to reconstitute the peripheral blood compartment of immunologically compromised bone marrow transplant (BMT) recipients. Recent data suggest that, among patients transplanted for leukemia, NK cells can prevent or delay disease relapse by mediating a cytotoxic graft vs leukemia (GvL) response. Although the major mechanism by which NK cells mediate target cell lysis involves degranulation and release of cytolytic effector molecules (granzymes, proteoglycans, perforin), accumulating evidence suggests that NK cells possess additional pathways to mediate target cell killing. In fact, it is well recognized that recombinant cytokines such as IL-2 enhance the in vitro cytolytic activity of NK cells. In this study, we observed that the lytic activity mediated by resting and IL-2 activated NK cells against the same target cell appears to occur via two distinct pathways, as distinguished by their differential response to R-verapamil. Specifically, we observed that 25 microM R-verapamil inhibited the lytic activity of resting NK cells against K562 targets by approximately 50%. However, the lytic activity of IL-2 activated NK cells was unaffected by this concentration of R-verapamil. Additional studies suggested that the inhibitory effect of R-verapamil on NK cytotoxic activity was associated with its ability to prevent degranulation of cytotoxic granules. Specifically, R-verapamil inhibited BLT esterase release from resting but not IL-2 activated NK cells. These data suggest that IL-2 activated NK cells can promote target cell lysis by a pathway (possibly degranulation independent) distinct from that used by resting NK cells. We speculate that the target of R-verapamil on resting NK cells is P-glycoprotein (Pgp), an ABC transporter that we recently reported was expressed on NK cells and whose functional activity is known to be inhibited by R-verapamil.

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.

Crime laboratory proficiency testing results, 1978-1991, II: Resolving questions of common origin.

A preceding article has examined the origins of crime laboratory proficiency testing and the performance of laboratories in the identification and classification of common types of physical evidence. Part II reviews laboratory proficiency in determining if two or more evidence samples shared a common source. Parts I and II together review the results of 175 separate tests issued to crime laboratories over the period 1978 to 1991. Laboratories perform best in determining the origin of finger and palm prints, metals, firearms (bullets and catridge cases), and footwear. Laboratories have moderate success in determining the source of bloodstains, questioned documents, toolmarks, and hair. A final category is of greater concern and includes those evidence categories where 10% or more of results disagree with manufacturers regarding the source of samples. This latter group includes paint, glass, fibers, and body fluid mixtures. The article concludes with a comparison of current findings with earlier LEAA study results, and a discussion of judicial and policy implications.

Crime laboratory proficiency testing results, 1978-1991, I: Identification and classification of physical evidence.

The proficiency testing of crime laboratories began in the mid-1970s and presently assumes an important role in quality assurance programs within most forensic laboratories. This article reviews the origins and early results of this testing program and also examines the progress of proficiency testing in allied scientific fields. Beginning in 1978, a fee-based crime laboratory proficiency testing program was launched and has grown to its present level involving almost 400 laboratories worldwide. This is the first of two articles that review the objectives, limitations and results of this testing from 1978 through 1991. Part I reviews the success of laboratories in the identification and classification of common evidence types: controlled substances, flammables, explosives, fibers, bloodstains, and hairs. Laboratories enjoy a high degree of success in identifying drugs and classifying (typing) bloodstains. They are moderately successful in identifying flammables, explosives, and fibers. Animal hair identification and human hair body location results are troublesome. The second paper will review the proficiency of crime laboratories in determining if two or more evidentiary samples shared a common origin.

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.

Human leukemia K562 cell mutant (K562/OA200) selected for resistance to okadaic acid (protein phosphatase inhibitor) lacks protein kinase C-epsilon, exhibits multidrug resistance phenotype, and expresses drug pump P-glycoprotein.

A human leukemia K562 cell mutant (K562/OA200) selected for resistance to okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A (PP1/PP2A), has been established. In wild type cells, the cytotoxicity of OA was associated with mitotic arrest and concentration- and time-dependent DNA fragmentation, a hallmark of apoptosis. The mutant was 100-fold more resistant to OA in terms of effects on these parameters. Although the synthesis of several proteins was altered, enzyme assay and immunoblot analysis indicated that the levels of PP1 and PP2A were unchanged in the mutant. Protein kinase C (PKC) assays and immunoblot analysis of calcium-dependent (cPKC) and calcium-independent (nPKC) isoforms revealed that nPKC-epsilon was strikingly absent in the mutant, which otherwise expressed in comparable amounts all other isotypes (cPKC-alpha, cPKC-beta, and nPKC-zeta) also present in the wild type. Northern blot analysis confirmed an absence of PKC-epsilon mRNA in the mutant cells. The OA200 cells were cross-resistant not only to another PP1/PP2A inhibitor, calyculin A, but also to structurally unrelated anticancer drugs (such as vinblastine and taxol) and furthermore, overexpressed the verapamil-sensitive drug pump P-glycoprotein at both the protein and mRNA levels. The mutant, however, was not cross-resistant to several PKC inhibitors tested including cardiotoxin, mastoparan, staurosporine, and an alkylphospholipid. Cardiotoxin, at a subtoxic concentration, enhanced by 6-fold vinblastine cytotoxicity in OA200 cells. These findings indicate that the multidrug resistance phenotype can be induced by cytotoxic agents other than conventional anticancer drugs, show that the development of multidrug resistance is not necessarily associated with increased cPKC activity, and identify certain PKC inhibitors that have potential as resistance modulators.

Diverse multidrug-resistance-modification agents inhibit cytolytic activity of natural killer cells.

Multidrug resistance (MDR) is the phenomenon in which cultured tumor cells selected for resistance to one chemotherapeutic agent simultaneously acquire resistance to several apparently unrelated drugs. MDR in tumor cells is associated with the over-expression of P-glycoprotein, an ATP-dependent cell-membrane transport molecule. P-glycoprotein is also expressed in several normal tissues but its physiological role(s) is unknown. We recently observed that a hierarchy of MDR-like activity exists among human peripheral blood lymphocytes in the order CD8 > CD4 > CD20 (cytotoxic/suppressor T cells, helper T cells and B cells respectively). In this study, we report that natural killer (NK) cells also express MDR-like activity. This activity could be inhibited with verapamil or solutol HS-15, two agents that reverse MDR in tumor cells. These, and four additional reversing agents, were used to investigate the possible role of P-glycoprotein in NK cells. We observed that at 10% of their IC50, five of six reversing agents inhibited NK-cell-mediated cytotoxicity; at higher (but non-toxic) doses, all six agents were inhibitory. These data suggest that NK-cell-mediated cytotoxicity may require the functional expression of an efflux molecule similar or identical to P-glycoprotein.

Multidrug resistance activity in human lymphocytes.

The multidrug resistance gene (mdr1) is a member of the recently described ATP binding cassette (ABC) superfamily of transporters. Family members include: (1) the cystic fibrosis transmembrane conductance regulator gene; (2) the hlyB gene of bacteria, and (3) the histocompatibility antigen modifier (HAM) gene. The level of expression of mdr1 correlates with multidrug resistance (MDR), the ability of cells to efflux otherwise toxic doses of several chemotherapeutic agents. MDR activity is also associated with the efflux of cationic lipophilic compounds such as the fluorescent dye rhodamine 123. Recently it was reported that normal lymphocytes efflux rhodamine 123, suggesting that these cells possess MDR-like activity due to the expression of mdr1. In this study, using two-color flow cytometric analysis, we observed that the ability to efflux rhodamine 123 was heterogeneous among human lymphocyte subsets in the order of CD8 greater than CD4 greater than CD2O. Rhodamine 123 efflux and accumulation in lymphocytes was sensitive to the known MDR reversing agents, verapamil and Solutol HS 15. Collectively, these data suggest that an MDR-like transport system is present in normal lymphocytes and may be important for trafficking of molecules involved in lymphocyte function.