VceR regulates the vceCAB drug efflux pump operon of Vibrio cholerae by alternating between mutually exclusive conformations that bind either drugs or promoter DNA.

VceR, a member of the TetR family of transcriptional regulators, is a repressor of the vceCAB operon, which encodes a multidrug efflux pump in Vibrio cholerae. VceR binds to a 28 bp inverted-repeat within the vceR-vceC intergenic region and is dissociated from this site with CCCP, a pump substrate. The rate of the CCCP-induced conformational change in VceR was determined by stopped-flow fluorescence spectroscopy, revealing a highly co-operative process that occurs with a Hill coefficient of approximately 4. The apparent affinity for CCCP decreased in a linear manner with increasing concentrations of DNA, indicative of competition between the CCCP and DNA for binding to VceR. These data are consistent with an equilibrium between mutually exclusive conformations that are supported by the binding of DNA and CCCP to the N and C termini of VceR, respectively. Size-exclusion chromatography and dynamic light-scattering studies indicate that VceR exists predominantly as a dimer; however, a pair of dimers binds to the DNA. In order to account for the fact that VceR is a dimer in the absence of DNA but binds CCCP with a Hill co-efficient of 4, implying that it has at least four binding-sites, we propose that the VceR monomer possesses a pair of binding sites that can be simultaneously occupied by CCCP. Using a gene-reporter system and stopped-flow spectroscopy, we established that the equilibrium between free VceR and VceR-CCCP plays a critical role in controlling expression of the pump. The co-operative transition between these states allows the repressor to respond to relatively small changes in drug concentration. Thus, repression and induction can be readily switched about a critical drug concentration which will prove toxic to the cell.

Microbial and viral drug resistance mechanisms.

Microorganisms and viruses have developed numerous resistance mechanisms that enable them to evade the effect of antimicrobials and antivirals. As a result, many have become resistant to almost every available means of treatment. This problem, although not new, is becoming increasingly acute and it is now clear that a fundamental understanding of the mechanisms that microbes and viruses deploy in the development of resistance is essential if we are to gain new insights into ways to combat this problem.

The structure and function of drug pumps: an update.

Our understanding of the exact mechanisms used by the transmembrane protein pumps that confer cellular resistance to cytotoxic drugs has improved enormously with the recent determination of the structures of three Escherichia coli transporters, two belonging to the ATP-binding cassette (ABC) superfamily and one to the resistance-nodulation-cell division (RND) family. Although these studies do not provide an insight into how drug pumps can recognize several structurally unrelated drugs, important advances have been also made in this area. Information on the molecular basis of multidrug recognition has been provided by determining the structure of transcriptional regulators that can bind, often structurally unrelated, cytotoxic drugs and control the expression of drug pumps.

The pathobiology of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis causes one of the most prevalent systemic mycoses in Latin America--paracoccidioidomycosis. It is a dimorphic fungus that undergoes a complex transformation in vivo, with mycelia in the environment producing conidia, which probably act as infectious propagules upon inhalation into the lungs, where they transform to the pathogenic yeast form. This transition is readily induced in vitro by temperature changes, resulting in modulation of the composition of the cell wall. Notably, the polymer linkages change from beta-glucan to alpha-glucan, possibly to avoid beta-glucan triggering the inflammatory response. Mammalian oestrogens inhibit this transition, giving rise to a higher incidence of disease in males. Furthermore, the susceptibility of individuals to paracoccidioidomycosis has a genetic basis, which results in a depressed cellular immune response in susceptible patients; resistance is conferred by cytokine-stimulated granuloma formation and nitric oxide production. The latency period and persistence of the disease and the apparent lack of efficacy of humoral immunity are consistent with P. brasiliensis existing as a facultative intracellular pathogen.

Structural understanding of efflux-mediated drug resistance: potential routes to efflux inhibition.

The active efflux of cytotoxic drugs mediated by multidrug transporters is the basis of multidrug resistance in prokaryotic and eukaryotic cells. Individual multidrug transporters can be extremely versatile, often exhibiting a staggering range of substrate specificity that can negate the effects of clinically relevant therapies. The effective treatment of bacterial, fungal and protozoan infections, along with certain cancer treatments, has been compromised by the presence of multidrug transporters. Traditionally, advances in the understanding of multidrug transporters have been made through biochemical analyses; more recently, however, fundamental advances have been made with the elucidation of several three dimensional structures of representative multidrug pumps. Biochemical and structural analysis of multidrug pumps could lead to the development of novel 'anti-efflux' therapies.

Structure and function of efflux pumps that confer resistance to drugs.

Resistance to therapeutic drugs encompasses a diverse range of biological systems, which all have a human impact. From the relative simplicity of bacterial cells, fungi and protozoa to the complexity of human cancer cells, resistance has become problematic. Stated in its simplest terms, drug resistance decreases the chance of providing successful treatment against a plethora of diseases. Worryingly, it is a problem that is increasing, and consequently there is a pressing need to develop new and effective classes of drugs. This has provided a powerful stimulus in promoting research on drug resistance and, ultimately, it is hoped that this research will provide novel approaches that will allow the deliberate circumvention of well understood resistance mechanisms. A major mechanism of resistance in both microbes and cancer cells is the membrane protein-catalysed extrusion of drugs from the cell. Resistant cells exploit proton-driven antiporters and/or ATP-driven ABC (ATP-binding cassette) transporters to extrude cytotoxic drugs that usually enter the cell by passive diffusion. Although some of these drug efflux pumps transport specific substrates, many are transporters of multiple substrates. These multidrug pumps can often transport a variety of structurally unrelated hydrophobic compounds, ranging from dyes to lipids. If we are to nullify the effects of efflux-mediated drug resistance, we must first of all understand how these efflux pumps can accommodate a diverse range of compounds and, secondly, how conformational changes in these proteins are coupled to substrate translocation. These are key questions that must be addressed. In this review we report on the advances that have been made in understanding the structure and function of drug efflux pumps.

MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.

Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled.

The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is a human pathogenic fungus that switches from a saprobic mycelium to a pathogenic yeast. Consistent with the morphological transition being regulated by the cAMP-signalling pathway, there is an increase in cellular cAMP levels both transiently at the onset (< 24 h) and progressively in the later stages (> 120 h) of the transition to the yeast form, and this transition can be modulated by exogenous cAMP. We have cloned the cyr1 gene encoding adenylate cyclase (AC) and established that its transcript levels correlate with cAMP levels. In addition, we have cloned the genes encoding three Galpha (Gpa1-3), Gbeta (Gpb1) and Ggamma (Gpg1) G proteins. Gpa1 and Gpb1 interact with one another and the N-terminus of AC, but neither Gpa2 nor Gpa3 interacted with Gpb1 or AC. The interaction of Gpa1 with Gpb1 was blocked by GTP, but its interaction with AC was independent of bound nucleotide. The transcript levels for gpa1, gpb1 and gpg1 were similar in mycelium, but there was a transient excess of gpb1 during the transition, and an excess of gpa1 in yeast. We have interpreted our findings in terms of a novel signalling mechanism in which the activity of AC is differentially modulated by Gpa1 and Gpb1 to maintain the signal over the 10 days needed for the morphological switch.

The crystal structure of the outer membrane protein VceC from the bacterial pathogen Vibrio cholerae at 1.8 A resolution.

Multidrug resistance in Gram-negative bacteria arises in part from the activities of tripartite drug efflux pumps. In the pathogen Vibrio cholerae, one such pump comprises the inner membrane proton antiporter VceB, the periplasmic adaptor VceA, and the outer membrane channel VceC. Here, we report the crystal structure of VceC at 1.8 A resolution. The trimeric VceC is organized in the crystal lattice within laminar arrays that resemble membranes. A well resolved detergent molecule within this array interacts with the transmembrane beta-barrel domain in a fashion that may mimic protein-lipopolysaccharide contacts. Our analyses of the external surfaces of VceC and other channel proteins suggest that different classes of efflux pumps have distinct architectures. We discuss the implications of these findings for mechanisms of drug and protein export.

Evidence for cooperativity between the four binding sites of dimeric ArsD, an As(III)-responsive transcriptional regulator.

ArsD is a trans-acting repressor of the arsRDABC operon that confers resistance to arsenicals and antimonials in Escherichia coli. It possesses two-pairs of vicinal cysteine residues, Cys(12)-Cys(13) and Cys(112)-Cys(113), that potentially form separate binding sites for the metalloids that trigger dissociation of ArsD from the operon. However, as a homodimer it has four vicinal cysteine pairs. Titration of the steady-state fluorescence of ArsD with metalloids revealed positive cooperativity, with a Hill coefficient of 2, between these sites. Disruption of the Cys(112)-Cys(113) site by mutagenesis of arsD, but not the Cys(12)-Cys(13) site, largely abolished this cooperativity, indicative of interactions between adjacent Cys(112)-Cys(113) sites within the dimer. The kinetics of metalloid binding were determined by stopped flow spectroscopy; the rate increased in a sigmoidal manner, with a Hill coefficient of 4, indicating that the pre-steady-state measurements reported cooperativity between all four sites of the dimer rather than just the intermolecular interactions reported by the steady-state measurements. The kinetics of Sb(III) displacement by As(III) revealed that the metalloid-binding sites behave differentially, with the rapid exchange of As(III) for Sb(III) at one site retarding the release of Sb(III) from the other sites. We propose a model involving the sequential binding and release of metalloids by the four binding sites of dimeric ArsD, with only one site releasing free metalloids.

Identification of oligomerization and drug-binding domains of the membrane fusion protein EmrA.

Many pathogenic Gram-negative bacteria possess tripartite transporters that catalyze drug extrusion across the inner and outer membranes, thereby conferring resistance. These transporters consist of inner (IMP) and outer (OMP) membrane proteins, which are coupled by a periplasmic membrane fusion (MFP) protein. However, it is not know whether the MFP translocates the drug between the membranes, by acting as a channel, or whether it brings the IMP and OMP together, facilitating drug transfer. The MFP EmrA has an elongated periplasmic domain, which binds transported drugs, and is anchored to the inner membrane by a single alpha-helix, which contains a leucine zipper dimerization domain. Consistent with CD and hydrodynamic analyses, the periplasmic domain is predicted to be composed of a beta-sheet subdomain and an alpha-helical coiled-coil. We propose that EmrA forms a trimer in which the coiled-coils radiate across the periplasm, where they could sequester the OMP TolC. The "free" leucine zipper in the EmrA trimer might stabilize the interaction with the IMP EmrB, which also possesses leucine zipper motifs in the putative N- and C-terminal helices. The beta-sheet subdomain of EmrA would sit at the membrane surface adjacent to the EmrB, from which it receives the transported drug, inducing a conformational change that triggers the interaction with the OMP.