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.

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.

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.

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.

The pfr1 gene from the human pathogenic fungus Paracoccidioides brasiliensis encodes a half-ABC transporter that is transcribed in response to treatment with fluconazole.

We have isolated a gene that encodes a half-ABC-transporter, designated Pfr1, from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis, which has high identity with members of the ABC-superfamily involved in multidrug resistance. The pfr1 gene is predicted to encode a 827 amino acid protein that, in common with mammalian Mdr1, has a TM-NBD topology. The transcription of the pfr1 gene is induced by the triazole drug fluconazole but not by amphotericin B, suggesting a role in transport-mediated azole resistance. However, Pfr1 has greatest identity to the mitochondrial ABC transporters Mdl1 and Mdl2 from Saccharomyces cerevisiae and mammalian ABC-me, with identities of 47.2%, 40.6% and 39.5%, respectively, over the length of these proteins. Furthermore, the N-terminus of Pfr1 is rich in positively charged residues, a feature of mitochondrial targeting sequences. Considering these features, it seems likely that Pfr1 is a mitochondrial protein. Previous studies have revealed that the acquisition of azole resistance in S. cerevisiae is linked to mitochondrial loss and, conversely, that mitochondrial dysfunction can lead to the upregulation of PDR transporters mediated by the transcription factor Pdr3. Our studies suggest that a mitochondrial ABC transporter is induced as part of the cellular response to drug treatment. The promoter region of pfr1 contains a PDRE-like consensus sequence to which Pdr3 binds, which may be the element responsible for the upregulation of Pfr1 in response to fluconazole. The nucleotide binding domain of Pfr1 was expressed and purified from Escherichia coli and shown to retain ATPase activity, consistent with Pfr1 functioning as a homodimeric transport ATPase.

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.

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.

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.

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.

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.

Antimonite regulation of the ATPase activity of ArsA, the catalytic subunit of the arsenical pump.

The ArsA ATPase is the catalytic subunit of the pump protein, coupling the hydrolysis of ATP to the movement of arsenicals and antimonials through the membrane-spanning ArsB protein. Previously, we have shown the binding and hydrolysis of MgATP to ArsA to be a multi-step process in which the rate-limiting step is an isomerization between different conformational forms of ArsA. This isomerization occurs after product release, at the end of the ATPase reaction, and involves the return of the ArsA to its original conformation, which can then bind MgATP. ArsA possesses an allosteric site for antimonite [Sb(III)], the binding of which elevates the steady-state ATPase activity. We have used a transient kinetics approach to investigate the kinetics of ternary complex formation that lead to an enhancement in the ATPase activity. These studies revealed that ArsA exists in at least two conformational forms that differ in their ligand binding affinities, and that ATP favours one form and Sb(III) the other. Ternary complex formation is rate-limited by a slow transition between these conformational forms, leading to a lag in attaining maximal steady-state activity. Sb(III) enhances the steady-state ATPase activity by inducing rapid product release, allowing ArsA to adopt a conformation that can bind MgATP for the next catalytic cycle. In the presence of Sb(III), ArsA avoids the rate-limiting isomerization at the end of the ATPase reaction and ATP hydrolysis becomes rate-limiting for the reaction. The binding of Sb(III) probably results in more effective pumping of the substrates from the cell by enhancing the rate of efflux.

The structure and function of drug pumps.

Resistance to drugs has emerged in biological systems as diverse as cancer cells undergoing chemotherapy and microbial pathogens undergoing treatment with antimicrobials. This medical problem is escalating and there is an urgent need for the development of new classes of drugs. In the case of pathogenic bacteria, we are rapidly approaching a scenario where there will be no effective antibiotics in the armoury of drugs available for treating the infectious diseases that these bacteria cause, returning us to the pre-antibiotic era when infectious diseases were rife because they were untreatable. One of the most frequently employed resistance strategies in both prokaryotes and eukaryotes is the transmembrane-protein-catalysed extrusion of drugs from the cell, with these proteins acting like bilge pumps, reducing the intracellular drug concentration to subtoxic levels. There is currently much scientific interest in understanding how these pumps operate, so that we might design transport inhibitors that would block them, allowing a renaissance for drugs that are no longer effective owing to their efflux.

A kinetic model for the action of a resistance efflux pump.

ArsA is the catalytic subunit of the arsenical pump, coupling ATP hydrolysis to the efflux of arsenicals through the ArsB membrane protein. It is a paradigm for understanding the structure-function of the nucleotide binding domains (NBD) of medically important efflux pumps, such as P-glycoprotein, because it has two sequence-related, interacting NBD, for which the structure is known. On the basis of a rigorous analysis of the pre-steady-state kinetics of nucleotide binding and hydrolysis, we propose a model in which ArsA alternates between two mutually exclusive conformations as follows: the ArsA(1) conformation in which the A1 site is closed but the A2 site open; and the ArsA(2) conformation, in which the A1 and A2 sites are open and closed, respectively. Antimonite elicits its effects by sequestering ArsA in the ArsA(1) conformation, which catalyzes rapid ATP hydrolysis at the A2 site to drive ArsA between conformations that have high (nucleotide-bound ArsA) and low affinity (nucleotide-free ArsA) for Sb(III). ArsA potentially utilizes this process to sequester Sb(III) from the medium and eject it into the channel of ArsB.

The quarternary molecular architecture of TetA, a secondary tetracycline transporter from Escherichia coli.

TetA, a tetracycline cation/proton antiporter, was expressed in Escherichia coli with a C-terminal tag of six histidines, solubilized in dodecyl maltoside and purified in a single step using Ni2+ affinity chromatography. Two-dimensional crystals were obtained after reconstitution of purified protein with lipids. Electron microscopy of negatively stained crystals revealed a trigonal symmetry, from which we infer that this secondary transporter has a trimeric structure. An overall molecular envelope can be described by a triangle of side approximately 100 A enclosing a central stain-filled depression. These dimensions are consistent with those obtained from projection views of single, isolated TetA particles that also display a trimeric architecture, confirming that the threefold symmetry is not simply a consequence of crystal-packing interactions. These data represent the first direct view of the quarternary arrangement of any antibiotic efflux pump. They are fully consistent with biochemical data on TetA, which indicate that it functions as a multimer and that the monomer consists of two domains, one of which plays the major part in oligomerization interactions.

Genetic diversity and dynamics of plasmodium falciparum and P. vivax populations in multiply infected children with asymptomatic malaria infections in Papua New Guinea.

We describe the dynamics of co-infections of Plasmodium falciparum and P. vivax in 28 asymptomatic children by genotyping these species using the polymorphic loci Msp2 and Msp3alpha, respectively. The total number of Plasmodium spp. infections detected using 3 day sampling over 61 days varied between 1 and 14 (mean 6.6). The dynamics of P. falciparum and P. vivax genotypes varied greatly both within and amongst children. Periodicity in the detection of P. falciparum infections is consistent with the synchronous replication of individual genotypes. Replication synchrony of multiple co-infecting genotypes was not detected. In 4-year-old children P. falciparum genotype complexity was reduced and episodes lasted significantly longer (median duration > 60 days) when compared to children aged 5-14 years (median duration 9 days). P. vivax genotype complexity was not correlated with age but the episode duration was also longer for this species in 4-year-olds than in older children but was not as long as P. falciparum episodes. Recurrence of P. falciparum and P. vivax genotypes over weeks was observed. We interpret these major fluctuations in the density of genotypes over time as the result of the mechanism of antigenic variation thought to be present in these Plasmodium species.

Distinct origins of adult and embryonic blood in Xenopus.

Whether embryonic and adult blood derive from a single (yolk sac) or dual (yolk sac plus intraembryonic) origin is controversial. Here, we show, in Xenopus, that the yolk sac (VBI) and intraembryonic (DLP) blood compartments derive from distinct blastomeres in the 32-cell embryo. The first adult hematopoietic stem cells (HSCs) are thought to form in association with the floor of the dorsal aorta, and we have detected such aortic clusters in Xenopus using hematopoietic markers. Lineage tracing shows that the aortic clusters derive from the blastomere that gives rise to the DLP. These observations indicate that the first adult HSCs arise independently of the embryonic lineage.

A role for GATA5 in Xenopus endoderm specification.

The endoderm gives rise to the gut and tissues that develop as outgrowths of the gut tube, including the lungs, liver and pancreas. Here we show that GATA5, a zinc-finger transcription factor, is expressed in the yolk-rich vegetal cells of Xenopus embryos from the early gastrula stage onwards, when these cells become committed to form endoderm. At mid-gastrula stages, GATA5 is restricted to the sub-blastoporal endoderm and is the first molecular marker for this subset of endodermal cells so far identified. We show that GATA4 and GATA5 are potent inducers of endodermal marker genes in animal cap assays, while other GATA factors induce these genes only weakly, if at all. When injected into the dorsal marginal zone, GATA5 respecifies prospective mesoderm towards an endodermal fate, thereby disrupting the convergence and extension movements normally undergone by the dorsal mesoderm. The resulting phenotype is very similar to those seen after injection of dominant negative versions of the FGF-receptor or the T-box transcription factor, Xbra and can be rescued by eFGF. The ability of GATA5 to respecify ectodermal and mesodermal cells towards endoderm suggests an important role for GATA5 in the formation of this germlayer. In animal cap assays, GATA5 is induced by concentrations of activin above those known to induce dorsal mesoderm and heart, in an FGF-independent manner. These data indicate that the emerging view for endodermal induction in general, namely that it is specified by high levels of TGF-beta in the absence of FGF signalling, is specifically true for sub-blastoporal endoderm.

A comparative study into the mechanisms of action of anti-tumor necrosis factor alpha, anti-CD4, and combined anti-tumor necrosis factor alpha/anti-CD4 treatment in early collagen-induced arthritis.

OBJECTIVE: Anti-tumor necrosis factor alpha (anti-TNFalpha) therapy is very effective in rheumatoid arthritis (RA), whereas depleting anti-CD4 therapy is relatively ineffective. To explain the differences in efficacy between these 2 therapies, we used an animal model of RA to compare their effects on different aspects of the disease process. METHODS: Mice with collagen-induced arthritis were treated with depleting anti-CD4 monoclonal antibodies (mAb), anti-TNFalpha mAb, or phosphate buffered saline. Another group was given a combination of anti-TNFalpha plus anti-CD4. The treatments were compared for their ability to down-regulate the expression of proinflammatory cytokines and adhesion molecules, reduce the cellularity of the joint, and inhibit Th1 activity. RESULTS: Anti-TNFalpha significantly reduced the numbers of cells expressing TNFalpha, interleukin-1beta (IL-1beta), very late activation antigen 4 (VLA-4), vascular cell adhesion molecule 1 (VCAM-1), and numbers of CD4+ T cells and macrophages in the joint. Anti-CD4 treatment led to a small reduction in the expression of TNFalpha, IL-1beta, VLA-4, and VCAM-1, but this did not reach statistical significance. Depleting anti-CD4 was also surprisingly ineffective in eliminating CD4+ T cells from the joint. Anti-TNFalpha therapy was also more effective than anti-CD4 in reducing Thl activity, as assessed by the production of interferon-gamma in lymph node cell cultures. There was a synergistic relationship between anti-TNFalpha and anti-CD4 in the reduction of histologic score and inhibition of TNFalpha/IL-1beta expression in the joints. CONCLUSION: The efficacy of the 3 treatments correlated with their ability to modulate the expression of inflammatory cytokines and adhesion molecules in the joint, reduce the cellularity of the joint, and inhibit Th1 activity. This kind of analysis may prove useful in the testing of novel therapies for RA.