Enhancing effect of taurohyodeoxycholate on ABCB4-mediated phospholipid efflux

Hydrophilic bile salts, ursodeoxycholate and hyodeoxycholate, have choleretic effects. ABCB4, a member of the ABC transporter family, is essential for the secretion of phospholipids from hepatocytes into bile. In this study, we assessed the effects of taurine- or glycine-conjugated cholate, ursodeoxycholate and hyodeoxycholate on the ABCB4-mediated phosphatidylcholine (PC) efflux using Abcb4 knockout mice and HEK293 cells stably expressing ABCB4. To evaluate the effects of bile salts on bile formation in Abcb4^+/+ or Abcb4^−/− mice, the bile was collected during intravenous infusion of saline or bile salts. The biliary PC secretion in Abcb4^+/+ mice was significantly increased by the infusions of all tested bile salts, especially taurohyodeoxycholate. On the other hand, Abcb4^−/− mice exhibited extremely low secretion of PC into bile, which was not altered by bile salt infusions. We also showed that the PC efflux from ABCB4-expressing HEK293 cells was stimulated by taurohyodeoxycholate much more strongly than the other tested bile salts. However, taurohyodeoxycholate did not restore the activities of ABCB4 mutants. Furthermore, light scattering measurements demonstrated a remarkable ability of taurohyodeoxycholate to form mixed micelles with PC. Therefore, the enhancing effect of taurohyodeoxycholate on the ABCB4-mediated PC efflux may be due to the strong mixed micelle formation ability.     

The Bifidobacterium longum NCIMB 702259T ctr gene codes for a novel cholate transporter

Preexposure of Bifidobacterium longum NCIMB 702259T to cholate caused increased resistance to cholate, chloramphenicol, and erythromycin. The B. longum ctr gene, encoding a cholate efflux transporter, was transformed into the efflux-negative mutant Escherichia coli KAM3, conferring resistance to bile salts and other antimicrobial compounds and causing the efflux of [14C]cholate.     

Cholic Acid Induces a Cftr Dependent Biliary Secretion and Liver Growth Response in Mice

The cause of Cystic fibrosis liver disease (CFLD), is unknown. It is well recognized that hepatic exposure to hydrophobic bile salts is associated with the development of liver disease. For this reason, we hypothesize that, CFTR dependent variations, in the hepatic handling of hydrophobic bile salts, are related to the development CFLD. To test our hypothesis we studied, in Cftr^-/- and control mice, bile production, bile composition and liver pathology, in normal feeding condition and during cholate exposure, either acute (intravenous) or chronic (three weeks via the diet). In Cftr^-/- and control mice the basal bile production was comparable. Intravenous taurocholate increased bile production to the same extent in Cftr^-/- and control mice. However, chronic cholate exposure increased the bile flow significantly less in Cftr^-/- mice than in controls, together with significantly higher biliary bile salt concentration in Cftr^-/- mice. Prolonged cholate exposure, however, did not induce CFLD like pathology in Cftr^-/- mice. Chronic cholate exposure did induce a significant increase in liver mass in controls that was absent in Cftr^-/- mice. Chronic cholate administration induces a cystic fibrosis-specific hepatobiliary phenotype, including changes in bile composition. These changes could not be associated with CFLD like pathological changes in CF mouse livers. However, chronic cholate administration induces liver growth in controls that is absent in Cftr^-/- mice. Our findings point to an impaired adaptive homeotrophic liver response to prolonged hydrophobic bile salt exposure in CF conditions.     

A simple method for the determination of resistance to gas diffusion in plant organs

A simple method was developed for the determination of resistance coefficients for ethylene diffusion in plant tissues based on the kinetic analysis of the efflux of preloaded ethane gas. Efflux curves were analyzed to obtain first-order rate constants and resistance coefficients. Resistance coefficients determined by the ethane efflux and steady-state methods were found to agree well. Employing the ethane efflux method, it was shown that over 97% of gas exchange of tomato (Lycopersicum esculentum Mill., cv. `Ace') fruits occurs through the stem scar. The resistances to diffusion of tomato skin and stem scar were found to be 280,000 and 300 seconds per centimeter, respectively; the combined resistance of intact tomato fruits was approximately 7,800 seconds per centimeter. The ethane efflux method was employed to show that plastic shrink-wrapping of English cucumbers (Cucumis sativus L. var anglicus Bailey) increased the resistance to ethane diffusion from 1.1 × 10³ to 23 × 10³ seconds per centimeter.     

Functionally Cloned pdrM from Streptococcus pneumoniae Encodes a Na+ Coupled Multidrug Efflux Pump

Multidrug efflux pumps play an important role as a self-defense system in bacteria. Bacterial multidrug efflux pumps are classified into five families based on structure and coupling energy: resistance−nodulation−cell division (RND), small multidrug resistance (SMR), major facilitator (MF), ATP binding cassette (ABC), and multidrug and toxic compounds extrusion (MATE). We cloned a gene encoding a MATE-type multidrug efflux pump from Streptococcus pneumoniae R6, and designated it pdrM. PdrM showed sequence similarity with NorM from Vibrio parahaemolyticus, YdhE from Escherichia coli, and other bacterial MATE-type multidrug efflux pumps. Heterologous expression of PdrM let to elevated resistance to several antibacterial agents, norfloxacin, acriflavine, and 4′,6-diamidino-2-phenylindole (DAPI) in E. coli KAM32 cells. PdrM effluxes acriflavine and DAPI in a Na⁺- or Li⁺-dependent manner. Moreover, Na⁺ efflux via PdrM was observed when acriflavine was added to Na⁺-loaded cells expressing pdrM. Therefore, we conclude that PdrM is a Na⁺/drug antiporter in S. pneumoniae. In addition to pdrM, we found another two genes, spr1756 and spr1877,that met the criteria of MATE-type by searching the S. pneumoniae genome database. However, cloned spr1756 and spr1877 did not elevate the MIC of any of the investigated drugs. mRNA expression of spr1756, spr1877, and pdrM was detected in S. pneumoniae R6 under laboratory growth conditions. Therefore, spr1756 and spr1877 are supposed to play physiological roles in this growth condition, but they may be unrelated to drug resistance.     

A single‐component multidrug transporter of the major facilitator superfamily is part of a network that protects Escherichia coli from bile salt stress

Resistance to high concentrations of bile salts in the human intestinal tract is vital for the survival of enteric bacteria such as Escherichia coli. Although the tripartite AcrAB–TolC efflux system plays a significant role in this resistance, it is purported that other efflux pumps must also be involved. We provide evidence from a comprehensive suite of experiments performed at two different pH values (7.2 and 6.0) that reflect pH conditions that E. coli may encounter in human gut that MdtM, a single‐component multidrug resistance transporter of the major facilitator superfamily, functions in bile salt resistance in E. coli by catalysing secondary active transport of bile salts out of the cell cytoplasm. Furthermore, assays performed on a chromosomal ΔacrB mutant transformed with multicopy plasmid encoding MdtM suggested a functional synergism between the single‐component MdtM transporter and the tripartite AcrAB–TolC system that results in a multiplicative effect on resistance. Substrate binding experiments performed on purified MdtM demonstrated that the transporter binds to cholate and deoxycholate with micromolar affinity, and transport assays performed on inverted vesicles confirmed the capacity of MdtM to catalyse electrogenic bile salt/H⁺ antiport.     

The interrelations of radiologic findings and mechanical ventilation in community acquired pneumonia patients admitted to the intensive care unit: a multicentre retrospective study

Background

Burkholderia cepacia complex (BCC) bacteria are highly virulent, typically multidrug-resistant, opportunistic pathogens in cystic fibrosis (CF) patients and other immunocompromised individuals. B. vietnamiensis is more often susceptible to aminoglycosides than other BCC species, and strains acquire aminoglycoside resistance during chronic CF infection and under tobramycin and azithromycin exposure in vitro, apparently from gain of antimicrobial efflux as determined through pump inhibition. The aims of the present study were to determine if oxidative stress could also induce aminoglycoside resistance and provide further observations in support of a role for antimicrobial efflux in aminoglycoside resistance in B. vietnamiensis.

Findings

Here we identified hydrogen peroxide as an additional aminoglycoside resistance inducing agent in B. vietnamiensis. After antibiotic and hydrogen peroxide exposure, isolates accumulated significantly less [³H] gentamicin than the susceptible isolate from which they were derived. Strains that acquired aminoglycoside resistance during infection and after exposure to tobramycin or azithromycin overexpressed a putative resistance-nodulation-division (RND) transporter gene, amrB. Missense mutations in the repressor of amrB, amrR, were identified in isolates that acquired resistance during infection, and not in those generated in vitro.

Conclusions

These data identify oxidative stress as an inducer of aminoglycoside resistance in B. vietnamiensis and further suggest that active efflux via a RND efflux system impairs aminoglycoside accumulation in clinical B. vietnamiensis strains that have acquired aminoglycoside resistance, and in those exposed to tobramycin and azithromycin, but not hydrogen peroxide, in vitro. Furthermore, the repressor AmrR is likely just one regulator of the putative AmrAB-OprM efflux system in B. vietnamiensis.     

A Linkage between SmeIJK Efflux Pump,Cell Envelope Integrity,and σE-Mediated Envelope Stress Response in Stenotrophomonas maltophilia

Resistance nodulation division (RND) efflux pumps, such as the SmeIJK pump of Stenotrophomonas maltophilia, are known to contribute to the multidrug resistance in Gram-negative bacteria. However, some RND pumps are constitutively expressed even though no antimicrobial stresses occur, implying that there should be some physical implications for these RND pumps. In this study, the role of SmeIJK in antimicrobials resistance, envelope integrity, and σ^E-mediated envelope stress response (ESR) of S. maltophilia was assessed. SmeIJK was involved in the intrinsic resistance of S. maltophilia KJ to aminoglycosides and leucomycin. Compared with the wild-type KJ, the smeIJK deletion mutant exhibited growth retardation in the MH medium, an increased sensitivity to membrane-damaging agents (MDAs), as well as activation of an σ^E-mediated ESR. Moreover, the expression of smeIJK was further induced by sub-lethal concentrations of MDAs or surfactants in an σ^E-dependent manner. These data collectively suggested an alternative physiological role of smeIJK in cell envelope integrity maintenance and σ^E-mediated ESR beyond the efflux of antibiotics. Because of the necessity of the physiological role of SmeIJK in protecting S. maltophilia from the envelope stress, smeIJK is constitutively expressed, which, in turn, contributes the intrinsic resistance to aminoglycoside and leucomycin. This is the first demonstration of the linkage among RND-type efflux pump, cell envelope integrity, and σ^E-mediated ESR in S. maltophilia.     

Premature Termination of MexR Leads to Overexpression of MexAB-OprM Efflux Pump in Pseudomonas aeruginosa in a Tertiary Referral Hospital in India

ObjectivesThe present study was undertaken to investigate the mutations that are present in mexR gene of multidrug resistant (MDR) isolates of Pseudomonas aeruginosa collected from a tertiary referral hospital of north east India.Methods76 MDR clinical isolates of P. aeruginosa were obtained from the patients who were admitted to or attended the clinics of Silchar medical college and hospital. They were screened phenotypically for the presence of efflux pump activity by an inhibitor based method. Acquired resistance mechanisms were detected by multiplex PCR. Real time PCR was performed to study the expression of mexA gene of MexAB-OprM efflux pump in isolates with increase efflux pump activity. mexR gene of the isolates with overexpressed MexAB-OprM efflux pump was amplified, sequenced and analysed.ResultsOut of 76 MDR isolates, 24 were found to exhibit efflux pump activity phenotypically against ciprofloxacin and meropenem. Acquired resistance mechanisms were absent in 11 of them and among those isolates, 8 of them overexpressed MexAB-OprM. All the 8 isolates possessed mutation in mexR gene. 11 transversions, 4 transitions, 2 deletion mutations and 2 insertion mutations were found in all the isolates. However, the most significant observation was the formation of a termination codon at 35^th position which resulted in the termination of the polypeptide and leads to overexpression of the MexAB-OprM efflux pump.ConclusionsThis study highlighted emergence of a novel mutation which is probably associated with multi drug resistance. Therefore, further investigations and actions are needed to prevent or at least hold back the expansion and emergence of newer mutations in nosocomial pathogens which may compromise future treatment options.     

Murein and the Outer Penetration Barrier of Escherichia coli K-12, Proteus mirabilis, and Pseudomonas aeruginosa

A penetration barrier operating outside the periplasmic enzyme penicillinase was studied in an ampicillin-resistant mutant of Escherichia coli K-12. Growth in the presence of lysozyme and sublethal concentrations of ampicillin partially opened the barrier. This could be recorded as an increased penetration of penicillin G, sodium cholate, and rifampin to their respective targets. Brief treatments with tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid and sodium cholate effectively impaired the barrier against penicillin and also caused leakage of penicillinase. Wild-type E. coli K-12, Proteus mirabilis, and Pseudomonas aeruginosa also showed an increased sensitivity to cholate after treatment with penicillins. Electron micrographs showed that lysis by cholate was due to a distortion of the cytoplasmic membrane causing a leakage of protein and RNA from the cells to the medium. Physiological data indicated that the increased sensitivity to cholate induced by growth in the presence of ampicillin or lysozyme was due to effects upon the murein. This was supported by measurement of the incorporation of ³H-diaminopimelic acid. These results indicate that the murein sacculus either is a part of the penetration barrier or is responsible for holding the structure of the outer membrane together.     

Targeting efflux pumps—In vitro investigations with acridone derivatives and identification of a lead molecule for MDR modulation

To search multi drug resistance modulators, acridones carrying hydroxyl amine substituent at N-10 and COOH/Cl at C-4 were investigated for their interactions with the three components of efflux pump viz. P-gp, ATP, and Mg²⁺. Experimental and theoretical results indicated that the compounds with COOH group at C-4 interact with P-gp and Mg²⁺ while other set of compounds with Cl at C-4 interact with ATP and Mg²⁺. Spot assay and R6G influx/efflux assay of compound 3 using Candida albicans showed decrease in the fungal growth and efflux of R6G, respectively, in presence of compound 3 suggesting the suitability of this compound for MDR modulation.     

Engineering greater aluminium resistance in wheat by over-expressing TaALMT1

MethodsParticle bombardment was used to transform wheat with TaALMT1, the Al³⁺ resistance gene from wheat, using the maize ubiquitin promoter to drive expression. TaALMT1 expression, malate efflux and Al³⁺ resistance were measured in the T₁ and T₂ lines and compared with the parental line and an Al³⁺-resistant reference genotype, ET8.ConclusionsThe Al³⁺ resistance of wheat was increased by enhancing TaALMT1 expression with biotechnology. This is the first report of a major food crop being stably transformed for greater Al³⁺ resistance. Transgenic strategies provide options for increasing food supply on acid soils.     

Cell potentials, cell resistance, and proton fluxes in corn root tissue: effects of dithioerythritol

Studies were made of the effect of dithioerythritol on net proton flux, potassium influx and efflux, cell potential, and cell resistance in fresh and washed corn (Zea mays L. WF9XM14) root tissue. Dithioerythritol induces equal proton influx and potassium efflux rates, decreases membrane resistance, and hyperpolarizes the cell potential. Greater effects on H⁺ and K⁺ fluxes are secured at pH 7 than at pH 5. Other sulfhydryl-protecting reagents produced the same responses. No evidence could be found that dithioerythritol affected energy metabolism or membrane ATPase, and proton influx was induced in the presence of uncoupling agents.     

Bacterial resistances to toxic metal ions - a review

Bacterial plasmids encode resistance systems for toxic metal ions, including Ag⁺, AsO₂^-, AsO₄^3-, Cd²⁺, Co²⁺, CrO₄^2-, Cu²⁺ Hg²⁺, Ni²⁺, Pb²⁺, Sb³⁺, TeO₃^2-, Tl⁺ and Zn²⁺. The function of most resistance systems is based on the energy-dependent efflux of toxic ions. Some of the efflux systems are ATPases and others are chemiosmotic cation/proton antiporters. The Cd²⁺-resistance ATPase of Gram-positive bacteria (CadA) is membrane cation pump homologous with other bacterial, animal and plant P-type ATPases. CadA has been labeled with ³²P from [α-³²p]ATP and drives ATP-dependent Cd²⁺ (and Zn²⁺) uptake by inside-out membrane vesicles (equivalent to efflux from whole cells). Recently, isolated genes defective in the human hereditary diseases of copper metabolism, namely Menkes syndrome and Wilson's disease, encode P-type ATPases that are more similar to bacterial CadA than to other ATPases from eukaryotes. The arsenic resistance efflux system transports arsenite [As(III)], alternatively using either a double-polypeptide (ArsA and ArsB) ATPase or a single-polypeptide (ArsB) functioning as a chemiosmotic transporter. The third gene in the arsenic resistance system, arsC, encodes an enzyme that converts intracellular arsenate [As(V)] to arsenite [As(III)], the substrate of the efflux system. The triple-polypeptide Czc (Cd²⁺, Zn²⁺ and Co²⁺) chemiosmotic efflux pump consists of inner membrane (CzcA), outer membrane (CzcC) and membrane-spanning (CzcB) proteins that together transport cations from the cytoplasm across the periplasmic space to the outside of the cell.     

Crystal Structure of the Membrane Fusion Protein CusB from Escherichia coli

Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes belonging to the resistance-nodulation-division family to expel diverse toxic compounds from the cell. These systems contain a periplasmic membrane fusion protein (MFP) that is critical for substrate transport. We here present the x-ray structures of the CusB MFP from the copper/silver efflux system of E. coli. This is the first structure of any MFPs associated with heavy-metal efflux transporters. CusB bridges the inner-membrane efflux pump CusA and outer-membrane channel CusC to mediate resistance to Cu⁺ and Ag⁺ ions. Two distinct structures of the elongated molecules of CusB were found in the asymmetric unit of a single crystal, which suggests the flexible nature of this protein. Each protomer of CusB can be divided into four different domains, whereby the first three domains are mostly β-strands and the last domain adopts an entirely helical architecture. Unlike other known structures of MFPs, the α-helical domain of CusB is folded into a three-helix bundle. This three-helix bundle presumably interacts with the periplasmic domain of CusC. The N- and C-termini of CusB form the first β-strand domain, which is found to interact with the periplasmic domain of the CusA efflux pump. Atomic details of how this efflux protein binds Cu⁺ and Ag⁺ were revealed by the crystals of the CusB-Cu(I) and CusB-Ag(I) complexes. The structures indicate that CusB consists of multiple binding sites for these metal ions. These findings reveal novel structural features of an MFP in the resistance-nodulation-division efflux system and provide direct evidence that this protein specifically interacts with transported substrates.     

Efflux systems in Serratia marcescens

A widespread bacterium Serratia marcescens (family Enterobacteriaceae) is an opportunistic pathogen and exhibits multiple drug resistance. Active removal of antibiotics and other antimicrobials from the cells by efflux systems is one of the mechanisms responsible for microbial resistance to these compounds. Among enterobacteria, efflux systems of Escherichia coli and Salmonella enterica ser. Typhimurium have been studied most extensively. Few efflux systems that belong to different families have been reported for S. marcescens. In this review, we analyzed available literature about S. marcescens efflux systems and carried out the comparative analysis of the genes encoding the RND type systems in different Serratia species and in other enterobacteria. Bioinformatical analysis of the S. marcescens genome allowed us to identify the previously unknown efflux systems based on their homology with the relevant E. coli genes. Identification of additional efflux systems in S. marcescens genome will promote our understanding of the physiology of these bacteria, will detect new molecular mechanisms of resistance, and will reveal their resistance potential.     

Calcium Efflux from Barnacle Muscle Fibers : Dependence on External Cations

Calcium-45 was injected into single giant barnacle muscle fibers, and the rate of efflux was measured under a variety of conditions. The rate constant (k) for ⁴⁵Ca efflux into standard seawater averaged 17 x 10^–4 min^–1 which corresponds to an efflux of about 1–2 pmol/cm²·s. Removal of external Ca (Ca_o) reduced the efflux by 50%. In most fibers about 40% of the ⁴⁵Ca efflux into Ca-free seawater was dependent on external Na (Na_o); treatment with 3.5 mM caffeine increased the magnitude of the Na_o-dependent efflux. In a few fibers removal of Na_o, in the absence of Ca_o, either had no effect or increased k; caffeine (2–3.5 mM) unmasked an Na_o-dependent efflux in these fibers. The Na_o-dependent Ca efflux had a Q₁₀ of about 3.7. The data are consistent with the idea that a large fraction of the Ca efflux may be carrier-mediated, and may involve both Ca-Ca and Na-Ca counterflow. The relation between the Na_o-dependent Ca efflux and the external Na concentration is sigmoid, and suggests that two, or more likely three, external Na⁺ ions may activate the efflux of one Ca⁺². With a three-for-one Na-Ca exchange, the Na electrochemical gradient may be able to supply sufficient energy to maintain the Ca gradient in these fibers. Other, more complex models are not excluded, however, and may be required to explain some puzzling features of the Ca efflux such as the variable Na_o-dependence.     

Effect of sulfhydryl reagents on k efflux from rose cells: relationship to ultraviolet-stimulated efflux

N-Ethylmaleimide causes a rapid efflux of K⁺ from suspension-cultured cells of Rosa damascena. This efflux shows many characteristics of the ultraviolet-induced efflux of K⁺, including the appearance of HCO₃⁻ together with the K⁺ and inhibition by respiratory inhibitors. Cysteine inhibits the ultraviolet-induced efflux of K⁺. These results are interpreted to mean that ultraviolet induces K⁺ efflux through an alteration of sulfhydryl residues.     

EZN-2208 (PEG-SN38) Overcomes ABCG2-Mediated Topotecan Resistance in BRCA1-Deficient Mouse Mammary Tumors

BRCA1 dysfunction in hereditary breast cancer causes defective homology-directed DNA repair and sensitivity towards DNA damaging agents like the clinically used topoisomerase I inhibitors topotecan and irinotecan. Using our conditional K14cre;Brca1^F/F;p53^F/F mouse model, we showed previously that BRCA1;p53-deficient mammary tumors initially respond to topotecan, but frequently acquire resistance by overexpression of the efflux transporter ABCG2. Here, we tested the pegylated SN38 compound EZN-2208 as a novel approach to treat BRCA1-mutated tumors that express ABCG2. We found that EZN-2208 therapy resulted in more pronounced and durable responses of ABCG2-positive tumors than topotecan or irinotecan therapy. We also evaluated tumor-specific ABCG2 inhibition by Ko143 in Abcg2^−/− host animals that carried tumors with topotecan-induced ABCG2 expression. Addition of Ko143 moderately increased overall survival of these animals, but did not yield tumor responses like those seen after EZN-2208 therapy. Our results suggest that pegylation of Top1 inhibitors may be a useful strategy to circumvent efflux transporter-mediated resistance and to improve their efficacy in the clinic.