Energy-dependent Efflux System Research Articles

Mathematical Modelling of Toxic Metal Uptake and Efflux Pump in Metal-Resistant Bacterium Bacillus cereus Isolated From Heavy Crude Oil

The aim of this study was to describe the mechanisms that native Bacillus cereus M6 isolated from heavy crude oil oAPI gravity 11.5 uses to tolerate and/or resist toxic metals. Metal tolerance and removal of Pb(II), Cr(VI), and As(V) was determined. In addition, we evidenced the subcellular distribution of metals, the efflux pump kinetics, and morphological changes in metal-tolerant bacteria. B. cereus M6 exhibited strong tolerance and resistance to the metals evaluated and efficiently removed the metal content by operating efflux pumps and accumulating mainly in membrane fraction. Also, it was found that the model that best fit the efflux corresponds to an equation for resonant oscillations. B. cereus M6 uses mechanisms, including efflux pumps, intracellular and extracellular accumulation in parallel in order to maintain metal levels below a toxic threshold and overcome the effects of high concentrations. These findings are an approach of an energy-dependent efflux system to eliminate excessive amounts of crude oil-associated metals in Bacillus. B. cereus M6 may potentially be useful in designing improved strategies for the bioremediation of soils polluted with metals. Additionally, the prediction model developed would be useful for improving the monitoring of in vitro and in vivo bioremediation processes.

Photodamage to Multidrug‐resistant Gram‐positive and Gram‐negative Bacteria by 870 nm/930 nm Light Potentiates Erythromycin, Tetracycline and Ciprofloxacin

We have previously shown that 870 nm/930 nm wavelengths cause photodamage at physiologic temperatures in methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli via generation of endogenous radical oxygen species (ROS) and decreased plasma membrane potentials (Delta Psi p). We tested MRSA (Strain HSJ216) in vitro with sublethal 870 nm/930 nm laser energy and subinhibitory concentrations of erythromycin, tetracycline, penicillin, rifampin and trimethoprim to surmise whether photodamage could potentiate these antimicrobials. We also tested patient isolates of fluoroquinolone-resistant MRSA and E. coli with subinhibitory concentrations of ciprofloxacin. In MRSA (Strain HSJ216) we observed 97% potentiation (a 1.5 log(10) CFU decrease) with erythromycin and tetracycline. In patient isolates of E. coli, we observed 100% potentiation (>3 log(10) CFU decrease) in all irradiated samples with ciprofloxacin. To assess whether staphyloxanthin pigment conferred protection against the generated ROS, we created an isogenic carotenoid-deficient mutant of S. aureus that was significantly less tolerant of 870 nm/930 nm exposure than the wild type strain (P < 0.0001). We suggest that antibiotic potentiation results from a photobiological attenuation of ATP-dependent macromolecular synthetic pathways, similar to that observed with daptomycin, via disruption of Delta Psi p and endogenous generation of ROS. With erythromycin, tetracycline and ciprofloxacin, attenuation of energy-dependent efflux systems is also a possibility.

VmeAB, an RND-type multidrug efflux transporter in Vibrio parahaemolyticus

Genes vmeA and vmeB, encoding a multidrug efflux transporter in the halophilic bacterium Vibrio parahaemolyticus, have been cloned using a drug-hypersusceptible Escherichia coli strain as the host. Cells of E. coli KAM33 (DeltaacrAB DeltaydhE) carrying the vmeAB region from V. parahaemolyticus conferred much higher MICs for a variety of antimicrobial agents than did control cells. Cells possessing VmeAB under energized conditions maintained very low intracellular concentrations of ethidium. This was as expected for an energy-dependent efflux system, and supports the notion--based on sequence homology--that VmeAB belongs to the resistance nodulation cell division (RND) family of multidrug efflux transporters. It is likely that VmeAB forms functional complexes with the outer-membrane protein TolC in E. coli, because introduction of vmeAB into cells of E. coli KAM43, which lacks the tolC gene, failed to elevate the MICs for any of the antimicrobial agents tested. Therefore, a V. parahaemolyticus homologue of tolC was also cloned, designated vpoC, and was introduced together with vmeAB into cells of E. coli KAM43. The MICs of all agents tested were raised and were comparable to the values observed in E. coli KAM33 harbouring a plasmid carrying vmeAB. Finally, a vmeAB-deficient mutant of V. parahaemolyticus was constructed (designated TM3). TM3 showed slightly higher susceptibility than the parental V. parahaemolyticus to some antimicrobial agents. Survival rate of the TM3 when exposed to deoxycholate decreased compared with that of the parent.

Acetobacter aceti Possesses a Proton Motive Force-Dependent Efflux System for Acetic Acid

Acetic acid bacteria are obligate aerobes able to oxidize ethanol, sugar alcohols, and sugars into their corresponding acids. Among them, Acetobacter and Gluconacetobacter species have very high ethanol oxidation capacity, leading to accumulation of vast amounts of acetic acid outside the cell. Since these bacteria are able to grow in media with high concentrations of acetic acid, they must possess a specific mechanism such as an efflux pump by which they can resist the toxic effects of acetic acid. In this study, the efflux pump of Acetobacter aceti IFO 3283 was examined using intact cells and membrane vesicles. The accumulation of acetic acid/acetate in intact cells was increased by the addition of a proton uncoupler and/or cyanide, suggesting the presence of an energy-dependent efflux system. To confirm this, right-side-out and inside-out membrane vesicles were prepared from A. aceti IFO 3283, and the accumulation of acetic acid/acetate in the vesicles was examined. Upon the addition of a respiratory substrate, the accumulation of acetic acid/acetate in the right-side-out vesicles was largely decreased, while its accumulation was very much increased in the inside-out vesicles. These respiration-dependent phenomena observed in both types of membrane vesicles were all sensitive to a proton uncoupler. Acetic acid/acetate uptake in the inside-out membrane vesicles was dependent not on ATP but on the proton motive force. Furthermore, uptake was shown to be rather specific for acetic acid and to be pH dependent, because higher uptake was observed at lower pH. Thus, A. aceti IFO 3283 possesses a proton motive force-dependent efflux pump for acetic acid.

Effect of bile on the cell surface permeability barrier and efflux system of Vibrio cholerae.

Gram-negative bacteria are inherently impermeable to hydrophobic compounds, due to the synergistic activity of the permeability barrier imposed by the outer membrane and energy dependent efflux systems. The gram-negative, enteric pathogen Vibrio cholerae appears to be deficient in both these activities; the outer membrane is not an effective barrier to hydrophobic permeants, presumably due to the presence of exposed phospholipids on the outer leaflet of the outer membrane, and efflux systems are at best only partially active. When V. cholerae was grown in the presence of bile, entry of hydrophobic compounds into the cells was significantly reduced. No difference was detected in the extent of exposed phospholipids on the outer leaflet of the outer membrane between cells grown in the presence or absence of bile. However, in the presence of energy uncouplers, uptake of hydrophobic probes was comparable between cells grown in the presence or absence of bile, indicating that energy-dependent efflux processes may be involved in restricting the entry of hydrophobic permeants into bile grown cells. Indeed, an efflux system(s) is essential for survival of V. cholerae in the presence of bile. Expression of acrAB, encoding an RND family efflux pump, was significantly increased in V. cholerae cells grown in vitro in the presence of bile and also in cells grown in rabbit intestine.

Involvement of adaptive mechanism and an energy-dependent efflux system of Pseudomonas sp. LE2 counteracting insecticide lindane

An adaptive mechanism and efflux-transport system of Pseudomonas sp. LE2 counteracting organochlorine insecticide lindane were investigated to understand the survival mechanism of this microorganism in lindane-contaminated environment. Changes in membrane fluidity and fatty acid composition of Pseudomonas sp. LE2 cells grown on glucose either in the presence or absence of lindane were determined. Considerably high ratio of total saturated to unsaturated fatty acids in cells grown on glucose in the presence of lindane was observed as compared to that in cells grown on glucose in the absence of lindane. Lindane significantly affected in vitro the membrane of Pseudomonas sp. LE2 cells by decreasing the fluorescence polarization of membrane probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) inserted into cytoplasmic membrane. Less fluidizing effect of lindane on the membranes of cells that had adapted to lindane was observed. Lindane transport was assessed by incubating cell suspensions with [ 14C]lindane either in the presence or absence of sodium azide, a membrane transport inhibitor, and by measuring the radioactivity in cell pellets. No time-dependent and steady-state increase in the radioactivity of [ 14C]lindane without sodium azide treatment was observed in cell pellets, suggesting simple diffusion mechanism for lindane accumulation in cells. Significant increase in the radioactivity was observed in cell pellets by sodium azide treatment. Sodium azide inhibited the efflux of lindane by Pseudomonas sp. LE2. These results suggest the view that Pseudomonas sp. LE2 capable of growing in the presence of lindane is able to adapt to the fluidizing action of lindane by incorporating the saturated fatty acid into membrane lipids, and eliminate lindane from cells by an energy-dependent efflux mechanism.

Effect of hemorrhagic shock on apoptosis and energy-dependent efflux system in the brain.

Recent findings suggest that apoptosis, which contributes to neuronal damage after ischemic injury, may play a role in sequelae associated with severe blood loss. This study examined the effect of hemorrhage and resuscitation on the expression (in situ hybridization and computerized image analysis) of bcl-2 mRNA, which codes for a protein that inhibits apoptosis, and mdr1 mRNA, which codes for a glycoprotein marker for drug efflux from the brain. Anaesthetized rats were subjected to volume-controlled (15 mL/kg) hemorrhage followed by resuscitation with shed blood (BR) or nonresuscitated (NR); control animals had femoral artery cannulation only (SHAM). Following 24 hr blood loss, distinctly lower levels of bcl-2 gene expression were observed in dentate gyrus of NR rats (0.25 +/- 0.04) as compared to SHAM rats (0.52 +/- 0.07); suscitation with shed blood prevented this reduction (0.58 +/- 0.05). Similar results were observed in cortex, striatum, and hypothalamus. Also, mdr1 mRNA levels were significantly reduced in all brain areas of the NR group as compared to the BR and SHAM groups. The findings suggest that blood resuscitation suppressed apoptosis and protected against loss of energy-dependent efflux system in the brain in response to hemorrhage.

Physiological characterization of Pseudomonas putida DOT-T1E tolerance to p-hydroxybenzoate.

Pseudomonas putida DOT-T1E was isolated as a toluene-tolerant strain. We show that it is also able to grow on high concentrations (up to 17 g/liter [123 mM]) of p-hydroxybenzoate (4HBA). Tolerance to this aromatic carboxylic acid (up to 30 g/liter [217 mM]) is improved by preexposing the cells to low 4HBA concentrations; the adaptation process is caused by the substrate itself rather than by products resulting from its metabolism. The mechanisms of 4HBA tolerance seem to involve increased rigidity of the cell membrane as a result of a decrease in the cis/trans ratio of unsaturated fatty acids. In addition, energy-dependent efflux systems seem to operate in the exclusion of 4HBA from the cell membranes.

Intestinal Absorption Mechanisms of .BETA.-Lactam Antibiotics.

There are remarkable differences in oral bioavailabilities among β-lactam antibiotics, ranging from poorly absorbed to completely absorbed, despite that most of these drugs posses similar degrees of hydrophilicity. Many of the well-absorbed β-lactam antibiotics have been shown to be substrates for the transport system by which peptides are absorbed (proton-coulpled transporter, Pep Tl), whereas the poorly absorbed analogs are not. Although the proton-coupled transport system has the greatest influence on intestinal absorption of β-lactam antibiotics, passive diffusion contributes significantly to the total transport. Considerling that passive diffusion of some β-lactam antibiotics is fairly rapid, the low oral bioavailability of other related compound, like cefazolin, could seem inconsistent. Recently, it has been found that the mechanisms which induce secretory-oriented permeation of orally inactive β-lactam antibiotics are factors limiting intestinal absorption of such antibiotics. This energy-demanding effiux system is distinct from P-glycoprotein-mediated transport. It should be noted that as long as a β-lactam antibiotic is the substrate with a high affinity for this intestiinal effiux system, the efflux system can pump it out efficiently from cytosol, even if an absorptive peptide transport system on the brush-border membrane mediates their translocation from the lumen into cytosol. Previously, we reported that the well-absorbed β-lactam antibiotics bind to the cytosolic component, fraction b, extensively, whereas the orally inactive analogs do not bind to this fraction. Therefore, it may be possible that the well-absorbed β-lactam antibiotics have a way to escape the intestinal effiux system; for example, binding to the fraction b could prevent the well-absorbed β-lactam antibiotics from interacting with the energy-dependent efflux system, stimulating absorption of these antibiotics. The role of the binding factor, fraction b, for intestinal absorption mechanisms should be evaluated in more detail.

NorM, a putative multidrug efflux protein, of Vibrio parahaemolyticus and its homolog in Escherichia coli.

We found that cells of Vibrio parahaemolyticus possess an energy-dependent efflux system for norfloxacin. We cloned a gene for a putative norfloxacin efflux protein from the chromosomal DNA of V. parahaemolyticus by using an Escherichia coli mutant lacking the major multidrug efflux system AcrAB as the host and sequenced the gene (norM). Cells of E. coli transformed with a plasmid carrying the norM gene showed elevated energy-dependent efflux of norfloxacin. The transformants showed elevated resistance not only to norfloxacin and ciprofloxacin but also to the structurally unrelated compounds ethidium, kanamycin, and streptomycin. These results suggest that this is a multidrug efflux system. The hydropathy pattern of the deduced amino acid sequence of NorM suggested the presence of 12 transmembrane domains. The deduced primary structure of NorM showed 57% identity and 88% similarity with that of a hypothetical E. coli membrane protein, YdhE. No reported drug efflux protein in the sequence databases showed significant sequence similarity with NorM. Thus, NorM seems to be a novel type of multidrug efflux protein. We cloned the ydhE gene from E. coli. Cells of E. coli transformed with the cloned ydhE gene showed elevated resistance to norfloxacin, ciprofloxacin, acriflavine, and tetraphenylphosphonium ion, but not to ethidium, when MICs were measured. Thus, it seems that NorM and YdhE differ somehow in substrate specificity.

Modulation of Neisseria gonorrhoeae susceptibility to vertebrate antibacterial peptides due to a member of the resistance/nodulation/division efflux pump family.

We have previously described the antibacterial capacity of protegrin-1 (PG-1), a cysteine-rich, cationic peptide from porcine leukocytes, against Neisseria gonorrhoeae. We now report genetic and biochemical evidence that gonococcal susceptibility to the lethal action of PG-1 and other structurally unrelated antibacterial peptides, including a peptide (LL-37) that is expressed constitutively by human granulocytes and testis and inducibly by keratinocytes, is modulated by an energy-dependent efflux system termed mtr. These results indicate that such efflux systems may enable mucosal pathogens like gonococci to resist endogenous antimicrobial peptides that are thought to act during infection.

Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon.

Multidrug resistance in bacteria is generally attributed to the acquisition of multiple transposons and plasmids bearing genetic determinants for different mechanisms of resistance (48, 62). However, descriptions of intrinsic mechanisms that confer multidrug resistance have begun to emerge. The first of these was a chromosomally encoded multiple antibiotic resistance (mar) locus (Fig. 1) in Escherichia coli (45, 46). Mar mutants of E. coli arose at a frequency of 10 to 10 and were selected by growth on subinhibitory levels of tetracycline or chloramphenicol (45, 46). These mutants exhibited resistance to tetracyclines, chloramphenicol, penicillins, cephalosporins, puromycin, nalidixic acid, and rifampin (45). Later, the resistance phenotype was extended to include fluoroquinolones (25, 105), oxidative stress agents (7, 51), and, more recently, organic solvents (8, 49, 144). The expression of the Mar phenotype is greater at 30°C than 37°C (45, 127). Continued growth in the same or higher antibiotic concentrations led to increased levels of resistance, thus demonstrating an amplifiable multiple antibiotic resistance phenotype (45). Both highand low-level resistances were decreased or completely reversed by a Tn5 insertion into a single locus at 34 min (1,636.7 kb) on the E. coli chromosome, called the mar locus (46). The genetic basis for high-level resistance is only partially attributed to the mar locus, since transduction of the locus from highor low-level mar mutants produced only a low level of multidrug resistance (94). The mar locus consists of two divergently positioned transcriptional units that flank the operator marO (Fig. 1) in E. coli (22, 24, 112) and Salmonella typhimurium (133). One operon encodes MarC, a putative integral inner membrane protein (Fig. 1) without any yet apparent function, but which appears to contribute to the Mar phenotype in some strains (see below) (49, 143, 144). The other operon comprises marRAB, encoding the Mar repressor (MarR), which binds marO and negatively regulates expression of marRAB (22, 90, 127), an activator (MarA), which controls expression of other genes on the chromosome, e.g., the mar regulon (22, 41, 126), and a putative small protein (MarB) of unknown function (Fig. 1). The marRAB operon responds to a variety of compounds (7, 24, 52, 54, 99, 119, 127) including tetracycline and chloramphenicol (54). Deletion or inactivation of the marRAB operon results in increased susceptibility to multiple antibiotics, a variety of oxidative stress agents, and organic solvents (22, 24, 46, 51, 54, 144). Since salicylate and acetylsalicylate induced a reversible “phenotypic antibiotic resistance” to multiple antibiotics (118), a connection was made between phenotypic antibiotic resistance and mar (24); salicylate induced expression of marRAB (24). These studies also extended the spectrum of inducers to include acetaminophen, sodium benzoate, 2,4-dinitrophenol (an uncoupling agent), and cinnamate (a salicylate precursor in plants) (24). The uncoupling agent carbonyl cyanide m-chlorophenylhydrazone and redox-cycling compounds, menadione and plumbagin, are also inducers of marRAB transcription (127). Initially it was thought that 7.8 kb of chromosomal DNA was needed to generate constitutive mar mutants (mar) in a strain bearing a large (39 kb) chromosomal deletion (54). However, this finding was shown to be strain specific, and ;1.1 kb of mar sequence, containing marO, marR, and marA sequences, was sufficient to select a mar mutant (91, 134). mar mediates tetracycline resistance through an energy-dependent efflux system (45). Mar mutants are resistant to fluoroquinolones through a combined decrease in cell influx, e.g., a decrease in the porin OmpF, and an intrinsic efflux system (25). Chloramphenicol resistance in Mar mutants is also attributed to active efflux, which is enhanced over an intrinsic efflux system (96). The Mar phenotype is linked to overexpression of the acrAB locus; deletion of acrAB confers increased susceptibility to multiple drugs (85, 106) and organic solvents (144) in wild-type or Mar strains (85, 106, 144). These findings suggest that the acrAB efflux system is a major mechanism of Mar-mediated resistance (106). However, since the tetracycline (45) and fluoroquinolone (26) efflux systems were only saturated by the respective drug and not by others, it is probable that other drug-specific efflux systems are involved, particularly in high-level multidrug-resistant mutants.

Restricted intestinal absorption of some beta-lactam antibiotics by an energy-dependent efflux system in rat intestine.

The purpose of this study was to examine factors limiting the intestinal absorption of orally inactive beta-lactam antibiotics. Permeation behaviors of various beta-lactam antibiotics across rat intestinal segments were evaluated in vitro using diffusion cells. Poorly absorbed beta-lactam antibiotics, like cephaloridine and cefoperazone, commonly exhibit greater serosal-to-mucosal permeation than mucosal-to-serosal permeation, while cephalexin permeation was greater in the mucosal-to-serosal direction. In the absence of D-glucose, secretory-oriented permeation of cephaloridine and cefoperazone disappeared. Addition of sodium azide into an experimental buffer including D-glucose significantly and selectively enhanced mucosal-to-serosal permeation of cephaloridine and cefoperazone. Although benzylpenicillin, ampicillin, and amoxicillin all showed secretory-oriented permeation, the tendency to permeation was greatest with benzylpenicillin and least with amoxicillin. Probenecid stimulated mucosal-to-serosal permeation of cephaloridine, but verapamil and p-aminohippuric acid had no significant effect on it. It has been suggested that mechanisms which induce secretory-oriented permeation of orally inactive beta-lactam antibiotics are factors limiting intestinal absorption of such antibiotics. This energy-demanding efflux system was distinct from P-glycoprotein-mediated transport. A free alpha-amino group in the molecule is an important factor for reducing an affinity with the efflux system.

Characteristics of an ethidium efflux system inEnterococcus hirae

An energy-dependent efflux system for ethidium and related cations has been detected in Enterococcus hirae ATCC 9790. The system was partially expressed when the organism was grown on a complex medium but was induced by the addition of phosphonium ions and related compounds. Mutants showing constitutive expression of the efflux system have been isolated on the basis of increased resistance to ethidium.

Characteristics of an ethidium efflux system in Enterococcus hirae

An energy-dependent efflux system for ethidium and related cations has been detected in Enterococcus hirae ATCC 9790. The system was partially expressed when the organism was grown on a complex medium but was induced by the addition of phosphonium ions and realated compounds. Mutants showing constitutive expression of the efflux system have been isolated on the basis of increased resistance to ethidium.

The accumulation of fluoroquinolones in Staphylococcus aureus during the postantibiotic effect.

The accumulation of radiolabelled pefloxacin, norfloxacin, and lomefloxacin was determined in Staphylococcus aureus during log phase growth, the postantibiotic effect (PAE), and at 1, 4 and 6 h post PAE. Uptake in actively growing cells was consistent with a non-saturable passive diffusion process. The addition of metabolic inhibitors significantly increased accumulation consistent with the presence of an energy dependent efflux system. Accumulation of all three fluoroquinolones in PAE phase cells was significantly increased compared to that which occurred in actively growing cells. In contrast to cells in the log phase, addition of metabolic inhibitors to PAE phase cells did not result in a significant increase in drug accumulation. Uptake kinetics 1 h and 4 h post PAE were similar to those found during the PAE phase. Organisms required 6 h after the end of the PAE phase before normal uptake kinetics of fluoroquinolones resumed.

P-Glycoprotein expression in malignant melanoma

In some human malignancies resistance to chemotherapy is caused by an energy-dependent efflux system, responsible for the removal of chemotherapeutics out of the resistant tumor cells. A major component of this efflux system is the permeability glycoprotein (p-glycoprotein), which depends on the multidrug-resistance gene MDR1. We have tested p-glycoprotein in primary and metastatic human melanoma by use of the monoclonal antibody C219; a substantial expression was only observed in 1/37 primary melanomas and in 1/27 melanoma metastases. None of the patients with negative metastases responded to chemotherapy. Moreover a complete remission of metastatic growth was observed in the patient with the metastasis significantly expressing the p-glycoprotein. Sequential studies revealed no significant increase of p-glycoprotein-positive cells during and after chemotherapy. We conclude that drug resistance in human melanoma does not usually depend on the p-glycoprotein-related efflux system. Other mechanisms are obviously responsible for drug resistance in this human malignancy.

Amplifiable resistance to tetracycline, chloramphenicol, and other antibiotics in Escherichia coli: involvement of a non-plasmid-determined efflux of tetracycline.

Increasing levels of resistance to tetracycline and to a number of other unrelated antibiotics, including chloramphenicol, beta-lactams, puromycin, and nalidixic acid, occurred in Escherichia coli after 50 to 200 generations of growth in the presence of subinhibitory concentrations of tetracycline or chloramphenicol. In the absence of selective pressure, resistances fell to low levels within 100 generations of growth. This amplification of resistance was observed in laboratory and naturally occurring E. coli strains as well as in polA and recA strains. With the exception of previously identified cmlA and cmlB mutations, tetracycline or chloramphenicol resistances were not P1 transducible. Coincident with the emergence of resistance was the appearance of a previously cryptic energy-dependent efflux system for tetracycline. The expression of resistance phenotypes and the tetracycline efflux system were temperature sensitive at 42 degrees C.