De-energized Cells Research Articles

Interaction of ethidium and tetraphenylphosphonium cations with Salmonella enterica cells

Background and objectiveOne of the main causes of bacterial resistance to antimicrobials is multidrug resistance induced by the increased efficiency of the efflux pumps. In this study we analyzed how the conditions of assay affect the efflux of indicator substrates ethidium (Et+) and tetraphenylphosphonium (TPP+) in Salmonella enterica ser. Typhimurium cells. Impact of the outer membrane permeability barrier, composition and temperature of the medium on accumulation of the indicator compounds also was analyzed. Materials and methodsThe fluorescence of Et+ and Nile Red was measured using 96-well plates and a plate reader. In parallel to traditional studies of fluorescence we applied a constructed selective electrode to follow the accumulation of Et+ in S. enterica cells. Simultaneously with monitoring of Et+ concentration in the cell incubation medium, electrochemical measurements of TPP+ accumulation were performed. Furthermore, Et+ and TPP+ were used within the same sample as agents competing for the interaction with the efflux pumps. An inhibitor phenylalanyl-arginyl-β-naphtylamide (PAβN) was applied to evaluate the input of RND-family pumps in the total efflux of these indicator compounds. ResultsS. enterica cells with the intact outer membrane (OM) bound very low amounts of Et+ or TPP+. Cells with the permeabilized OM accumulate considerably higher amounts of the indicator compounds at pH 8.0, but only Et+ was considerably accumulated at pH 6.5. At conditions of electrochemical monitoring accumulation of Et+ by the permeabilized cells at 37°C was considerably faster than at 23°C, but at the higher temperature most of the cell-accumulated Et+ was extruded back to the medium. The fluorescence of Et+ in suspension of cells incubated in 400mmol/L Tris buffer was about twice higher compared to 100mmol/L one. The inhibitory action of TPP+ on Et+ efflux was evident only in 400mmol/L Tris although PAβN effectively increased Et+ fluorescence at both buffer concentrations. ConclusionsResults of our experiments indicate that ionic strength of the incubation medium influence the selectivity, the medium temperature and the assay conditions impact the kinetics of efflux. The lower accumulated amount and the weaker fluorescence of Et+ registered in slightly acidic medium indicate that ΔΨ plays a role in the accumulation of this indicator cation. The bound amount of Et+ to the de-energized or permeabilized cells considerably varies depending on the conditions and methods of de-energization or permeabilization of cells. Tris/EDTA permeabilization of the cells does not inhibit the efflux.

Azole Drugs Are Imported By Facilitated Diffusion in Candida albicans and Other Pathogenic Fungi

Despite the wealth of knowledge regarding the mechanisms of action and the mechanisms of resistance to azole antifungals, very little is known about how the azoles are imported into pathogenic fungal cells. Here the in-vitro accumulation and import of Fluconazole (FLC) was examined in the pathogenic fungus, Candida albicans. In energized cells, FLC accumulation correlates inversely with expression of ATP-dependent efflux pumps. In de-energized cells, all strains accumulate FLC, suggesting that FLC import is not ATP-dependent. The kinetics of import in de-energized cells displays saturation kinetics with a Km of 0.64 uM and Vmax of 0.0056 pmol/min/108 cells, demonstrating that FLC import proceeds via facilitated diffusion through a transporter rather than passive diffusion. Other azoles inhibit FLC import on a mole/mole basis, suggesting that all azoles utilize the same facilitated diffusion mechanism. An analysis of related compounds indicates that competition for azole import depends on an aromatic ring and an imidazole or triazole ring together in one molecule. Import of FLC by facilitated diffusion is observed in other fungi, including Cryptococcus neoformans, Saccharomyces cerevisiae, and Candida krusei, indicating that the mechanism of transport is conserved among fungal species. FLC import was shown to vary among Candida albicans resistant clinical isolates, suggesting that altered facilitated diffusion may be a previously uncharacterized mechanism of resistance to azole drugs.

Salivary histatin 5 internalization by translocation, but not endocytosis, is required for fungicidal activity in Candida albicans

Salivary histatin 5 (Hst 5) is a cationic salivary protein with high fungicidal activity against Candida albicans. Binding to the cell wall followed by intracellular translocation is required for killing; however, specific binding components and critical toxic events are not understood. In this study, laminarin (β-1,3-glucan) but not sialic acid, mannan or pustulan mediated Hst 5 binding to C. albicans, and was disassociated by 100 mM NaCl. Time-lapse confocal microscopy revealed a dose-dependent rate of cytosolic uptake of Hst 5 that invariably preceded propidium iodide (PI) entry, demonstrating that translocation itself does not disrupt membrane integrity. Cell toxicity was manifest by vacuolar expansion followed by PI entrance; however, loss of endocytotic vacuolar trafficking of Hst 5 did not reduce killing. Extracellular NaCl (100 mM), but not sorbitol, prevented vacuolar expansion and PI entry in cells already containing cytosolic Hst 5, thus showing a critical role for ionic balance in Hst 5 toxicity. Hst 5 uptake, but not cell wall binding, was blocked by pretreatment with azide or carbonyl cyanide m-chlorophenylhydrazone; however, 10% of de-energized cells had membrane disruption. Thus, Hst 5 is capable of heterogeneous intracellular entry routes, but only direct cytosolic translocation causes cell death as a result of ionic efflux.

Multiple physiological states of a Pseudomonas fluorescens DR54 biocontrol inoculant monitored by a new flow cytometry protocol

A new fluorescence staining and flow cytometry protocol was developed to monitor several physiological states in biocontrol strain Pseudomonas fluorescens DR54 during storage survival in a stationary-phase culture, preparation of clay carrier for seed formulation, and establishment in a sugar beet spermosphere. The high load of impurities in the environmental samples was dealt with by adding a density-gradient purification step to the staining protocol. Staining by SYBR Green, combined with either propidium iodide or ethidium bromide (EB)+DiBAC((4))3, was used to quantify the total cell population and further divide this population into: (1) intact cells with an unaffected membrane and energy metabolism. (2) De-energized cells unable to maintain membrane export (EB exclusion). (3) Depolarized cells unable to maintain membrane potential. (4) Permeabilized cells with a damaged membrane. During both stationary-phase storage and steps for preparation of formulation carrier, loss of intact P. fluorescens DR54 cells was quantitatively accounted for by depolarized and permeabilized states. Surviving inoculum cells subsequently proliferated on the germinating seeds, but with a surprisingly high abundance of de-energized cells. The new protocol is the first for flow cytometry to include a recording of both intact and several subpopulations of physiologically affected bacteria in complex, environmental samples with high impurity loads.

Transport of glycerol by Pediococcus pentosaceus isolated from wine

Pediococcus pentosaceus N 5p is a strain isolated from wine that uses glycerol as its sole carbon source, mainly via the glycerol kinase pathway. The transport of glycerol was investigated in resting cells of this strain. Glycerol uptake followed a Michaelis–Menten relationship with an observed apparent K m of 33 μM and a V max of 2.5 nmol/min/mg of cell protein. The transport system was specific for glycerol, which was present in the cells grown either on glycerol or glucose suggesting its constitutive nature. The presence of uptake when resting cells were treated with HgCl 2 and the absence of counterflow indicate that facilitated diffusion is not involved in glycerol transport. On the other hand, glycerol uptake was inhibited by the metabolic poisons that affect ATP availability by acting on either electron transport or ATPase activity, and by the proton-conducting uncouplers without any effect on glycerol kinase activity. The restoration of glycerol uptake in de-energized cells by the addition of glucose and low concentration of cyanide- m-chlorophenyl hydrazone was achieved. These results, the first in the genus Pediococcus, provide evidence for an energy-dependent uptake of glycerol that involves the proton motive force directly or coupled with ATP synthesis.

BspA (CyuC) in Lactobacillus fermentum BR11 Is a Highly Expressed High-Affinity L-Cystine-Binding Protein

The BspA protein of Lactobacillus fermentum BR11 (BR11) is a cell envelope constituent that is similar to known solute-binding proteins and putative adhesins. BspA is required for L-cystine uptake and oxidative defense and is likely to be an L-cystine-binding protein. The aim of this study was to directly measure L-cystine-BspA binding and BspA expression. De-energized BR11 cells bound radiolabelled L-cystine with a Kd of 0.2 microM. A bspA mutant could not bind L-cystine. L-cystine-BR11 binding was unaffected by large excesses of L-glutamine, L-methionine, or collagen, indicating L-cystine specificity. BR11 and the bspA mutant were identical in their abilities to bind L-cysteine, indicating that L-cysteine is not a BspA ligand. BspA expression levels were deduced from radiolabelled L-cystine binding and it was found that there are 1-2 x 10(5) BspA molecules per cell, and that expression is slightly higher under oxidizing conditions. It is proposed that BspA be renamed CyuC.

Metabolism of fructooligosaccharides by Lactobacillus paracasei 1195.

Fermentation of fructooligosaccharides (FOS) and other oligosaccharides has been suggested to be an important property for the selection of bacterial strains used as probiotics. However, little information is available on FOS transport and metabolism by lactic acid bacteria and other probiotic bacteria. The objectives of this research were to identify and characterize the FOS transport system of Lactobacillus paracasei 1195. Radiolabeled FOS was synthesized enzymatically from [(3)H]sucrose and purified by column and thin-layer chromatography, yielding three main products: glucose (G) alpha-1,2 linked to two, three, or four fructose (F) units (GF(2), GF(3), and GF(4), respectively). FOS hydrolysis activity was detected only in cell extracts prepared from FOS- or sucrose-grown cells and was absent in cell supernatants, indicating that transport must precede hydrolysis. FOS transport assays revealed that the uptake of GF(2) and GF(3) was rapid, whereas little GF(4) uptake occurred. Competition experiments showed that glucose, fructose, and sucrose reduced FOS uptake but that other mono-, di-, and trisaccharides were less inhibitory. When cells were treated with sodium fluoride, iodoacetic acid, or other metabolic inhibitors, FOS transport rates were reduced by up to 60%; however, ionophores that abolished the proton motive force only slightly decreased FOS transport. In contrast, uptake was inhibited by ortho-vanadate, an inhibitor of ATP-binding cassette transport systems. De-energized cells had low intracellular ATP concentrations and had a reduced capacity to accumulate FOS. These results suggest that FOS transport in L. paracasei 1195 is mediated by an ATP-dependent transport system having specificity for a narrow range of substrates.

Rhamnolipid stimulates uptake of hydrophobic compounds by Pseudomonas aeruginosa.

The biodegradation of hexadecane by five biosurfactant-producing bacterial strains (Pseudomonas aeruginosa UG2, Acinetobacter calcoaceticus RAG1, Rhodococcus erythropolis DSM 43066, R. erythropolis ATCC 19558, and strain BCG112) was determined in the presence and absence of exogenously added biosurfactants. The degradation of hexadecane by P. aeruginosa was stimulated only by the rhamnolipid biosurfactant produced by the same organism. This rhamnolipid did not stimulate the biodegradation of hexadecane by the four other strains to the same extent, nor was degradation of hexadecane by these strains stimulated by addition of their own biosurfactants. This suggests that P. aeruginosa has a mode of hexadecane uptake different from those of the other organisms. Rhamnolipid also enhanced the rate of epoxidation of the aliphatic hydrocarbon alpha,omega-tetradecadiene by a cell suspension of P. aeruginosa. Furthermore, the uptake of the hydrophobic probe 1-naphthylphenylamine by cells of P. aeruginosa was enhanced by rhamnolipid, as indicated by stopped-flow fluorescence experiments. Rhamnolipid did not stimulate the uptake rate of this probe in de-energized cells. These results indicate that an energy-dependent system is present in P. aeruginosa strain UG2 that mediates fast uptake of hydrophobic compounds in the presence of rhamnolipid.

PDR16 and PDR17, Two Homologous Genes ofSaccharomyces cerevisiae, Affect Lipid Biosynthesis and Resistance to Multiple Drugs

The Saccharomyces cerevisiae open reading frame YNL231C was recently found to be controlled by the multiple drug resistance regulator Pdr1p. Here we characterize YNL231C (PDR16) and its homologue YNL264C (PDR17). Deletion of PDR16 resulted in hypersensitivity of yeast to azole inhibitors of ergosterol biosynthesis. While no increase in drug sensitivity was found upon deletion of PDR17 alone, a Deltapdr16,Deltapdr17 double mutant was hypersensitive to a broad range of drugs. Both mutations caused significant changes of the lipid composition of plasma membrane and total cell extracts. Deletion of PDR16 had pronounced effects on the sterol composition, whereas PDR17 deletion mainly affected the phospholipid composition. Thus, Pdr16p and Pdr17p may regulate yeast lipid synthesis like their distant homologue, Sec14p. The azole sensitivity of the PDR16-deleted strain may be the result of imbalanced ergosterol synthesis. Impaired plasma membrane barrier function resulting from a change in the lipid composition appears to cause the increased drug sensitivity of the double mutant strain Deltapdr16,Deltapdr17. The uptake rate of rhodamine-6-G into de-energized cells was shown to be almost 2-fold increased in a Deltapdr16,Deltapdr17 strain as compared with wild-type and Deltapdr5 strains. Collectively, our results indicate that PDR16 and PDR17 control levels of various lipids in various compartments of the cell and thereby provide a mechanism for multidrug resistance unrecognized so far.

Listeria monocytogenes Scott A transports glucose by high-affinity and low-affinity glucose transport systems.

Listeria monocytogenes transported glucose by a high-affinity phosphoenolpyruvate-dependent phosphotransferase system and a low-affinity proton motive force-mediated system. The low-affinity system (Km = 2.9 mM) was inhibited by 2-deoxyglucose and 6-deoxyglucose, whereas the high-affinity system (Km = 0.11 mM) was inhibited by 2-deoxyglucose and mannose but not 6-deoxyglucose. Cells and vesicles artificially energized with valinomycin transported glucose or 2-deoxyglucose at rates greater than those of de-energized cells, indicating that a membrane potential could drive uptake by the low-affinity system.

Lactic acid excretion by Streptococcus mutans.

Lactic acid is the major end-product of glycolysis by Streptococcus mutans under conditions of sugar excess or low environmental pH. However, the mechanism of lactic acid excretion by S. mutans is unknown. To characterize lactic acid efflux in S. mutans the transmembrane movement of radiolabelled lactate was monitored in de-energized cells. Lactate was found to equilibrate across the membrane in accordance with artificially imposed transmembrane pH gradient (Δψ). The imposition of a transmembrane electrical potential (Δψ) upon de-energized cells did not cause an accumulation of lactate within the cell. The efflux of lactate from lactate-loaded, deenergized cells created a ΔpH, but did not create a Δψ, indicating that lactate crosses the cell membrane in an electroneutral process, as lactic acid. ΔpH and Δψ were determined by the transmembrane equilibration of [14C]benzoic acid and [14C]tetraphenylphosphonium ion (TPP), respectively. The presence of a membrane carrier for lactic acid in S. mutans was suggested by counterflow. Enzymic determination of the intra- and extracellular lactate concentrations of S. mutans cells glycolysing at pHo 6.8 and 5.5 showed that lactate distributed across the cell membrane in accordance with the equation ΔpH = log[lact]i/[lact]o. The addition of high extracellular concentrations of lactate to glycolysing S. mutans at acidic pH resulted in a fall in ΔpH and a subsequent decrease in glycolysis. The fall in ΔpH was attributed to the F1F0 ATPase being unable to raise the pHi back to its initial level due to the build up of lactate anion within the cell creating a large Δψ. The increase in Δψ resulted in the overall proton motive force remaining constant at about -110 mV. The results demonstrate that lactate is transported across the cell membrane of S. mutans as lactic acid in an electroneutral process that is independent of metabolic energy and as such has important bioenergetic implications for the cell.

Characterization of glutamine transport in Streptococcus mutans.

Glutamine transport in glucose-energized cells of Streptococcus mutans Ingbritt exhibited Michaelis-Menten-type kinetics with a Vmax of 13.4 nmol/mg dry weight/min and a Kt of 4.1 microM. Diffusion of glutamine into de-energized cells of S. mutans displayed similar type kinetics, with a Kt of 6.8 microM but with a markedly reduced Vmax of 53.9 pmol/mg dry weight/min. Glutamine transport in S. mutans is not proton motive force-driven, as the intracellular accumulation of glutamine by energized cells far exceeded the thermodynamic limits of the proton motive force, and the dissipation of this proton motive force by gramicidin in a high K+ medium did not decrease the intracellular glutamine concentration. Glutamine transport is therefore likely to be energized by ATP hydrolysis. The activity of the transporter was maximal between pH 6.0 and 7.0 and decreased rapidly above pH 7.0. The transport of glutamine was not competitively inhibited by asparagine, glutamate or aspartate, indicating a specific glutamine transport system. Reversed-phase high-pressure liquid chromatography of cell extracts revealed that approximately 26% of the glutamine taken into the cell was converted to glutamate within 10 min. The results are consistent with transported glutamine being converted to glutamate and ammonia by the action of an intracellular glutaminase. Glutamine therefore may be an important source of nitrogen for the cell.

Na + and K + transport by 4-chlorophenylurethane-monensin in Enterococcus hirae de-energized and energized cells studied by 23Na-NMR and K + atomic absorption

Na + and K + movements induced by 4-chlorophenylurethane-monensin, which presents an inverted ion selectivity ( K + > Na + ) in model systems compared with monensin, were followed on Enterococcus hirae cells by 23Na-NMR and K + atomic absorption. For de-energized cells, the urethane derivative is much more selective for K + than monensin, but only at low concentrations (10 −3–10 −4 mM). For higher concentrations, as previously shown for monensin, the sodium and potassium movements are driven by the ion gradients present. On energized cells, both K + and Na + gradients were highly perturbed, and this can be related to the higher toxicity in mice and bacteria for this derivative.

Conditions modulating the ionic selectivity of transport by monensin examined on Enterococcus hirae (Streptococcus faecalis) by 23Na-NMR and K + atomic absorption

Factors likely to modulate the ionic selectivity of monensin were examined on Enterococcus hirae (Streptococcus faecalis) in two states previously characterized: the resting (de-energized) cell and the active (energized) cell. Internal and external Na + were followed by corresponding 23Na-NMR resonances, K + concentrations were measured by atomic absorption. For a given cellular population of de-energized cells, the apparent transport rates and the final cationic concentrations reached at the steady state were decreasing with the ionophore dose. Monensin was selective for sodium only at low concentrations, in the range 1 mM-10 −4 mM the transport was depending on the effective cationic gradients. Comparison of the activity curves for two cell populations (7·10 9 and 7·10 10 cells/ml) showed the importance of the ratios of monensin/mg phospholipid and also of the ratios of external/internal volumes. On energized cells, except for low monensin concentrations, the main effect was a K +-induced efflux and not a Na + influx. Two factors were modulating the resulting selectivity of this ionophore: the response of the intrinsic bacterial carriers and the generation of the gradients (mainly the external pH) which were favourable to a K +/Na + transport. Once again the results obtained for two cell populations could be compared, the determining factors were the ratio external/internal volume and the generation of the pH gradient.

Monensin-induced modifications of ionic gradients on Streptococcus faecalis. A 23Na and 31P NMR study

The study of Na+ (23Na NMR), H+ (31P NMR) and K+ (atomic absorption) movements was performed on Streptococcus faecalis cells, with or without monensin addition. Our approach with energized or de-energized cells allowed the distinction between the ionophore effect and the bacterial carriers response. At high monensin concentration the intrinsic systems of the bacteria were unable to re-establish normal ionic gradients. The high selectivity of transport of the ionophore for Na+ / K+ as measured in model physico-chemical sytems, was not determining “in vivo” as the most important damage caused to the bacteria was a large K+ efflux.

Phosphate-dependent sodium transport in S. faecalis investigated by 23Na and 31P NMR

Na+ movements in S. faecalis were studied by 23Na NMR. They proved to be dependent on phosphate concentration in the buffer during the de-energization step. K+ and H+ were also studied respectively by potentiometry and 31P NMR and were shown not to be implicated. For de-energized cells the internal phosphate concentration, on the contrary, was directly linked to the external phosphate contained in the buffer. The experiments showed a Na+/Pi dependence in this prokaryote so far known only in eukaryotes.

Electrogenic L-malate transport by Lactobacillus plantarum: a basis for energy derivation from malolactic fermentation

L-Malate transport in Lactobacillus plantarum was inducible, and the pH optimum was 4.5. Malate uptake could be driven by an artificial proton gradient (delta pH) or an electroneutral lactate efflux. Because L-lactate efflux was unable to drive L-malate transport in the absence of a delta pH, it did not appear that the carrier was a malate-lactate exchanger. The kinetics of malate transport were, however, biphasic, suggesting that the external malate concentration was also serving as a driving force for low-affinity malate uptake. Because the electrical potential (delta psi, inside negative) inhibited malate transport, it appeared that the malate transport-lactate efflux couple was electrogenic (net negative) at high concentrations of malate. De-energized cells that were provided with malate only generated a large proton motive force (greater than 100 mV) when the malate concentration was greater than 5 mM, and malate only caused an increase in cell yield (glucose-limited chemostats) when malate accumulated in the culture vessel. The use of the malate gradient to drive malate transport (facilitated diffusion) explains how L. plantarum derives energy from malolactic fermentation, a process which does not involve substrate-level phosphorylation.

Study by NMR of the mode of action of monensin on Streptococcus faecalis de-energized and energized cells

Streptococcus faecalis was used as a bacterial model for studying the mode of action of monensin by NMR investigations. Experiments were carried out in two states, characterized by several complementary methods: (i) the resting (de-energized) cell which was considered as an inert biological membrane, on which cationic transport induced by the ionophore alone can be investigated; (ii) the active (energized) cell where the ionophore-sensitive response of the living organism, particularly the cation pumps and the glycolysis, is probed. Studies of resting cells were performed, with changing external ionic concentrations, in the presence of monensin, which is preferentially a sodium carrier. Internal and external Na + and H + were followed by corresponding 23Na and 31P 9inorganic phosphate) NMR resonances, K + fluxes were measured by atomic absorption. It was shown that the induced cationic movements were linked to the existing ionic gradients for K + and Na +. 31P and 13C NMR spectra for the intermediary metabolites detected in active cells showed that glycolysis is dramatically modified in the presence of monensin.

Transport of amino acids in Lactobacillus casei by proton-motive-force-dependent and non-proton-motive-force-dependent mechanisms

Lactobacillus casei 393 cells which were energized with glucose (pH 6.0) took up glutamine, asparagine, glutamate, aspartate, leucine, and phenylalanine. Little or no uptake of several essential amino acids (valine, isoleucine, arginine, cysteine, tyrosine, and tryptophan) was observed. Inhibition studies indicated that there were at least five amino acid carriers, for glutamine, asparagine, glutamate/aspartate, phenylalanine, or branched-chain amino acids. Transport activities had pH optima between 5.5 and 6.0, but all amino acid carriers showed significant activity even at pH 4.0. Leucine and phenylalanine transport decreased markedly when the pH was increased to 7.5. Inhibitors which decreased proton motive force (delta p) nearly eliminated leucine and phenylalanine uptake, and studies with de-energized cells and membrane vesicles showed that an artificial electrical potential (delta psi) of at least -100 mV was needed for rapid uptake. An artificial delta p was unable to drive glutamine, asparagine, or glutamate uptake, and transport of these amino acids was sensitive to a decline in intracellular pH. When intracellular pH was greater than 7.7, glutamine, asparagine, or glutamate was transported rapidly even though the proton motive force had been abolished by inhibitors.

Mechanism and energetics of dipeptide transport in membrane vesicles of Lactococcus lactis

Alanyl-alpha-glutamate transport has been studied in Lactococcus lactis ML3 cells and in membrane vesicles fused with liposomes containing beefheart cytochrome c oxidase as a proton-motive-force-generating system. The uptake of Ala-Glu observed in de-energized cells can be stimulated 26-fold upon addition of lactose. No intracellular dipeptide pool could be detected in intact cells. In fused membranes, a 40-fold accumulation of Ala-Glu was observed in response to a proton motive force. Addition of ionophores and uncouplers resulted in a rapid efflux of the accumulated dipeptide, indicating that Ala-Glu accumulation is directly coupled to the proton motive force as a driving force. Ala-Glu uptake is an electrogenic process and the dipeptide is transported in symport with two protons. In both fused membranes and intact cells the same affinity constant (0.70 mM) for Ala-Glu uptake was found. Accumulated Ala-Glu is exchangeable with externally added alanyl-glutamate, glutamyl-glutamate, and leucyl-leucine, while no exchange occurred upon addition of the amino acid glutamate or alanine. These results indicate that the Ala-Glu transport system has a broad substrate specificity.