Role of calcium in the efflux system of Escherichia coli

https://doi.org/10.1016/j.ijantimicag.2011.01.010Get rights and content

Abstract

Efflux of antibiotics by Escherichia coli AG100 is performed by a variety of efflux pumps, ensuring survival of the bacterium in widely diverse media. At pH 5, efflux is independent of metabolic energy during the period of time the assay is conducted; at pH 8 it is totally dependent upon metabolic energy. Because calcium ions (Ca2+) are important for membrane transport channels and the activity of ATPases that provide energy functions, the role of Ca2+ in the extrusion of an efflux pump substrate under conditions that challenge the bacterium was investigated. Real-time accumulation and efflux of ethidium bromide (EtBr) by E. coli K-12 AG100 strain [argE3 thi-1 rpsL xyl mtl Δ(gal-uvrB) supE44] was determined by a semi-automated fluorometric method in the presence and absence of Ca2+ and agents that are known to inhibit access of calcium to enzymes that provide energy. Chlorpromazine (CPZ), an inhibitor of calcium binding to proteins (calcium-dependent enzymes), and ethylene diamine tetra-acetic acid (EDTA), a chelator of Ca2+, increased accumulation and efflux of EtBr at pH 8 but not at pH 5. Ca2+ reverses these effects when the assay is conducted at pH 8. In conclusion, the activity of the efflux pump system of E. coli is dependent upon metabolic energy at pH 8. Because at pH 8 hydrolysis of ATP is favoured and contributes protons for activation of the AcrAB–TolC efflux pump, CPZ is suspected of having its effects on accumulation/efflux of EtBr by indirectly affecting ATPase activity that is dependent upon Ca2+.

Introduction

The cell envelope of Gram-negative bacteria consists of an outer cell wall, a periplasmic space and the plasma membrane. Permeability of the cell envelope to antibiotics involves the interaction between antibiotics and components of the cell envelope. Antibiotics that are hydrophobic or contain hydrophobic components may penetrate the lipopolysaccharide (LPS) layer of the outer cell wall, which is a barrier to hydrophilic antibiotics [1], [2]. Hydrophilic antibiotics gain access to targets beyond the outer cell wall via conduits that begin at the surface of the cell and end at the plasma membrane. These conduits, or porins, are selective diffusion channels that can be highly specialised for a given molecule, such as maltose, or allow diffusion of many unrelated compounds to reach the periplasmic space and even the cytoplasm [3]. Noxious substances that gain access to the periplasm or cytoplasm may be recognised and extruded to the outside of the cell by means of efflux pumps that are able to recognise a wide range of different molecular structures [2]. The degree of permeability to a given noxious agent such as an antibiotic is therefore a balance between LPS, general porins and the activity of the main efflux pump of the organism [2], [3].

The majority of Gram-negative clinical isolates that exhibit a multidrug-resistant phenotype owe their multidrug resistance to overexpressed efflux pumps [2], [4]. In recent years, many compounds have been shown to be effective efflux pump inhibitors (EPIs); however, with the exception of some naturally occurring compounds [5], [6], a derivative of an antibiotic [7], a designed EPI [8] and some compounds used for therapy of psychosis [8], [9], [10], [11], most EPIs show serious cytotoxicity [2]. Because phenothiazines have been shown to express EPI activity against overexpressed efflux pumps of Gram-negative bacteria [8], [9], [10], [11], [12], [13], they have been used in our investigations.

Phenothiazines have been shown to non-specifically inhibit enzymes that are involved in metabolism [14] as well as specifically by inhibiting binding of calcium to calcium-dependent enzymes [15]. The efflux pumps of Gram-negative bacteria, which are involved in the extrusion of a variety of non-related antibiotics, obtain their energy from the proton-motive force (PMF) [16]. The PMF is maintained by the metabolic activity of the bacterium and is the result of protons generated from the hydrolysis of ATP that are transported via channels to the surface of the bacterium, distributed on its surface and bound to reactive groups of the outer cell wall (LPS and –CO2) [17], [18], [19]. The difference in proton concentration between the surface of the cell and the medial side of the plasma membrane establishes a pH gradient, resulting in an electrochemical potential that drives protons from the surface to the periplasm of the cell [17], [18], [19]. The protons that are mobilised via channels to the periplasm are used by efflux pump(s) of the bacterium for extrusion of antibiotics prior to reaching their intended targets. Although it was initially proposed that these protons activate the efflux pump, it was demonstrated that the protons create a localised pH that is acidic enough to promote the dissociation of the substrate from the transporter component of the pump [19], [20], [21], [22]. Binding of protons on the surface of the bacterium results in a pH that is 2 to 3 units lower than that of the bulk medium [19]. Consequently, when the pH of the medium is near or slightly above 7, protons do not readily dissociate into the bulk medium and therefore the PMF is maintained [19], [20]. However, if the bacterium is challenged with a noxious substance, more protons are needed in the periplasm and, because the concentration of protons at the surface of the cell must be maintained if the PMF is to be maintained, additional protons must be made available by metabolic activity for subsequent transport to the cell surface. Therefore, when the pH of the medium is near neutral and the cell is exposed to a noxious agent for a prolonged period of time, any interference with the process of metabolic energy would be expected to decrease the effectiveness of the efflux pump [16]. It is under these conditions that phenothiazine is expected to indirectly express its effects on the activity of the efflux pump and hence render the bacterium increasingly susceptible to the antibiotic to which it was initially resistant as a consequence of an overexpressed efflux pump. Because the targets of phenothiazines are Ca2+-dependent, we have studied the effect of phenothiazines on the accumulation and efflux of ethidium bromide (EtBr) as well as the role of calcium on modulation of that effect.

Section snippets

Materials

Mueller–Hinton and trypticase soy in powder form for the preparation of broth and agar were purchased from Oxoid Ltd. (Basingstoke, UK). Phosphate-buffered saline (PBS), glucose, EtBr, chlorpromazine (CPZ), CaCl2 and ethylene diamine tetra-acetic acid (EDTA) were purchased from Sigma-Aldrich Quimica S.A. (Madrid, Spain).

Bacteria

Wild-type Escherichia coli K-12 AG100 strain [argE3 thi-1 rpsL xyl mtl Δ(gal-uvrB) supE44] [22] was kindly provided by Hiroshi Nikaido (Department of Molecular and Cell Biology

Results

Phenothiazines such as CPZ and thioridazine (TZ) inhibit the binding of Ca2+ to enzymes involved in furnishing energy from the hydrolysis of ATP [24]. Ca2+ plays a crucial role in the biochemical pathways of the cell and is of extreme importance for cell signalling, membrane transport channels and also for the activity of some types of ATPases [25], [26]. Because the phenothiazines CPZ and TZ have major effects on the accumulation and efflux of EtBr at pH 7 [23], and these effects are modified

Discussion

Calcium is important for signalling [25] and for activating genetic systems [26] as well as for a wide variety of metabolic and energy-deriving pathways within the cell. Central to these pathways are ATPases that hydrolyse ATP and furnish protons for the activation of ABC-type transporters.

CPZ is noted for its inhibitory effects on the binding of Ca2+ to enzymes that are involved in the provision of energy (ATP) [14]. The results of this study demonstrate that CPZ, a phenothiazine noted for its

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    Permanent address: Institute of Pharmacognosy, Faculty of Pharmacy, University of Szeged, Eötvös u. 6, 6720 Szeged, Hungary.

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