Tetracycline diffusion through phospholipid bilayers and binding to phospholipids

Abstract

The ability of tetracycline to pass through phospholipid bilayers by diffusion was investigated. Liposomes did not retain enclosed tetracycline. Accumulation of tetracycline was observed with liposomes containing entrapped Tet repressor protein. These results indicate that the drug can pass through lipid bilayers. The antibiotic was also shown to bind to liposomes and isolated phospholipids.Incubation of Escherichia coli cells in medium containing tetracycline leads to uptake and accumulation of the antibiotic by these cells (1,4,5). To date no involvement of proteins in these processes has been demonstrated (5, 6). However, only a fraction of the antibiotic accumulation appears to be mediated by an active process: the treatment of cells with uncouplers reduces accumulation but does not abolish it. Active uptake of tetracycline has been shown to be driven by the proton motive force of the cell (5), but the basis for its accumulation by de-energized cells has so far eluded explanation. The present study was initiated to investigate the interaction of tetracycline with dissolved phospholipids and with phospholipid vesicles in the absence of membrane proteins.To test whether tetracycline can pass through phospholipid bilayers via passive diffusion, the permeability properties of liposomes to tetracycline were investigated. Two series of experiments were carried out. (i) The release of [3H]tetracycline enclosed in liposomes was determined by dialysis of liposomes against tetracycline-free buffer. Liposomes which contained radioactive rubidium, which is known not to pass through the lipid bilayer (8), served as a control. Tetracycline concentration in liposomes decreased gradually, whereas the concentration of liposome-enclosed 86Rb remained constant (after the first 30 min) over a period of 27 h (compare Fig. 1A and B). The rapid initial decrease seen with 86Rb is due to diffusion of 86Rb not enclosed in liposomes. The kinetics of diffusion of tetracycline and 86Rb from the dialysis tubes (without liposomes) is also shown in Fig. 1A and B, respectively. The data suggest that tetracycline present in the internal aqueous volume of the liposomes diffused passively through the phospholipid bilayers, whereas 86Rb was retained. The retention during the initial phase of dialysis of (nonencapsulated) tetracycline in the presence of liposomes (Fig. 1A) (3) (without alkali treatment) in the presence and absence of 0.5% sodium dodecyl sulfate. Protein could not be detected when the assay was carried out in the absence of the detergent. Equilibrium dialysis of Tet repressor protein encapsulated in liposomes (see below) in the presence of 100 ,ug each of trypsin and pronase per ml (Boehringer Mannheim Corp.) did not affect the results, confirming the absence of Tet repressor protein on the outside of liposomes. Equilibrium dialysis was performed with small dialysis tubes (VisKing; diameter, 1 cm; length, 2 cm). Either 2 ml of a liposome suspension (4.9 ,ul internal aqueous volume) containing 3 jig of Tet repressor protein per ml...

Delta pH-dependent accumulation of tetracycline in Escherichia coli

“…In order to reveal whether ApH-dependent tetracycline accumulation requires a carrier, accumulation of tetracycline in protein-free liposomes was investigated. The downhill diffusion of tetracycline through phospholipid bilayer membranes had previously been reported by Argast and Beck (1). We succeeded in showing that tetracycline accumulation in liposomes is driven by an artificially imposed ApH.…”

“…In contrast, Argast and Beck (1,2) showed that tetracycline enters the cytoplasm through simple diffusion because of the nonsaturability of tetracycline uptake and presented evidence for diffusion through phospholipid bilayers. The question is how such simple diffusion leads to the accumulation of tetracycline in cells.…”

Permeation of tetracyclines through membranes of liposomes and Escherichia coli

“…Despite their wide application, only little is known about their mode of action, including both their uptake and inhibition of translation [1]. It is assumed that the ability of tetracycline (tc) to diffuse through lipid bilayers [4] and its protonation behaviour along the pH gradient of the cell is sufficient to explain accumulation in Escherichia coli [5,6].…”

Overproduction of transposon Tn10-encoded tetracycline resistance protein results in cell death and loss of membrane potential

“…According to current concepts, TetA protein functions as a carrier for tetracycline in the excretion of the antibiotic (which enters the cell presumably by passive diffusion [1,2]). This transport of tetracycline was shown to be energy dependent (15) …”