Ampicillin permeation across OmpF, the major outer-membrane channel in Escherichia coli

Abstract

The outer cell wall of the Gram-negative bacteria is a crucial barrier for antibiotics to reach their target. Here, we show that the chemical stability of the widely used antibiotic ampicillin is a major factor in the permeation across OmpF to reach the target in the periplasm. Using planar lipid bilayers we investigated the interactions and permeation of OmpF with ampicillin, its basic pH-induced primary degradation product (penicilloic acid), and the chemically more stable benzylpenicillin. We found that the solute-induced ion current fluctuation is 10 times higher with penicilloic acid than with ampicillin. Furthermore, we also found that ampicillin can easily permeate through OmpF, at an ampicillin gradient of 10 μm and a conductance of G_amp ≅ 3.8 fS, with a flux rate of roughly 237 molecules/s of ampicillin at V_m = 10 mV. The structurally related benzylpenicillin yields a lower conductance of G_amp ≅ 2 fS, corresponding to a flux rate of ≈120 molecules/s. In contrast, the similar sized penicilloic acid was nearly unable to permeate through OmpF. MD calculations show that, besides their charge difference, the main differences between ampicillin and penicilloic acid are the shape of the molecules, and the strength and direction of the dipole vector. Our results show that OmpF can impose selective permeation on similar sized molecules based on their structure and their dipolar properties.

Keywords: NMR; OmpF; ampicillin; antibiotics; bacteria; electrophysiology; membrane reconstitution; membrane transport; peniciollic acid; planar bilayer.

Figure 1.

Alkaline-induced ampicillin degradation by raising the pH to ≅12.5 (45 min) to induce degradation. See supporting data for details.

Figure 2.

Effect of ampicillin on the ion current across a single active reconstituted trimeric OmpF channel (left) and the corresponding all point ion current amplitude histogram (right). OmpF was added to the cis = ground side, the applied voltage was ±100 mV, and the buffer contained 1 m KCl, buffered with 20 mm MES, pH 6.0. Cis/trans ampicillin addition was measured in the same experiments separated by an intensive buffer exchange. A, ion current in the absence of substrate. B, addition of 20 mm ampicillin on the cis side. C, addition of 20 mm ampicillin trans after intensive volume exchange.

Figure 3.

Effect of ampicillin-degradation products on the ion current across a single active reconstituted trimeric OmpF channel (left) and the corresponding all point ion current amplitude histogram (right). OmpF was added to the cis = ground side, the applied voltage was ±100 mV, and the buffer contained 1 m KCl, buffered with 20 mm MES, pH 6.0. Cis/trans ampicillin-degradation product addition was measured separately. Note that 20 mm ampicillin-degradation product corresponds to 20 mm ampicillin. A, ion current in the absence of substrate. B, addition of 20 mm ampicillin-degradation product (DG) on the cis side (cis side is connected to the electrical ground). C, addition of 20 mm ampicillin-degradation product on the trans side.

Figure 4.

A, current–voltage relationship of reconstituted OmpF channels under symmetrical 30 mm KCl (cis/trans) bi-ionic conditions (control) and under tri-ionic conditions with 80 mm ampicillin (cis) at pH 8 (see Table 1), V_rev = 21.5 ±4.8. B, calculated current–voltage relationship for a single trimeric OmpF channel (i(Σ)) with the cis/trans tri-ionic concentrations given in Table 1 and the separated currents carried by K⁺ or Cl⁻ ions (i(KCl)) and i_ampicillin = (i(Σ)) − (i(KCl)) (for more details see supporting data). Conditions: 30 mm KCl, buffered with 10 mm MES, pH 6.0. OmpF and ampicillin was added to cis = GND side.

Figure 5.

A, current–voltage relationship of reconstituted OmpF under symmetrical 30 mm KCl (cis/trans), pH 6, buffered with 10 mm MES, bi-ionic conditions (control), and under tri-ionic, pH 6, conditions with 80 mm benzylpenicillin (cis, see Table 1). B, calculated current–voltage relationship for a single OmpF pore bathed in 100 mm KCl symmetrical (cis/trans) and 10 μm (theoretical) benzylpenicillin (cis) (for details see supporting data).