Molecular Basis of Filtering Carbapenems by Porins from β-Lactam-resistant Clinical Strains of Escherichia coli

Abstract

Integral membrane proteins known as porins are the major pathway by which hydrophilic antibiotics cross the outer membrane of Gram-negative bacteria. Single point mutations in porins can decrease the permeability of an antibiotic, either by reduction of channel size or modification of electrostatics in the channel, and thereby confer clinical resistance. Here, we investigate four mutant OmpC proteins from four different clinical isolates of Escherichia coli obtained sequentially from a single patient during a course of antimicrobial chemotherapy. OmpC porin from the first isolate (OmpC20) undergoes three consecutive and additive substitutions giving rise to OmpC26, OmpC28, and finally OmpC33. The permeability of two zwitterionic carbapenems, imipenem and meropenem, measured using liposome permeation assays and single channel electrophysiology differs significantly between OmpC20 and OmpC33. Molecular dynamic simulations show that the antibiotics must pass through the constriction zone of porins with a specific orientation, where the antibiotic dipole is aligned along the electric field inside the porin. We identify that changes in the vector of the electric field in the mutated porin, OmpC33, create an additional barrier by "trapping" the antibiotic in an unfavorable orientation in the constriction zone that suffers steric hindrance for the reorientation needed for its onward translocation. Identification and understanding the underlying molecular details of such a barrier to translocation will aid in the design of new antibiotics with improved permeation properties in Gram-negative bacteria.

Keywords: Porin; antibiotic resistance; electrophysiology; gram-negative bacteria; membrane biophysics; membrane transport; metadynamics; molecular dynamics simulations.

FIGURE 1.

The typical starting position of the antibiotic in the presented computer simulations is shown in the first monomer of OmpC20 (a, side view; b, top view). The antibiotic (imipenem in the figure) was placed between the extracellular loops. The typical starting value of the two collective variables used in metadynamics simulations were +20 Å and 0 Å for position and orientation, respectively. In c, one monomer of OmpC33 is shown where the positively charged residues comprising the basic ladder at the constriction region are highlighted in blue, the negatively charged residues of the loop L3 in red, and the three residues mutated in the clinical series from OmpC20 to OmpC26 (D18E) to OmpC28 (D18E and S271F) and OmpC33 (D18E, S271F, and R124H), in green. The chemical structures of imipenem (d) and meropenem (e) are also shown, together with the color-coded electrostatic potential corresponding to the atomic partial charges. For the sake of completion, the pK_a values of ionizable groups and their net charge at neutral pH are reported.

FIGURE 2.

Liposome permeation assay. Relative diffusion rates of the reference sugars arabinose and raffinose and the antibiotics, imipenem and meropenem, into liposomes reconstituted with OmpC20 and OmpC33 are shown.

FIGURE 3.

Ion current traces of single channels of OmpC20 in 1 m KCl (a), without any antibiotic (b), with 2.5 mm imipenem on cis side (c) OmpC20 with 2.5 mm meropenem cis side; OmpC33 (d) without any antibiotic (e) with 2.5 mm imipenem on cis side (f) with 2.5 mm meropenem cis side. Electrolyte conditions: 1 m KCl, 20 mm MES, pH 6, at room temperature and −100 mV.

FIGURE 4.

Free energy surface of carbapenem translocation, imipenem (a and c) and meropenem (b and d) in the OmpC20 (a and b) and OmpC33 (c and d) porin are shown. The antibiotic orientation is defined as the difference of the z-coordinate between the lactam carbonyl carbon and the sulfur bonded carbon of the antibiotic two-ring core. The difference of the z-coordinate between the center of mass of the antibiotic two-ring core and that of the monomer of the porin (Δz) represents the antibiotic depth inside the porin lumen (Δz = 0 corresponds to the constriction region). Each iso contour corresponds to a free energy increase of 2 kcal mol⁻¹. Free energy values were rescaled to have the absolute minimum equal to zero. Different labels are used to indicate specific regions analyzed and discussed in the text. Water ordering inside (e) the first monomer of OmpC20 is shown together with the net electric dipole of the water molecules calculated at different depth inside the lumen, according to a recent theoretical investigation (20). The loop L3 is highlighted to provide a reference. Meropenem is shown at the constriction region, i.e. the representative conformer for the free energy minimum 3 in Fig. 4b. Net electric charges are indicated for clarity. In f, the system is rotated to show the xy projection. The electric dipole of the antibiotic (purple) is shown together with the net dipole of water molecules (cyan) in the absence of antibiotic at the same level inside the channel.

FIGURE 5.

Meropenem translocation through OmpC20 (a) and OmpC33 (b). The representative conformers of the different free energy states, labeled in Fig. 4, b and d, are reported in different colors: red, orange, blue, and purple for the minimum 1, 2, 3, and 4, respectively. The position of the positively charged nitrogen is highlighted by a sphere, to provide a reference for antibiotic orientation inside the channel. Protein backbone is gray but the loop L3, which has been colored green as a reference.

FIGURE 6.

Imipenem translocation through OmpC20 (a, front view; b, top view) and OmpC33 (c). The representative conformers of the different FES regions, labeled in Fig. 4, a and c, are reported in different colors: red, orange, blue, and purple for the minimum 1, 2, 3, and 4, respectively. Yellow is used for the representative conformer of the main energy barrier. The position of the positively charged nitrogen of the antibiotic is highlighted by a sphere, to provide a reference for its orientation inside the channel. Protein backbone is gray but the loop L3, which has been colored green.

FIGURE 7.

Porin cross-sectional area profile along the channel axis is reported as the radius of the equivalent circle. The profiles due to the presence of the representative conformers of the different free energy states, labeled in Fig. 4, are reported with different colored solid lines. The results pertaining to the porin in the absence of antibiotic are reported with the black dashed line as reference.

FIGURE 8.

Ion current trace of single channel of OmpC20 at 5 °C (a) 2.5 mm imipenem on cis side at −100 mV and (b) 2.5 mm meropenem cis side at −100 mV. Electrolyte conditions were: 1 m KCl, 20 mm MES, pH 6.