The A226D Mutation of OmpC Leads to Increased Susceptibility to β-Lactam Antibiotics in Escherichia coli

Abstract

Bacterial resistance to antibiotics can lead to long-lasting, hard-to-cure infections that pose significant threats to human health. One key mechanism of antimicrobial resistance (AMR) is to reduce the antibiotic permeation of cellular membranes. For instance, the lack of outer membrane porins (OMPs) can lead to elevated AMR levels. However, knowledge on whether mutations of OMPs can also influence antibiotic susceptibility is limited. This work aims to address this question and identified an A226D mutation in OmpC, a trimeric OMP, in Escherichia coli. Surveillance studies found that this mutation is present in 50 E. coli strains for which whole genomic sequences are available. Measurement of minimum inhibition concentrations (MICs) found that this mutation leads to a 2-fold decrease in MICs for β-lactams ampicillin and piperacillin. Further survival assays confirmed the role this mutation plays in β-lactam susceptibility. With molecular dynamics, we found that the A226D mutation led to increased overall flexibility of the protein, thus facilitating antibiotic uptake, and that binding with piperacillin was weakened, leading to easier antibiotic penetration. This work reports a novel mutation that plays a role in antibiotic susceptibility, along with mechanistic studies, and further confirms the role of OMPs in bacterial tolerance to antibiotics.

Keywords: OmpC; antimicrobial resistance; molecular dynamics; outer membrane porin; β-lactam.

Figure 1

MIC values of E. coli strains. OMPA226D (black), E. coli OMP_A226D; OMPWT (red), E. coli OMP_WT; BW25113 (blue), E. coli BW25113. ∆OmpC (green), E. coli BW25113 ∆ompC. **, p < 0.01; ns, no statistical difference. Five parallels were performed in each group. The horizontal lines represent the average MIC value, vertical lines represent the SDs of the MIC averages.

Figure 2

Survival assays for AMP. OMPA226D, E. coli OMP_A226D; OMPWT, E. coli OMP_WT; BW25113, E. coli BW25113. ∆OmpC, E. coli BW25113 ∆ompC. Error bars indicate standard deviations. Survival rate is the ratio of CFU numbers of the measured time point to time zero. (A), 1 μg/mL AMP; (B), 4 μg/mL AMP; (C), 10 μg/mL AMP; (D), 16 μg/mL AMP.

Figure 3

Survival assays for PIP. OMPA226D, E. coli OMP_A226D; OMPWT, E. coli OMP_WT; BW25113, E. coli BW25113. ∆OmpC, E. coli BW25113 ∆ompC. Survival rate is the ratio of CFU numbers of the measured time point to time zero. (A), 1 μg/mL PIP; (B), 4 μg/mL PIP; (C), 10 μg/mL PIP; (D), 16 μg/mL PIP.

Figure 4

Expression ompC levels in all strains. OMPA226D, E. coli OMP_A226D; OMPWT, E. coli OMP_WT; BW25113, E. coli BW25113. Error bars indicate standard deviations. Expression levels were normalized with 16S rDNA levels.

Figure 5

Simulated structures of OmpC wildtype and A226D proteins. Panel (A), alignment of wildtype and A226D OmpC proteins, numbers indicate the pore number. Panel (B), RMSD curves of the simulation. WT, OmpC; A226D, OmpC_A226D. Panel (C), alignment of key residues in tunnel of pore 1. Panel (D), alignment of key residues in tunnel of pore 2. Panel (E), alignment of key residues in tunnel of pore 3. Gray color indicates wildtype OmpC. Orange color indicates OmpC_A226D mutant.

Figure 6

Changes of RMSF following A226D mutation. Red color indicates increased RMSF (in nm). Blue color indicates decreased RMSF (in nm). EC, view from the extracellular side. PP, view from the periplasmic side. The numbers 1,2, 3 represent pore 1, pore 2, and pore 3.

Figure 7

Kinetic and potential energies of simulated systems. Panel (A), kinetic energy. Panel (B), potential energy. A226D, OmpC A226D mutant. WT, wildtype OmpC.