Distinct Roles of Outer Membrane Porins in Antibiotic Resistance and Membrane Integrity in Escherichia coli

Abstract

A defining characteristic of Gram-negative bacteria is the presence of an outer membrane, which functions as an additional barrier inhibiting the penetration of toxic chemicals, such as antibiotics. Porins are outer membrane proteins associated with the modulation of cellular permeability and antibiotic resistance. Although there are numerous studies regarding porins, a systematic approach about the roles of porins in bacterial physiology and antibiotic resistance does not exist yet. In this study, we constructed mutants of all porins in Escherichia coli and examined the effect of porins on antibiotic resistance and membrane integrity. The OmpF-defective mutant was resistant to several antibiotics including β-lactams, suggesting that OmpF functions as the main route of outer membrane penetration for many antibiotics. In contrast, OmpA was strongly associated with the maintenance of membrane integrity, which resulted in the increased susceptibility of the ompA mutant to many antibiotics. Notably, OmpC was involved in both the roles. Additionally, our systematic analyses revealed that other porins were not involved in the maintenance of membrane integrity, but several porins played a major or minor role in the outer membrane penetration for a few antibiotics. Collectively, these results show that each porin plays a distinct role in antibiotic resistance and membrane integrity, which could improve our understanding of the physiological function and clinical importance of porins.

Keywords: OmpA; OmpC; OmpF; antibiotic resistance; membrane integrity; porins.

FIGURE 1

A systematic examination of the effects of porins on antibiotic resistance. (A) The MICs of various antibiotics were measured in the wild-type or mutant strain defective for OmpA, OmpC, or OmpF. The relative MIC value in the mutant cells compared to the wild-type cells is shown. (B) The MICs of various antibiotics were measured in the wild-type or mutant strain defective for the indicated specific porin. The relative MIC value in the mutant cells compared to the wild-type cells is shown.

FIGURE 2

Complemetation of the antibiotic-resistant phenotype of the yddB or lamB mutant. (A) The MICs of novobiocin were examined in 0.01% arabinose-containing agar plates with the agar dilution method according to the Clinical Laboratory Standards Institute guidelines in the wild-type or yddB mutant with or without a pBAD-YbbB plasmid. After incubation at 37°C for 20 h, photographs of the plates were taken with a digital camera. (B) The MICs of vancomycin were examined in 0.01% arabinose-containing agar plates with the agar dilution method in the wild-type or lamB mutant with or without a pBAD-LamB plasmid.

FIGURE 3

The effect of non-specific porins on envelope stress responses and membrane integrity. (A) The wild-type, ompA, ompC, or ompF mutant cells were serially diluted from 10⁸ to 10⁴ cells/ml in 10-fold steps and spotted onto a LB plate or LB plates with the addition of 2% SDS, 6% ethanol, 750 mM NaCl, or 20 μg/ml CPRG as indicated. (B) The wild-type, ompA ompC, ompA ompF, ompC ompF, or ompA ompC ompF mutant cells were serially diluted 10-fold from 10⁸ to 10⁴ cells/ml and spotted onto a LB plate or LB plates with the addition of 2% SDS, 6% ethanol, 750 mM NaCl, or 20 μg/ml CPRG as indicated. (C) The wild-type or ompA ompC ompF triple mutant cells grown in LB medium were stained with FM4-64 (red), DAPI (blue), and SYTOX green (green), and then spotted on a 1% agarose pad. Cells were visualized using a Nikon Eclipse Ni microscope. Bars, 0.5 μm.

FIGURE 4

The importance of the C-terminal region of OmpA on its function. The wild-type, ompA, or ompAΔC mutant cells were serially diluted from 10⁸ to 10⁴ cells/ml in 10-fold steps and spotted onto a LB plate or LB plates with the addition of 2% SDS, 6% ethanol, 750 mM NaCl, 20 μg/ml CPRG, 6 μg/ml ampicillin, 2.5 μg/ml cefalotin, 6 μg/ml choramphenicol, 150 μg/ml clindamycin, or 5 μg/ml rifampicin as indicated.

FIGURE 5

The effects of single-, double-, and triple-mutants of non-specific porins on antibiotic resistance. (A) The MICs of β-lactam antibiotics were measured in the wild-type strain or single-, double-, and triple-mutants of OmpA, OmpC, and OmpF. The relative MIC value in the mutant cells compared to the wild-type cells was shown. (B) The MICs of non-β-lactam antibiotics were measured in the wild-type strain or single-, double-, and triple-mutants of OmpA, OmpC, and OmpF. The relative MIC value in the mutant cells compared to the wild-type cells was shown: WT, MG1655; ΔA, ΔompA; ΔC, ΔompC; ΔF, ΔompF, ΔAC, ΔompA ompC; ΔAF, ΔompA ompF; ΔCF, ΔompC ompF; ΔACF, ΔompA ompC ompF.