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. 2021 Aug 3;118(31):e2107644118.
doi: 10.1073/pnas.2107644118.

Outer membrane permeability: Antimicrobials and diverse nutrients bypass porins in Pseudomonas aeruginosa

Johanna Ude  1 , Vishwachi Tripathi  1 , Julien M Buyck  1 , Sandra Söderholm  1 , Olivier Cunrath  1 , Joseph Fanous  1 , Beatrice Claudi  1 , Adrian Egli  2   3 , Christian Schleberger  1 , Sebastian Hiller  4 , Dirk Bumann  4
Affiliations

Outer membrane permeability: Antimicrobials and diverse nutrients bypass porins in Pseudomonas aeruginosa

Johanna Ude et al. Proc Natl Acad Sci U S A. .

Abstract

Gram-negative bacterial pathogens have an outer membrane that restricts entry of molecules into the cell. Water-filled protein channels in the outer membrane, so-called porins, facilitate nutrient uptake and are thought to enable antibiotic entry. Here, we determined the role of porins in a major pathogen, Pseudomonas aeruginosa, by constructing a strain lacking all 40 identifiable porins and 15 strains carrying only a single unique type of porin and characterizing these strains with NMR metabolomics and antimicrobial susceptibility assays. In contrast to common assumptions, all porins were dispensable for Pseudomonas growth in rich medium and consumption of diverse hydrophilic nutrients. However, preferred nutrients with two or more carboxylate groups such as succinate and citrate permeated poorly in the absence of porins. Porins provided efficient translocation pathways for these nutrients with broad and overlapping substrate selectivity while efficiently excluding all tested antibiotics except carbapenems, which partially entered through OprD. Porin-independent permeation of antibiotics through the outer-membrane lipid bilayer was hampered by carboxylate groups, consistent with our nutrient data. Together, these results challenge common assumptions about the role of porins by demonstrating porin-independent permeation of the outer-membrane lipid bilayer as a major pathway for nutrient and drug entry into the bacterial cell.

Keywords: antimicrobial resistance; bacterial outer membrane; diffusion; lipid bilayer; membrane transport.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Porin involvement in P. aeruginosa antimicrobial susceptibility. (A) Model of OprF with a large majority of two-domain conformer with a narrow outer-membrane β-barrel and a C-terminal domain linking the outer membrane with peptidoglycan and a minority of one-domain conformer with a large pore. (B) MIC (Upper) and antibiograms (Lower) of wild-type P. aeruginosa UCBPP-PA14 and various porin mutants. Means and SD of three experiments are shown. (C) Porin abundance in clinical P. aeruginosa strains in vitro and in UCBPP-PA14 in two rodent pneumonia models as determined by targeted proteomics.
Fig. 2.
Fig. 2.
Porin dependency of P. aeruginosa nutrient uptake. (A) Growth rates of P. aeruginosa PA14 wild-type and porin-free PA14 Δ40 in BM2 minimal media containing a single energy/carbon source. (B) One-dimensional 1H-NMR spectrum of modified BM2 medium containing 16 different nutrients before and after 5 h growth of P. aeruginosa PA14 wild type or porin-free PA14 Δ40. (C and D) PA14 nutrient consumption as measured by one-dimensional [1H] NMR spectroscopy. Each dot represents individual data for 1 of 21 independent cultures. (E) PA14 Δ40 nutrient consumption as measured by one-dimensional [1H] NMR spectroscopy. Each dot represents individual data for 1 of 21 independent cultures. Uptake rates for individual nutrients based on data shown in C and D. Means and SDs are shown.
Fig. 3.
Fig. 3.
Nutrient uptake through single porins in absence of other porins. (A) Nutrient consumption rates of PA14 Δ40 strains expressing a single porin from a low-copy-number plasmid. Means and averages for three independent cultures (single-porin strains) or 21 cultures (PA14, PA14 Δ40) are shown. (B) Substrate selectivity of 15 porins determined in single-porin strains. The capacity of each porin to boost nutrient consumption from baseline levels in PA14 Δ40 to wild-type levels is shown. Porins and nutrients are grouped based on unsupervised clustering. (C) Permeability of 472 antimicrobial compounds with detectable antipseudomonal activity (15). Following the published analysis, compounds are grouped in permeability classes according to the MIC values for efflux-deficient P. aeruginosa strains with intact (P Δ6) or abolished (P Δ6-pore) outer membrane permeability barrier (class 0, MIC of PA Δ6-pore is less than 20% of PA Δ6; 1, 20 to 40%; 2, 40 to 60%, 3, 60 to 80%; 4 more than 80%). Statistical significance was analyzed with the χ2 test.
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