Antibiotic Uptake through Membrane Channels: Role of <i>Providencia stuartii</i> OmpPst1 Porin in Carbapenem Resistance

Bajaj, Harsha; Tran, Que‐Tien; Mahendran, Kozhinjampara R.; Nasrallah, Chady; Colletier, J.P.; Davin-Régli, Anne; Bolla, Jean‐Michel; Pagès, Jean‐Marie; Winterhalter, Mathias

doi:10.1021/bi301398j

Cited by 31 publications

(41 citation statements)

References 27 publications

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“…4a and b). The blockage events were fitted into a kinetic binding model with an on rate, k on , of ʋ/3[c] (ʋ = number of blockages per second; [c] is concentration) and an off rate, k off , of 1/τ (τ = dwell time) [28]. The average dwell time did not depend on the concentration of the ciprofloxacin and was found to be in the range of 60 μs for the voltages applied between 125 and 199 mV (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…We conclude that in the absence of the Ca 2 + , antibiotic translocation occurs too rapidly to see individual events under our experimental conditions. Metal-ion-dependent blockages were observed with imipenem added to OmpPst1 channel from Providencia stuartii [28] in the presence of La 3 + and with enrofloxacin added to OmpF channel in the presence of Mg 2 + [30]. In both these cases, the ion does not co-purify with the protein or seem to have any role in stability.…”

Section: Discussionmentioning

confidence: 99%

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MOMP from Campylobacter jejuni Is a Trimer of 18-Stranded β-Barrel Monomers with a Ca 2+ Ion Bound at the Constriction Zone

Ferrara

Wallat

Moynié

et al. 2016

Journal of Molecular Biology

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The Gram-negative organism Campylobacter jejuni is the major cause of food poisoning. Unlike Escherichia coli, which has two major porins, OmpC and OmpF, C. jejuni has one, termed major outer membrane protein (MOMP) through which nutrients and antibiotics transit. We report the 2.1-Å crystal structure of C. jejuni MOMP expressed in E. coli and a lower resolution but otherwise identical structure purified directly from C. jejuni. The 2.1-Å resolution structure of recombinant MOMP showed that although the protein has timeric arrangement similar to OmpC, it is an 18-stranded, not 16-stranded, β-barrel. The structure has identified a Ca2 + bound at the constriction zone, which is functionally significant as suggested by molecular dynamics and single-channel experiments. The water-filled channel of MOMP has a narrow constriction zone, and single-molecule studies show a monomeric conductivity of 0.7 ± 0.2 nS and a trimeric conductance of 2.2 ± 0.2 nS. The ion neutralizes negative charges at the constriction zone, reducing the transverse electric field and reversing ion selectivity. Modeling of the transit of ciprofloxacin, an antibiotic of choice for treating Campylobacter infection, through the pore of MOMP reveals a trajectory that is dependent upon the presence metal ion.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

MOMP from Campylobacter jejuni Is a Trimer of 18-Stranded β-Barrel Monomers with a Ca 2+ Ion Bound at the Constriction Zone

Ferrara

Wallat

Moynié

et al. 2016

Journal of Molecular Biology

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“…Liposome Swelling Assays-Swelling assays were performed as described previously with a slight modification (24,25). In brief, 2 mg of E. coli total lipid extract (Avanti Polar Lipids) were used to form a lipid film that was dried for several hours.…”

Section: Methodsmentioning

confidence: 99%

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

Bajaj

Scorciapino

Moynié

et al. 2016

Journal of Biological Chemistry

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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.

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“…The functions of OMPs are versatile and often essential as they include transport of metabolites and toxins as well as membrane biogenesis (2). Alterations of outer membranes and outer membrane proteins can lead to the development of antibiotic-resistance in pathogenic bacteria, and dysfunction of OMPs in outer membranes of mitochondria plays a role in diabetes and neurodegenerative diseases, among other life-threatening illnesses (3)(4)(5)(6)(7). How OMPs attain their native fold in their natural lipid environment is therefore a fundamental question in biological and biomedical research.…”

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confidence: 99%

Outer membrane β-barrel protein folding is physically controlled by periplasmic lipid head groups and BamA

Gessmann

Chung

Danoff

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

175

222

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Outer membrane β-barrel proteins (OMPs) are crucial for numerous cellular processes in prokaryotes and eukaryotes. Despite extensive studies on OMP biogenesis, it is unclear why OMPs require assembly machineries to fold into their native outer membranes, as they are capable of folding quickly and efficiently through an intrinsic folding pathway in vitro. By investigating the folding of several bacterial OMPs using membranes with naturally occurring Escherichia coli lipids, we show that phosphoethanolamine and phosphoglycerol head groups impose a kinetic barrier to OMP folding. The kinetic retardation of OMP folding places a strong negative pressure against spontaneous incorporation of OMPs into inner bacterial membranes, which would dissipate the proton motive force and undoubtedly kill bacteria. We further show that prefolded β-barrel assembly machinery subunit A (BamA), the evolutionarily conserved, central subunit of the BAM complex, accelerates OMP folding by lowering the kinetic barrier imposed by phosphoethanolamine head groups. Our results suggest that OMP assembly machineries are required in vivo to enable physical control over the spontaneously occurring OMP folding reaction in the periplasm. Mechanistic studies further allowed us to derive a model for BamA function, which explains how OMP assembly can be conserved between prokaryotes and eukaryotes.membrane protein folding | beta-barrel transmembrane protein

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Antibiotic Uptake through Membrane Channels: Role of Providencia stuartii OmpPst1 Porin in Carbapenem Resistance

Cited by 31 publications

References 27 publications

MOMP from Campylobacter jejuni Is a Trimer of 18-Stranded β-Barrel Monomers with a Ca 2+ Ion Bound at the Constriction Zone

MOMP from Campylobacter jejuni Is a Trimer of 18-Stranded β-Barrel Monomers with a Ca 2+ Ion Bound at the Constriction Zone

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

Outer membrane β-barrel protein folding is physically controlled by periplasmic lipid head groups and BamA

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