Expression in Escherichia coli and function of Pseudomonas aeruginosa outer membrane porin protein F

Woodruff, Wendy A.; Parr, Thomas; Hancock, Robert E. W.; Hanne, Larry F.; Nicas, Thalia I.; Iglewski, B H

doi:10.1128/jb.167.2.473-479.1986

Cited by 89 publications

(78 citation statements)

References 32 publications

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“…The outer member protein fraction, although complex, was predominated by a protein that we identified as OprF. OprF represents ϳ15% of P. aeruginosa outer member protein fraction (ϳ2 ϫ 10 5 copies/cell), making it a major protein of the pathogen (45). Strikingly, NE degraded OprF, coinciding with death of bacteria.…”

Section: Discussionmentioning

confidence: 99%

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Neutrophil Elastase Mediates Innate Host Protection against Pseudomonas aeruginosa

Hirche

Benabid

Deslée

et al. 2008

The Journal of Immunology

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According to the widely accepted view, neutrophil elastase (NE), a neutrophil-specific serine protease, is a major contributor to Pseudomonas aeruginosa infection-associated host tissue inflammation and damage, which in severe cases can lead to death. Herein, we provide for the first time compelling evidence that the host rather employs NE to protect itself against P. aeruginosa infection. Using a clinically relevant model of pneumonia, targeted deficiency in NE increased the susceptibility of mice to P. aeruginosa. We found that NE was required for maximal intracellular killing of P. aeruginosa by neutrophils. In investigating the mechanism of NE-mediated killing of P. aeruginosa, we found that NE degraded the major outer membrane protein F, a protein with important functions, including porin activity, maintenance of structural integrity, and sensing of host immune system activation. Consistent with this, the use of an isogenic mutant deficient in outer membrane protein F negated the role of NE in host defense against P. aeruginosa infection.

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Section: Discussionmentioning

confidence: 99%

“…OprF is associated with the underlying peptidoglycan and contributes to the stabilization of the Om as well as exhibiting physiologically relevant porin activity (45)(46)(47). Degradation of OprF by NE would therefore explain the observed phenotypic alterations and ensuing death of P. aeruginosa.…”

Section: Discussionmentioning

confidence: 99%

Neutrophil Elastase Mediates Innate Host Protection against Pseudomonas aeruginosa

Hirche

Benabid

Deslée

et al. 2008

The Journal of Immunology

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“…We also introduced a decahistidine tag at the N terminus of the protein. OprF, when produced in E. coli, is known to fold correctly and to become inserted in the outer membrane (22,23,32). The modified OprF was expressed in E. coli; it was found nearly exclusively in the outer membrane as long as its expression took place at 30°C (results not shown).…”

Section: Conformation Of the Majority Of Oprf Population-e Colimentioning

confidence: 99%

Pseudomonas aeruginosa Porin OprF Exists in Two Different Conformations

Sugawara

Nestorovich

Bezrukov

et al. 2006

Journal of Biological Chemistry

110

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The major nonspecific porin of Pseudomonas aeruginosa, OprF, produces a large channel yet allows only a slow diffusion of various solutes. Here we provide an explanation of this apparent paradox. We first show, by introduction of tobacco etch virus protease cleavage site in the middle of OprF protein, that most of OprF population folds as a two-domain protein with an N-terminal ␤-barrel domain and a C-terminal periplasmic domain rich in ␣-helices. However, sedimentation of unilamellar proteoliposomes through an iso-osmotic gradient showed that only about 5% of the OprF population produced open channels. Gel filtration showed that the open channel conformers tended to occur in oligomeric associations. Because the open channel conformer is likely to fold as a single domain protein with a large ␤-barrel, we reasoned that residues near the C terminus may be exposed on cell surface in this conformer. Introduction of a cysteine residue at position 312 produced a functional mutant protein. By using bulky biotinylation reagents on intact cells, we showed that this cysteine residue was not exposed on cell surface in most of the OprF population. However, the minority OprF population that was biotinylated in such experiments was enriched for the conformer with pore-forming activity and had a 10-fold higher pore-forming specific activity than the bulk OprF population. Finally trypsin treatment, which preferentially cleaves the C-terminal domain of the two-domain conformer, did not affect the pore-forming activity of OprF nor did it digest the minority conformer whose residue 312 is exposed on cell surface.Structure and functions of the now classical, trimeric bacterial porins, such as OmpF, PhoE, and OmpC of Escherichia coli, are well understood. Each monomer forms a ␤-barrel composed of 16 membranetraversing ␤-strands, and the central channel in the barrel is narrowed by the infolding of an external loop, L3 (for reviews, see Refs.

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“…Small channels were observed, with an average single-channel conductance of 0.28 nS from 100 recorded events (compared with an average single-channel conductance of 0.35 nS for the small OprF [39]). Larger channels were also observed, but no extensive attempts were made to characterize these.…”

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confidence: 99%

Conservation of the gene for outer membrane protein OprF in the family Pseudomonadaceae: sequence of the Pseudomonas syringae oprF gene

et al. 1991

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The conservation of the oprF gene for the major outer membrane protein OprF was determined by restriction mapping and Southern blot hybridization with the Pseudomonas aeruginosa oprF gene as a probe. The restriction map was highly conserved among 16 of the 17 serotype type strains and 42 clinical isolates of P. aeruginosa. Only the serotype 12 isolate and one clinical isolate showed small differences in restriction pattern. Southern probing of PstI chromosomal digests of 14 species from the family Pseudomonadaceae revealed that only the nine members of rRNA homology group I hybridized with the oprF gene. To reveal the actual extent of homology, the oprF gene and its product were characterized in Pseudomonas syringae. Nine strains of P. syringae from seven different pathovars hybridized with the P. aeruginosa gene to produce five different but related restriction maps. All produced an OprF protein in their outer membranes with the same apparent molecular weight as that of P. aeruginosa OprF. In each case the protein reacted with monoclonal antibody MA4-10 and was similarly heat and 2-mercaptoethanol modifiable. The purified OprF protein of the type strain P. syringae pv. syringae ATCC 19310 reconstituted small channels in lipid bilayer membranes. The oprF gene from this latter strain was cloned and sequenced. Despite the low level of DNA hybridization between P. aeruginosa and P. syringae DNA, the OprF gene was highly conserved between the species with 72% DNA sequence identity and 68% amino acid sequence identity overall. The carboxy terminus-encoding region of P. syringae oprF showed 85 and 33% identity, respectively, with the same regions of the P. aeruginosa oprF and Escherichia coli ompA genes.

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Expression in Escherichia coli and function of Pseudomonas aeruginosa outer membrane porin protein F

Cited by 89 publications

References 32 publications

Neutrophil Elastase Mediates Innate Host Protection against Pseudomonas aeruginosa

Neutrophil Elastase Mediates Innate Host Protection against Pseudomonas aeruginosa

Pseudomonas aeruginosa Porin OprF Exists in Two Different Conformations

Conservation of the gene for outer membrane protein OprF in the family Pseudomonadaceae: sequence of the Pseudomonas syringae oprF gene

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