Pseudomonas aeruginosa porin OprF exists in two different conformations

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 beta-barrel domain and a C-terminal periplasmic domain rich in alpha-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 beta-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.     

Conservation of surface epitopes in Pseudomonas aeruginosa outer membrane porin protein OprF

Abstract The outer membrane proteins of several prominent bacterial pathogens demonstrate substantial variation in their surface antigenic epitopes. To determine if this was also true for Pseudomonas aeruginosa outer membraine protein OprF, gene sequencing of a serotype 5 isolate was performed to permit comparison with the published serotype 12 oprF gene sequence. Only 16 nucleotide substitutions in the 1053 nucleotide coding region were observed; none of these changed the amino acid sequence. A panel of 10 monoclonal antibodies (mAbs) reacted with each of 46 P. aeruginosa strains representing all 17 serotype strains, 12 clinical isolates, 15 environmental isolates and 2 laboratory isolates. Between two and eight of these mAbs also reacted with proteins from representatives of the rRNA homology group I of the Pseudomonadaceae . Nine of the ten mAbs recognized surface antigenic epitopes as determined by indirect immunofluorescence techniques and their ability to opsonize P. aeuroginosa for phagocytosis. These epitopes were partially masked by lipopolysacharide side chains as revealed using a side chain-deficient mutant. It is concluded that OprF is a highly conserved protein with several conserved surface antigenic epitopes.     

Exogenous siderophore-mediated iron uptake in Pseudomonas aeruginosa: possible involvement of porin OprF in iron translocation.

In addition to the two siderophores pyoverdine and pyochelin synthesized by Pseudomonas aeruginosa ATCC 15692 (strain PAO1), several siderophores produced by other bacteria or fungi, namely cepabactin, salicylic acid, desferriferrichrysin, desferriferricrocin, desferriferrioxamine B, desferriferrioxamine E and coprogen, were able to promote iron uptake with variable efficiencies into this bacterium. For most of these siderophores, these results were consistent with the growth stimulation produced by the same compounds in a plate bioassay. Desferriferrichrome A, enterobactin and desferriferrirubin, however, did not promote iron uptake, although enterobactin and desferriferrirubin stimulated bacterial growth. These paradoxical data are discussed in view of siderophore-inducible iron uptake systems, as demonstrated recently for enterobactin. Among the strains tested, including the wild-type PAO1, the pyoverdine-less mutant PAO6606 and the two porin-mutants P. aeruginosa H636 (oprF::omega) and P. aeruginosa H673 (oprD::Tn501), only for the porin-OprF mutant were fewer siderophores able to promote iron uptake compared to the other strains. Such results suggest that beside specific routes for iron uptake P. aeruginosa is also able to take up siderophore-liganded iron through OprF.     

Linker-insertion mutagenesis of Pseudomonas aeruginosa outer membrane protein OprF

The oprF gene, expressing Pseudomonas aeruginosa major outer membrane protein OprF, was subjected to semi-random linker mutagenesis by insertion of a 1.3 kb Hincll kanamycin-resistance fragment from plasmid pUC4KAPA into multiple blunt-ended restriction sites in the oprF gene. The kanamycin-resistance gene was then removed by Pstl digestion, which left a 12 nucleotide pair linker residue. Nine unique clones were identified that contained such linkers at different locations within the oprF gene and were permissive for the production of full-length OprF variants. In addition, one permissive site-directed insertion, one non-permissive insertion and one carboxy-terminal insertion leading to proteolytic truncation were also identified. These mutants were characterized by DNA sequencing and reactivity of the OprF variants with a bank of 10 OprF-specific monoclonal antibodies. Permissive clones produced OprF variants that were shown to be reactive with the majority of these monoclonal antibodies, except where the insertion was suspected of interrupting the epitope for the specific monoclonal antibody. In addition, these variants were shown to be 2-mercaptoethanol modifiable, to be resistant to trypsin cleavage in intact cells and partly cleaved to a high-molecular-weight core fragment in outer membranes and, where studied, to be accessible to indirect immunofluorescenee labelling in intact cells by monoclonal antibodies specific for surface epitopes. Based on these data, a revised structural model for OprF is proposed.     

Evaluation of properties of Pseudomonas aeruginosa recombinant outer membrane protein F (OprF)

Study showed that synthesis of specific IgG occurs in rabbits immunized with recombinant outer membrane protein F (OprF) of Pseudomonas aeruginosa and that these antibodies inhibit grow of P. aeruginosa in vitro. In vivo studies on mice showed that rabbit hyperimmune sera and recombinant OprF are both able to protect animals from intraperitoneal challenge with P. aeruginosa.     

Reevaluation, using intact cells, of the exclusion limit and role of porin OprF in Pseudomonas aeruginosa outer membrane permeability.

Earlier studies that used model membrane reconstitution methods have come to different conclusions regarding the exclusion limit of the outer membrane of Pseudomonas aeruginosa and whether OprF is the major channel-forming protein in the outer membrane. In this study, a 6.2-kbp SalI fragment, encoding only two cytoplasmic enzymes, alpha-galactosidase and sucrose hydrolase, and the inner membrane raffinose permease, was cloned behind the m-toluate-inducible tol promoter of vector pNM185 to create plasmid pFB71. P. aeruginosa strains harboring pFB71, when grown with inducer, produced both enzymes encoded by the insert and had acquired the ability to grow on the disaccharide melibiose and the trisaccharide raffinose. The rate of growth was dependent on the concentration and size of the saccharide and was decreased three- to fivefold by the absence of OprF, as examined by measuring the growth on melibiose and raffinose of an isogenic OprF-deficient omega insertion derivative, H636(pFB71). At high concentrations, di-, tri-, and tetrasaccharides could pass across the outer membrane to plasmolyze P. aeruginosa, as measured by light scattering and confirmed by electron microscopy. The initial rate kinetics of light-scattering changes were dependent on the size of the saccharide being used. Furthermore, the rates of change in light scattering due to raffinose and stachyose uptake across the outer membrane for strain H636 were fivefold or more lower than for its OprF-sufficient parent H103. These data are consistent with model membrane studies showing that OprF is the most predominant porin for compounds larger than disaccharides in P. aeruginosa and suggest that the exclusion limit for this porin and the outer membrane is greater than the size of a tetrasaccharide. In addition, these data confirmed the existence of other porins with a predominant function in monosaccharide uptake and a more minor function in the uptake of larger saccharides.     

Enlightening mineral iron sensing in Pseudomonas fluorescens by surface active maghemite nanoparticles: Involvement of the OprF porin

BackgroundMineral iron(III) recognition by bacteria is considered a matter of debate. The peculiar surface chemistry of novel naked magnetic nanoparticles, called SAMNs (surface active maghemite nanoparticles) characterized by solvent exposed Fe^3 + sites on their surface, was exploited for studying mineral iron sensing in Pseudomonas fluorescens.MethodsSAMNs were applied for mimicking Fe^3 + ions in solution, acting as magnetically drivable probes to evaluate putative Fe^3 + recognition sites on the microorganism surface. Culture broths and nano-bio-conjugates were characterized by UV–Vis spectroscopy and mass spectrometry.ResultsThe whole heritage of a membrane porin (OprF) of P. fluorescens Ps_22 cells was recognized and firmly bound by SAMNs. The binding of nanoparticles to OprF porin was correlated to a drastic inhibition of a siderophore (pyoverdine) biosynthesis and to the stimulation of the production and rate of formation of a secondary siderophore. The analysis of metabolic pathways, based on P. fluorescens Ps_22 genomic information, evidenced that this putative secondary siderophore does not belong to a selection of the most common siderophores.ConclusionsIn the scenario of an adhesion mechanism, it is plausible to consider OprF as the biological component deputed to the mineral iron sensing in P. fluorescens Ps_22, as well as one key of siderophore regulation.General significanceThe present work sheds light on mineral iron sensing in microorganisms. Peculiar colloidal naked iron oxide nanoparticles offer a useful approach for probing the adhesion of bacterial surface on mineral iron for the identification of the specific recognition site for this iron uptake regulation in microorganisms.     

铜绿假单胞菌OprF/I融合蛋白疫苗研制现状

铜绿假单胞菌是一种革兰阴性非发酵和需氧的机会性致病菌,是医院获得性感染的常见病原体。铜绿假单胞菌治疗面临巨大挑战,研制疫苗是防治铜绿假单胞菌感染的有效替代方法。融合外膜蛋白OprF/I是一种保护性抗疫苗以及重组沙门菌疫苗的研制现状进行综述。     

Insertion mutagenesis of the Pseudomonas aeruginosa phosphate-specific porin OprP.

The gene encoding the Pseudomonas aeruginosa phosphate-specific porin OprP was subjected to both linker and epitope insertion mutageneses. Nine of the 13 linker mutant genes expressed protein at levels comparable to those obtained with the wild-type gene. These mutant proteins were shown, by indirect immunofluorescence with an OprP-specific antiserum, to be properly exposed at the cell surface. Four of the linker mutant genes expressed protein at reduced levels which were not detectable at the cell surface. A foreign epitope from the circumsporozoite form of the malarial parasite Plasmodium falciparum was cloned into the linker sites of 12 of the 13 mutant genes. Seven of the resultant epitope insertion mutant genes expressed surface-exposed protein. Two of these mutant genes presented the foreign epitope at surface-accessible regions as assessed by indirect immunofluorescence with a malarial epitope-specific monoclonal antibody. The data from these experiments were used to create a topological model of the OprP monomer.     

Analysis of the Pseudomonas aeruginosa major outer membrane protein OprF by use of truncated OprF derivatives and monoclonal antibodies.

TnphoA mutagenesis of the cloned oprF gene was utilized to generate 16 classes of fusions encoding differing lengths of the amino terminus of OprF fused to either alkaline phosphatase or to peptide tags of 1 to 20 amino acids, depending on the orientation and reading frame into which TnphoA was inserted. Representatives of each of the 16 classes were sequenced to determine the precise fusion joint. Four of these 16 representatives which produced in-frame fusions to alkaline phosphatase and another 8 with fusion joints in the amino-terminal half of OprF failed to react with a panel of 10 specific monoclonal antibodies. In contrast, OprF derivatives with predicted fusion joints at amino acids 180, 204, 289, and 299 reacted with one to five of the monoclonal antibodies. Four other immunoreactive OprF derivatives were created by subcloning and encoded amino acids 1 to 187, 188 to 326, 1 to 273 and 1 to 170 plus 301 to 326. On the basis of reactivity with the TnphoA-truncated derivatives and subclones of oprF, the epitopes for all 10 monoclonal antibodies were localized, in part, to specific regions of OprF. Nine of the 10 monoclonal antibodies, 8 of which recognize surface-exposed epitopes, mapped within the carboxy-terminal region of OprF that is homologous to the Escherichia coli outer membrane protein OmpA. Thus, we concluded that parts of the carboxy terminus of OprF are exposed on the external face of the outer membrane. In addition, a clone containing only the first two cysteine residues of OprF demonstrated reactivity with monoclonal antibodies MA4-4 and MA7-8 that was destroyed by 2-mercaptoethanol treatment, as was reactivity with intact OprF. Thus, we conclude that this first pair of cysteines at residues 176 and 185 of mature OprF form a disulfide bond.     

Modeling and simulations of a bacterial outer membrane protein: OprF from Pseudomonas aeruginosa

OprF is a major outer membrane protein from Pseudomonas aeruginosa, a homolog of OmpA from Escherichia coli. The N-terminal domains of both proteins have been demonstrated to form low conductance channels in lipid bilayers. Homology models, consisting of an eight-stranded beta-barrel, of the N-terminal domain OprF have been constructed based on the crystal structure of the corresponding domain from E. coli OmpA. OprF homology models have been evaluated via a set (6 x 10 ns) of simulations of the beta-barrel embedded within a solvated dimyristoyl-phosphatidylcholine (DMPC) bilayer. The conformational stability of the models is similar to that of the crystal structure of OmpA in comparable simulations. There is a degree of water penetration into the pore-like center of the OprF barrel. The presence of an acidic/basic (E8/K121) side-chain interaction within the OprF barrel may form a gate able to close/open a central pore. Lipid-protein interactions within the simulations were analyzed and revealed that aromatic side-chains (Trp, Tyr) of OprF interact with lipid headgroups. Overall, the behavior of the OprF model in simulations supports the suggestion that this molecule is comparable to OmpA. The simulations help to explain the mechanism of formation of low conductance pores within the outer membrane.     

Polyphosphate-selective porin OprO of Pseudomonas aeruginosa: expression, purification and sequence

The oprO gene of Pseudomonas aeruginosa codes for a polyphosphate-specific porin and terminates 458 bp upstream of the start codon for the phosphate-specific porin OprP. OprO was found to be expressed only under phosphate-starvation conditions in both wild-type and oprP::Tn501 mutant P. aeruginosa strains. However, unlike the rest of the genes of the Pho regulon, including oprP, expression of oprO required cells to be in the stationary growth phase in addition to phosphate starvation. Wild-type P. aeruginosa cells were grown in fermentor culture under these conditions and fractionated by selective solubilization in octylpolyoxyethylene detergent solution. Solubilized OprO was separated from OprP by application to a Mono Q FPLC column and elution with a salt gradient and shown to be functionally identical to cloned OprO produced in Escherichia coli. DNA sequencing of oprO showed the gene product to be highly homologous to OprP, with 76% identity and 16% conserved substitutions. Most genes of the Pho regulon possess a modified -35 region called the Pho box. Two such elements, separated by 4 bp were found in oprO. DNA sequencing also revealed a second Pho box in the oprP gene with the same spacing.     

Membrane topology and site-specific mutagenesis of Pseudomonas aeruginosa porin OprD

Pseudomonas aeruginosa OprD is a 420-amino-acid protein that facilitates the uptake of basic amino acids, imipenem and gluconate across the outer membrane. OprD was the first specific porin that could be aligned with members of the non-specific porin super-family. Utilizing multiple alignments in conjugation with structure predictions and amphipathicity calculations, an OprD-topology model was proposed. Sixteen β-strands were predicted, connected by short loops at the periplasmic side. The eight external loops were of variable length but tended to be much longer than the periplasmic ones. Polymerase chain reaction (PCR)-based site-specific mutagenesis was performed to delete separately short stretches (4-8 amino acid residues) from each of the predicted external loops. The mutants with deletions in the predicted external loops L1, L2, L5, L6, L7 and L8 were tolerated in both Escherichia coli and P. aeruginosa. The expressed mutant proteins maintained substantial resistance to trypsin treatment in the context of isolated outer membranes. Proteins with deletions in loops L1, L5, L6, L7 and L8 reconstituted similar imipenem supersusceptibility in a P. aeruginosa OprD::Ω background. The L2-deletion mutant only partially reconstituted supersusceptibility, suggesting that loop L2 is involved in imipenem binding. These data were generally consistent with the topology model.     

Expression in Caulobacter crescentus of the phosphate-starvation-inducible porin OprP of Pseudomonas aeruginosa

The gene for the phosphate-starvation-inducible outer membrane protein OprP, of Pseudomonas aeruginosa was introduced into Caulobacter crescentus CB2A on a plasmid vector. As is the case in P. aeruginosa and Escherichia coli the oprP gene was inducible under conditions of limiting phosphate in C. crescentus. However, the maximal medium concentration of phosphate which still permitted induction of OprP was lower in C. crescentus (50 microM) than in P. aeruginosa (200 microM). Induction of OprP was coincident with the process of stalk elongation, known to occur in C. crescentus under phosphate starvation conditions. When induced, OprP was localized to the cell envelope and became a major membrane protein, indicating that the Pseudomonas promoter was efficiently recognized in C. crescentus and that the gene product was targeted to the appropriate region of the cell. Our data provide support for the hypothesis that the mechanism for regulation of phosphate-starvation-inducible genes is highly conserved amongst the eubacteria.     

Specific Association of Lectin LecB with the Surface of Pseudomonas aeruginosa: Role of Outer Membrane Protein OprF

The fucose binding lectin LecB affects biofilm formation and is involved in pathogenicity of Pseudomonas aeruginosa. LecB resides in the outer membrane and can be released specifically by treatment of an outer membrane fraction with fucose suggesting that it binds to specific ligands. Here, we report that LecB binds to the outer membrane protein OprF. In an OprF-deficient P. aeruginosa mutant, LecB is no longer detectable in the membrane but instead in the culture supernatant indicating a specific interaction between LecB and OprF.     

Pseudomonas aeruginosa outer membrane permeability: isolation of a porin protein F-deficient mutant.

A mutant of Pseudomonas aeruginosa severely deficient in outer membrane protein F levels was isolated by screening heavily mutagenized strains for membrane protein alterations on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. To provide a basis for phenotypic comparison, three independent spontaneous revertants with normal protein F levels were isolated. Neither the protein F-deficient mutant nor its revertants had gross surface alterations as judged by their sensitivities to 31 phages with diverse receptors and their low degrees of leakage of periplasmic beta-lactamase into the supernatant. Outer membrane permeability was measured in whole cells by examining the rates of hydrolysis of a chromogenic beta-lactam, nitrocefin, by periplasmic RP1-encoded beta-lactamase. It was found that the outer membrane permeabilities of wild-type and protein F revertant strains were similar, but low when compared with those of Escherichia coli and an antibiotic-supersusceptible mutant Z61 of P. aeruginosa. The loss of protein F caused a further significant decrease in outer membrane permeability. The results suggest that protein F is a pore-forming protein in vivo and that only a small proportion, as few as 1 in 400, of the protein F molecules form active functional channels in vivo.     

Characterization of OpdH, a Pseudomonas aeruginosa porin involved in the uptake of tricarboxylates

The Pseudomonas aeruginosa outer membrane is intrinsically impermeable to many classes of antibiotics, due in part to its relative lack of general uptake pathways. Instead, this organism relies on a large number of substrate-specific uptake porins. Included in this group are the 19 members of the OprD family, which are involved in the uptake of a diverse array of metabolites. One of these porins, OpdH, has been implicated in the uptake of cis-aconitate. Here we demonstrate that this porin may also enable P. aeruginosa to take up other tricarboxylates. Isocitrate and citrate strongly and specifically induced the opdH gene via a mechanism involving derepression by the putative two-component regulatory system PA0756-PA0757. Planar bilayer analysis of purified OpdH demonstrated that it was a channel-forming protein with a large single-channel conductance (230 pS in 1 M KCl; 10-fold higher than that of OprD); however, we were unable to demonstrate the presence of a tricarboxylate binding site within the channel. Thus, these data suggest that the requirement for OpdH for efficient growth on tricarboxylates was likely due to the specific expression of this large-channel porin under particular growth conditions.     

Secondary structure of the outer membrane proteins OmpA of Escherichia coli and OprF of Pseudomonas aeruginosa.

When purified without the use of ionic detergents, both OmpA and OprF proteins contained nearly 20% alpha-helical structures, which disappeared completely upon the addition of sodium dodecyl sulfate. This result suggests that the proteins fold in a similar manner, with an N-terminal, membrane-spanning beta-barrel domain and a C-terminal, globular, periplasmic domain.