Identification and characterization of porins in Pseudomonas aeruginosa.

Earlier studies have shown that the major porin species in Pseudomonas aeruginosa outer membrane is protein F (OprF), which produces channels wider than those produced by Escherichia coli porins. In contrast, Yoshihara and Nakae ((1989) J. Biol. Chem. 264, 6297-6301) reported that protein F has no pore-forming activity as measured by the flux of L-arabinose, and that the channels in P. aeruginosa outer membrane, being produced by proteins C, "D," and "E," are much narrower than E. coli porin channels. In this study, we followed the protein purification scheme of Yoshihara and Nakae as closely as possible, and found that protein F had a specific activity for pore formation similar to that of proteins D1, D2, and E2. Furthermore, proteoliposome reconstitution assays showed conclusively that the channels formed by protein F, as well as by unfractionated outer membranes, allowed the diffusion of a tetrasaccharide, stachyose, at a significant rate, indicating that these channels are much larger than E. coli porin channels. It appears likely that in the study of Yoshihara and Nakae protein F was inadvertently inactivated during purification. We further suggest a hypothesis that resolves the apparent conflict between the presence of large diameter channels and the low permeability of the outer membrane in P. aeruginosa.     

Porin activity of Anaplasma phagocytophilum outer membrane fraction and purified P44

Anaplasma phagocytophilum, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, has significantly less coding capacity for biosynthesis and central intermediary metabolism than do free-living bacteria. Thus, A. phagocytophilum needs to usurp and acquire various compounds from its host. Here we demonstrate that the isolated outer membrane of A. phagocytophilum has porin activity, as measured by a liposome swelling assay. The activity allows the diffusion of L-glutamine, the monosaccharides arabinose and glucose, the disaccharide sucrose, and even the tetrasaccharide stachyose, and this diffusion could be inhibited with an anti-P44 monoclonal antibody. P44s are the most abundant outer membrane proteins and neutralizing targets of A. phagocytophilum. The P44 protein demonstrates characteristics consistent with porins of gram-negative bacteria, including detergent solubility, heat modifiability, a predicted structure of amphipathic and antiparallel beta-strands, an abundance of polar residues, and a C-terminal phenylalanine. We purified native P44s under two different nondenaturing conditions. When reconstituted into proteoliposomes, both purified P44s exhibited porin activity. P44s are encoded by approximately 100 p44 paralogs and go through extensive antigenic variation. The 16-transmembrane-domain beta-strands consist of conserved P44 N- and C-terminal regions. By looping out the hypervariable region, the porin structure is conserved among diverse P44 proteins yet enables antigenic variation for immunoevasion. The tricarboxylic acid (TCA) cycle of A. phagocytophilum is incomplete and requires the exogenous acquisition of L-glutamine or L-glutamate for function. Efficient diffusion of L-glutamine across the outer membrane suggests that the porin feeds the Anaplasma TCA cycle and that the relatively large pore size provides Anaplasma with the necessary metabolic intermediates from the host cytoplasm.     

Porin from bacterial and mitochondrial outer membranes

The outer membrane of gram-negative bacteria acts as a molecular filter with defined exclusion limit for hydrophilic substances. The exclusion limit is dependent on the type of bacteria and has for enteric bacteria like Escherichia coli and Salmonella typhimurium a value between 600 and 800 Daltons, whereas molecules with molecular weights up to 6000 can penetrate the outer membrane of Pseudomonas aeruginosa. The molecular sieving properties result from the presence of a class of major proteins called porins which form trimers of identical subunits in the outer membrane. The porin trimers most likely contain only one large but well-defined pore with a diameter between 1.2 and 2 nm. Mitochondria are presumably descendents of gram-negative bacteria. The outer membrane of mitochondria contains in agreement with this hypothesis large pores which are permeable for hydrophilic substances with molecular weights up to 6000. The mitochondrial porins are processed by the cell and have molecular weights around 30,000 Daltons. There exists some evidence that the pore is controlled by electric fields and metabolic processes.     

Isolation and characterization of a conserved porin protein from Helicobacter pylori.

Helicobacter pylori is a causative agent of gastritis in humans and is correlated with gastric ulcer formation. Infections with this bacterium have proven difficult to treat with antimicrobial agents. To better understand how this bacterium transports compounds such as antimicrobial agents across its outer membrane, identification of porin proteins is important. We have recently identified a family of H. pylori porins (HopA to HopD) (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995). Here, we report on an unrelated porin species (HopE) from this bacterium. This protein had a apparent molecular mass of 31 kDa and was seen to form 50- and 90-kDa aggregates that were designated putative dimeric and trimeric forms, respectively. The protein was purified to homogeneity and, with a model planar lipid membrane system, was shown to act as a nonselective pore with a single channel conductance in 1.0 M KCl of 1.5 nS, similarly to other bacterial nonspecific porins. An internal peptide sequence of HopE shared homology with the P2 porin of Haemophilus influenzae. HopE was also shown to be antigenic in vivo as assessed by sera taken from H. pylori-infected individuals and was immunologically conserved with both patient sera and specific monoclonal antibodies. From these data, it appears that HopE is a major nonselective porin of H. pylori. The implications of these findings are discussed.     

Molecular and functional characterisation of the Serratia marcescens outer membrane protein Omp1

Serratia marcescens outer membrane contains three different general diffusion porins: Omp1, Omp2 and Omp3. Omp1 was cloned and sequenced and it shows a great homology to the family of outer membrane porins that comprises the general porins of enteric bacteria. The gene for Omp1 was transferred into an expression plasmid and was expressed in Escherichia coli UH302 (E. coli UH302 pOM100), a porin deficient strain. Its expression confers a higher susceptibility towards different antibiotics to this strain. Omp1 was purified to homogeneity from outer membrane of E. coli UH302 pOM100. Reconstitution of the purified protein into black lipid bilayers demonstrated that it is a channel-forming component with a single-channel conductance of approximately 2 nS in 1 M KCl similar to that of other porins from enteric bacteria. Omp1 is slightly cation-selective. Its homology to already crystallised members of the family of enteric porins whose three-dimensional-structures are known and allowed the design of a topology model for Omp1. The charge distribution within a porin monomer is similar as in other general diffusion pores. The positively charged amino acids localised at the beta-strands opposite the external loop L3, which restrict the pore diameter in the porin monomer.     

Molecular and Structural Characterization of the HMP-AB Gene Encoding a Pore-Forming Protein from a Clinical Isolate of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis>

The major outer membrane protein of Acinetobacter baumannii is the heat-modifiable protein HMP-AB, a porin with a large pore size allowing the penetration of solutes having a molecular weight of up to approximately 800 Da. Cross-linking experiments with glutardialdehyde failed to show any cross-linking between the monomers, a fact that proves again that this porin protein functions as a monomeric porin. The specific activity of this porin was found to be similar to that of other monomeric porins. Tryptic digestion of the outer membrane yielded a 23-kDa fragment of the HMP-AB protein that was resistant to further trypsin treatment. This observation indicates that HMP-AB is assembled in the membrane in a manner similar to monomeric porins. Cloning of the HMP-AB gene revealed an open reading frame of 1038 bp encoding a protein of 346 amino acids and a calculated molecular mass of 35,636 Da. The amino acid sequence and composition were typical of Gram-negative bacterial porins: a highly negative hydropathy index, absence of hydrophobic residue stretches, a slightly negative total charge, low instability index, high glycine content, and an absence of cysteine residues. Sequence comparison of HMP-AB with other outer membrane proteins revealed a clear homology with the monomeric outer membrane proteins, outer membrane protein A (OmpA) of Enterobacteria, and outer membrane protein F (OprF) of Pseudomonas sp. Secondary structure analysis indicated that HMP-AB has a 172-amino acid N-terminal domain that spans the outer membrane by eight amphiphilic beta strands and a C-terminal domain that apparently serves as an anchoring protein to the peptidoglycan layer. The results also indicate that HMP-AB belongs to the eight transmembrane beta-strand family of outer membrane proteins.     

Reduced outer membrane permeability of Escherichia coli O157:H7: suggested role of modified outer membrane porins and theoretical function in resistance to antimicrobial agents

Outer membrane permeability of Escherichia coli O157:H7 was determined by an in vivo kinetic model with the periplasmic enzyme alkaline phosphatase [Martinez et al. (1996) Biochemistry 35, 1179-1186]. p-Nitrophenyl phosphate (PNPP) substrate, added to intact bacteria, must diffuse through the outer membrane to reach the enzyme. At low substrate concentration the bacterium was in the perfectly reactive state where all molecules that entered the periplasm were captured and converted to product. Transmembrane diffusion was rate limiting, and the permeability of the outer membrane was determined from kinetic properties. The O157:H7 strain grown at 30 degrees C showed one-sixth the permeability of wild-type E. coli grown at 30 degrees C. Wild-type bacteria grown at >/=37 degrees C show a physiological response with a shift in expression of outer membrane porins that lowered permeability to PNPP by approximately 70%. The O157:H7 strain did not display this temperature-sensitive shift in permeability even though a change in porin expression could be visualized by staining intensity of Omp F and Omp C on acrylamide gels. Altered behavior of the O157:H7 membrane was also indicated by a several thousand-fold lower response to transformation relative to wild-type E. coli. Matrix-assisted laser desorption ionization time of flight mass spectrometry and electrospray ionization mass spectrometry confirmed the expression of the Omp F and Omp C variants that are unique to E. coli O157:H7. This reduced outer membrane permeability can contribute to enhanced resistance of O157:H7 to antimicrobial agents.     

Outer membrane protein-specific monoclonal antibodies protect SCID mice from fatal infection by the obligate intracellular bacterial pathogen Ehrlichia chaffeensis

Previous studies of Ehrlichia chaffeensis infection in the mouse have demonstrated that passive transfer of polyclonal Abs from resistant immunocompetent mice to susceptible SCID mice ameliorated infection and disease, even when Abs were administered during established infection. To identify particular Abs that could mediate bacterial clearance in vivo, E. chaffeensis-specific mAbs were generated and administered to infected SCID mice. Bacterial infection in the livers was significantly lowered after administration of either of two Abs of different isotypes (IgG2a and IgG3). Moreover, repeated administration of one Ab (Ec56.5; IgG2a) rescued mice from an otherwise lethal infection for at least 5 wk. Both protective Abs recognized the E. chaffeensis major outer membrane protein (OMP)-1g. Further studies revealed that both Abs recognized closely related epitopes within the amino terminus of the first hypervariable region of OMP-1g. Analyses of human sera showed that E. chaffeensis-infected patients also generated serological responses to OMP-1g hypervariable region 1, indicating that humans and mice recognize identical or closely related epitopes. These studies demonstrate that OMP-specific mAbs can mediate bacterial elimination in SCID mice, and indicate that Abs, in the absence of cell-mediated immunity, can play a significant role in host defense during infection by this obligate intracellular bacterium.     

Ion selectivity of gram-negative bacterial porins.

Twelve different porins from the gram-negative bacteria Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Yersinia pestis were reconstituted into lipid bilayer membranes. Most of the porins, except outer membrane protein P, formed large, water-filled, ion-permeable channels with a single-channel conductance between 1.5 and 6 nS in 1 M KCl. The ions used for probing the pore structure had the same relative mobilities while moving through the porin pore as they did while moving in free solution. Thus the single-channel conductances of the individual porins could be used to estimate the effective channel diameters of these porins, yielding values ranging from 1.0 to 2.0 nm. Zero-current potential measurements in the presence of salt gradients across lipid bilayer membranes containing individual porins gave results that were consistent with the conclusions drawn from the single-channel experiments. For all porins except protein P, the channels exhibited a greater cation selectivity for less mobile anions and a greater anion selectivity for less mobile cations, which again indicated that the ions were moving inside the pores in a fashion similar to their movement in the aqueous phase. Three porins, PhoE and NmpC of E. coli and protein P of P. aeruginosa, formed anion-selective pores. PhoE and NmpC were only weakly anion selective, and their selectivity was dependent on the mobility of the ions. In contrast, cations were unable to enter the selectivity filter of the protein P channel. This resulted in a high anion selectivity for all salts tested in this study. The other porins examined, including all of the known constitutive porins of the four gram-negative bacteria studied, were cation selective with a 3- to 40-fold preference for K+ ions over Cl- ions.     

Investigation of the thermal stability of porin from Paracoccus denitrificans by site-directed mutagenesis and Fourier transform infrared spectroscopy

The folding of membrane proteins was addressed using outer membrane protein porin from the soil bacterium Paracoccus denitrificans (P. den.). IR spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were used to probe the effect of mutagenesis on the thermal stability of the protein. Secondary structure analysis by amide I ir spectroscopy showed that the wild-type protein was predominantly composed of beta-sheet, which supports the x-ray crystal structure information (A. Hirsch, J. Breed, K. Saxena, O.-M. H. Richter, B. Ludwig, K. Diederichs, and W. Welte, FEBS Letters, 1997, Vol. 404, pp. 208-210). The mutants E81Q, W74C, and E81Q/D148N were shown to have similar secondary structure composition as the wild type. Wild-type protein and the mutants in detergent micelles underwent irreversible denaturation as a result of heating. Transition temperature calculated from the amide I analysis revealed that mutant porins were slightly less stable compared to the wild type. The protein in micelles showed complete monomerization of the trimer above 85 degrees C. In native-like conditions (provided by liposomes), no change was observed in the secondary structure of the protein until 95 degrees C. This is supported by SDS-PAGE as no change in quaternary structure was observed, proving that the proteins are structurally thermostable in liposomes as compared to micelles. Our studies demonstrated that porins resistant to detergents and proteases are highly thermostable as well.     

Purification, amino acid composition and N-terminal sequence of the major protein (protein H) of the outer membrane of Pasteurella multocida]

The major protein (protein H) of the outer membrane of Pasteurella multocida was purified by size-exclusion chromatography after selective extraction with detergents. The protein forms homotrimers which are stable in the presence of SDS at room temperature. Upon treatment at 100 degrees C, the protein is fully dissociated by the detergent into monomers exhibiting an apparent molecular mass of 37 kDa as estimated by electrophoresis. The amino acid composition of protein H is characterized by a low hydropathy index (HI = -0.40) and is strongly related to the compositions of bacterial porins, notably porins P2 (Haemophilus influenzae), PIA (Neisseria gonorrhoeae) and Cl.2 ("class 2 porin" of N. meningitidis). The N-terminal amino acid sequence of protein H shares a strong homology with those of porins OmpC (Escherichia coli) and P2. These data indicate that protein H of P. multocida is a porin belonging to the superfamily of the non-specific porins of Gram-negative eubacteria outer membrane.     

Borrelia burgdorferi BBA74, a periplasmic protein associated with the outer membrane, lacks porin-like properties

The outer membrane of Borrelia burgdorferi, the causative agent of Lyme disease, contains very few integral membrane proteins, in contrast to other gram-negative bacteria. BBA74, a Borrelia burgdorferi plasmid-encoded protein, was proposed to be an integral outer membrane protein with putative porin function and designated as a 28-kDa outer membrane-spanning porin (Oms28). In this study, the biophysical properties of BBA74 and its subcellular localization were investigated. BBA74 is posttranslationally modified by signal peptidase I cleavage to a mature 25-kDa protein. The secondary structure of BBA74 as determined by circular dichroism spectroscopy consists of at least 78% alpha-helix with little beta-sheet structure. BBA74 in intact B. burgdorferi cells was insensitive to proteinase K digestion, and indirect immunofluorescence microscopy showed that BBA74 was not exposed on the cell surface. Triton X-114 extraction of outer membrane vesicle preparations indicated that BBA74 is not an integral membrane protein. Taken together, the data indicate that BBA74 is a periplasmic, outer membrane-associated protein that lacks properties typically associated with porins.     

Outer membrane porin proteins F, P, and D1 of Pseudomonas aeruginosa and PhoE of Escherichia coli: chemical cross-linking to reveal native oligomers.

Native oligomers of three Pseudomonas aeruginosa outer membrane porin proteins and one Escherichia coli porin were demonstrated by using a chemical cross-linking technique. P. aeruginosa protein F, the major constitutive outer membrane porin, was cross-linked to dimers in outer membrane and whole-cell cross-linking experiments. Purified preparations of P. aeruginosa proteins F, D1 (glucose induced), and P (phosphate starvation induced) and E. coli protein PhoE (Ic) were also cross-linked to reveal dimers and trimers upon two-dimensional sodium dodecyl sulfate-polyacrylamide electrophoretic analysis. Cross-linking of protein F was abolished by pretreatment of the protein with sodium dodecyl sulfate, indicating that the cross-linked products were due to native associations in the outer membrane.     

Structural relatedness of enteric bacterial porins assessed with monoclonal antibodies to Salmonella typhimurium OmpD and OmpC.

The immunochemistry and structure of enteric bacterial porins are critical to the understanding of the immune response to bacterial infection. We raised 41 monoclonal antibodies (MAbs) to Salmonella typhimurium OmpD and OmpC porin trimers and monomers. Enzyme-linked immunosorbent assays, immunoprecipitations, and/or Western immunoblot techniques indicated that 39 MAbs (11 anti-trimer and 28 anti-monomer) in the panel are porin specific and one binds to the lipopolysaccharide; the specificity of the remaining MAb probably lies in the porin-lipopolysaccharide complex. Among the porin-specific MAbs, 10 bound cell-surface-exposed epitopes, one reacted with a periplasmic epitope, and the remaining 28 recognized determinants that are buried within the outer membrane bilayer. Many of the MAbs reacting with surface-exposed epitopes were highly specific, recognizing only the homologous porin trimers; this suggests that the cell-surface-exposed regions of porins tends to be quite different among S. typhimurium OmpF, OmpC, and OmpD porins. Immunological cross-reaction showed that S. typhimurium OmpD was very closely related to Escherichia coli NmpC and to the Lc porin of bacteriophage PA-2. Immunologically, E. coli OmpG and protein K also appear to belong to the family of closely related porins including E. coli OmpF, OmpC, PhoE, and NmpC and S. typhimurium OmpF, OmpC, and OmpD. It appears, however, that S. typhimurium "PhoE" is not closely related to this group. Finally, about one-third of the MAbs that presumably recognize buried epitopes reacted with porin domains that are widely conserved in 13 species of the family Enterobacteriaceae, but apparently not in the seven nonenterobacterial species tested. These data are evaluated in relation to host immune response to infection by gram-negative bacteria.     

Immunological cross-reactivity between outer membrane pore proteins of Campylobacter jejuni and Escherichia coli

Immunocrossreactivity between the major outer membrane protein (MOMP) of Campylobacter jejuni 85H and the OmpC porin of Escherichia coli K-12 was observed. These results indicate that a common antigenic domain is conserved in both MOMP and OmpC. This antigenic region is detected only after a 96 degrees C treatment suggesting that it is buried in the native conformation of the respective porins. In addition, differences were observed between the major outer membrane proteins from various C. jejuni strains. About 60% of the C. jejuni pathogenic strains tested contained a protein exhibiting a similar electrophoretic profile to the 85H porin.     

Nonspecific porins of the outer membrane of Gram-negative bacteria: Structure and functions

The structure and functional properties of nonspecific porins (β-structured integral proteins of the outer membrane of Gram-negative bacteria) are overviewed. The characteristic features of porin spatial structure related to the principles of β-barrel construction and pore geometry are considered. The data concerning nonspecific diffusion of low-molecular substances and dynamic behavior of porin channels dependent on the distribution of charged amino acid residues in different structural domains of the porin molecule are presented. The methods and approaches used in the study of functional activity of porins are surveyed. The data on modulation of pore-forming activity of these proteins by external factors and membrane components are considered separately.     

Effects of magnesium and sodium ions on the outer membrane permeability of cephalosporins in Escherichia coli

Both Mg2+ and Na+ stimulated the outer membrane permeation of negatively charged cephalosporins in Escherichia coli without any significant alteration of the permeation of a zwitterionic cephalosporin. Such stimulation was not observed in an E. coli mutant lacking porins. The stimulation was caused by the direct interaction between the cations and the porin pores, which resulted in a decrease in cation selectivity of both the Omp F and Omp C porin pores.     

Phenotypic characterization of pore mutants of the Vibrio cholerae porin OmpU

General-diffusion porins form large β-barrel channels that control the permeability of the outer membrane of gram-negative bacteria to nutrients, some antibiotics, and external signals. Here, we have analyzed the effects of mutations in the OmpU porin of Vibrio cholerae at conserved residues that are known to affect pore properties in the Escherichia coli porins OmpF and OmpC. Various phenotypes were investigated, including sensitivity to β-lactam antibiotics, growth on large sugars, and sensitivity to and biofilm induction by sodium deoxycholate, a major bile component that acts as an external signal for multiple cellular responses of this intestinal pathogen. Overall, our results indicate that specific residues play different roles in controlling the passage of various compounds. Mutations of barrel wall arginine residues that protrude in the pore affect pore size and growth in the presence of large sugars or sodium deoxycholate. Sensitivity to large cephalosporins is mostly affected by D116, located on the L3 loop, whose homolog in E. coli, OmpF, is a known binding determinant for these drugs. L3 loop residues also affect biofilm induction. The results are interpreted in terms of a homology model based on the structures of E. coli porins.