Pseudomonas aeruginosa lipopolysaccharide induction of keratinocyte proliferation, NF-kappa B, and cyclin D1 is inhibited by indomethacin

NF-kappaB is activated during acute inflammatory states as well as in other injury response disease states. Several pathologic states in squamous tissue injury response are characterized by increased squamous proliferation. This study was performed to investigate the hypothesis that Pseudomonas aeruginosa LPS is able to activate a proliferative phenotype in squamous cells via NF-kappaB induction and that this NF-kappaB-mediated response may be abrogated with the classic anti-inflammatory agent indomethacin. EMSA, luciferase reporter gene experiments, Western blots, and cellular proliferation assays were performed in normal and transformed human keratinocytes after stimulation with P. aeruginosa LPS. EMSA and luciferase reporter gene assays showed a 3- to 5-fold induction of active NF-kappaB in human keratinocyte cell lines after stimulation with P. aeruginosa LPS. The stimulation correlated with significantly increased cellular proliferation. As one potential mechanism for this increase in proliferation, an NF-kappaB-specific activation of cyclin D1 was observed. Both the NF-kappaB induction and proliferation response were inhibited with indomethacin and in dominant negative stable transfection clones. P. aeruginosa LPS activates proliferation of human keratinocytes, potentially through the induction of NF-kappaB and cyclin D1. These findings suggest that bacterial components can contribute to proliferative disease states in squamous epithelium through NF-kappaB activation.     

Spontaneous release of lipopolysaccharide by Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO grown in glucose mineral salts medium released lipopolysaccharide which was chemically and immunologically similar to the cellular lipopolysaccharide. In addition, it possessed identical phage E79-inactivating properties. Through neutralization of phage activity and hemolysis inhibition assays, the organism was found to liberate lipopolysaccharide at a constant rate during log-phase growth equivalent to 1.3 to 2.2 ng/10(8) cells over a growth temperature range of 25 to 42 degrees C. At 19 degrees C, a lipopolysaccharide was released which was deficient in phage-inactivating activity but retained its immunological properties. Chemical analysis of lipopolysaccharide extracted from cells grown at 19 degrees C showed a deficiency in the O-side-chain component fucosamine. Gel exclusion chromatography of the polysaccharide fraction derived from lipopolysaccharide isolated from cells grown at 19 degrees C exhibited a decreased content of side-chain polysaccharide as well as a difference in the hexosamine:hexose ratio. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis confirmed these results as well as establishing that an essentially normal distribution of side-chain repeating unit lengths were to be found in the 19 degrees C preparation. These results suggest a decrease in the frequency of capping R-form lipopolysaccharide at 19 degrees C.     

Analysis of a common-antigen lipopolysaccharide from Pseudomonas aeruginosa.

Lipopolysaccharide isolated from Pseudomonas aeruginosa PAO1 (O5 serotype) was separated into two antigenically distinct fractions. A minor fraction, containing shorter polysaccharide chains, reacted with a monoclonal antibody to a P. aeruginosa common antigen but did not react with antibodies specific to O5-serotype lipopolysaccharide. In contrast, fractions containing long polysaccharide chains reacted only with the O5-specific monoclonal antibodies. The shorter, common-antigen fraction lacked phosphate and contained stoichiometric amounts of sulfate, and the fatty acid composition of this fraction was similar to that of the O-antigen-specific fraction. The lipid A derived from the serotype-specific lipopolysaccharide cross-reacted with monoclonal antibodies against lipid A from Escherichia coli, while the lipid A derived from the common antigen did not react. We propose that many serotypes of P. aeruginosa produce two chemically and antigenically distinct lipopolysaccharide molecules, one of which is a common antigen with a short polysaccharide and a unique core-lipid A structure.     

Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length.

Lipopolysaccharide (LPS) from smooth strains of Pseudomonas aeruginosa 503, PAZ1, PAO1715, PAO1716, and Z61 was fractionated by gel filtration chromatography. LPS samples from the first four strains, all PAO1 derivatives, separated into three major size populations, whereas LPS from strain Z61, a Pac K799/WT mutant strain, separated into two size populations. When column fractions were applied to sodium dodecyl sulfate-polyacrylamide gels in their order of elution, molecules of decreasing size were resolved, and the ladder of molecules with different-length O antigens formed a diagonal across the gel. The LPS from the PAO1 derivatives contained two distinct sets of bands, distinguished on the gels as two sets of diagonals. The set of bands with the faster mobility, the B bands, was found in column fractions comprising the three major amino sugar-containing peaks. In the sample from strain 503, a fourth minor peak which contained B bands was resolved. The slower-moving set of bands, the A bands, were recovered in a minor peak. LPS from strain Z61 contained only one set of bands, with the higher-molecular-weight molecules eluting from the column in a volume similar to that of the B bands of the PAO1 strains. Analysis of the fractions of LPS from all strains indicated that less than 8% of the LPS molecules had a long, attached O antigen. Analysis of the peak that contained mainly A bands indicated a lack of reactive amino sugar and phosphate, although heptose and 2-keto-3-deoxyoctulosonic acid were detected. Reaction of isolated fractions with monoclonal antibody specific for the PAO1 O-antigen side chain indicated that only the B bands from the PAO1 strains were antigenically reactive. The bands from strain Z61 showed no reactivity. The data suggest that the A and B bands from the PAO1 strains are antigenically distinct. We propose that PAO1 strains synthesize two types of molecules that are antigenically different.     

Characterization of guanosine diphospho-D-mannose dehydrogenase from Pseudomonas aeruginosa. Structural analysis by limited proteolysis.

Alginate is believed to be a major virulence factor in the pathogenicity of Pseudomonas aeruginosa in the lungs of patients suffering from cystic fibrosis. Guanosine diphospho-D-mannose dehydrogenase (GDPmannose dehydrogenase, EC 1.1.1.132) is a key enzyme in the alginate biosynthetic pathway which catalyzes the oxidation of guanosine diphospho-D-mannose (GDP-D-mannose) to GDP-D-mannuronic acid. In this paper, we report the structural analysis of GMD by limited proteolysis using three different proteases, trypsin, submaxillary Arg-C protease, and chymotrypsin. Treatment of GMD with these proteases indicated that the amino-terminal part of this enzyme may fold into a structural domain with an apparent molecular mass of 25-26 kDa. Multiple proteolytic cleavage sites existed at the carboxyl-terminal end of this domain, indicating that this segment may represent an exposed region of the protein. Initial proteolysis also generated a carboxyl-terminal fragment with an apparent molecular mass of 16-17 kDa which was further digested into smaller fragments by trypsin and chymotrypsin. The proteolytic cleavage sites were localized by partial amino-terminal sequencing of the peptide fragments. Arg-295 was identified as the initial cleavage site for trypsin and Tyr-278 for chymotrypsin. Catalytic activity of GMD was totally abolished by the initial cleavage. However, binding of the substrate, GDP-D-mannose, increased stability toward proteolysis and inhibited the loss of enzyme activity. GMP and GDP (guanosine 5'-mono- and diphosphates) also blocked the initial cleavage, but NAD and mannose showed no effect. These results suggest that binding of the guanosine moiety at the catalytic site of GMD may induce a conformational change that reduces the accessibility of the cleavage sites to proteases. Binding of [14C]GDP-D-mannose to the amino-terminal domain was not affected by the removal of the carboxyl-terminal 16-kDa fragment. Furthermore, photoaffinity labeling of GMD with [32P]arylazido-beta-alanine-NAD followed by proteolysis demonstrated that the radioactive NAD was covalently linked to the amino-terminal domain. These observations imply that the amino-terminal domain (25-26 kDa) contains both the substrate and cofactor binding sites. However, the carboxyl-terminal fragment (16-17 kDa) may possess amino acid residues essential for catalysis. Thus, proteolysis had little effect on substrate binding, but totally eliminated catalysis. These biochemical data are in complete agreement with amino acid sequence analysis for the existence of substrate and cofactor sites of GMD. A linear peptide map of GMD was constructed for future structure/functional studies.     

Polycrylamide gel electrophoresis analysis of lipopolysaccharide from Pseudomonas aeruginosa growing planktonically and as biofilm

Abstract Lipopolysaccharide (LPS, endotoxin) was extracted from biofilm and planktonically grown monoagglutinable (1118) and polyagglutinable (258 and 15703) strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients with chronic pulmonary infections. Analysis by polyacrylamide gel electrophoresis (PAGE) followed by immune-detection of LPS fractions showed an S-form appearance of strain 1118 and 258 with three distinct clusters of high molecular weight bands, whereas 15703 appeared semi-rough. LPS of semi-rough cells grown planktonically and as biofilm showed a very similar PAGE pattern; however, the core/lipid A R-LPS fraction was more prominent in biofilm-LPS than in planktonic-LPS extracted from the S-form bacteria (1118 and 258). The apparent change in LPS sub-unit components of the bacteria when grown as biofilm may reflect changes in the outer membrane structure that contribute to the altered physico-chemical properties of biofilm bacteria in foreign-device associated infections and chronic P. aeruginosa lung infection in cystic fibrosis patients.     

1H NMR and ESR studies of oxidized cytochrome c551 from Pseudomonas aeruginosa.

Near neutral pH, Fe(III) cytochrome c551 exhibits an ESR absorption due primarily to a single species with g values of 3.24, 2.06, and 1.48. These g values are somewhat different from those of horse heart cytochrome c and can be interpreted by the generalizations of Brautigan et al. [(1977) J. Biol. Chem. 252, 574] to be due to Fe binding by the imidazole anion of histidine rather than by neutral imidazole. The NMR spectrum of Fe(III) cytochrome c551 exhibits a number of hyperfine-shifted peaks whose pattern shows similarities to but many differences from that of horse heart cytochrome c. Variation in shifts of some of the peaks in the pH range 5--9 is ascribed to ionization of a somewhat buried propionic acid side chain (pK = 5.8) and to ionization of the N-terminal NH3+ group (pK = 7.7). At alkaline pH greater than 9.4, as shown by a variety of optical and ESR spectral changes, the Met-61 S ligand is replaced by other ligands.     

Studies of lipid A fractions from the lipopolysaccharides of Pseudomonas aeruginosa and Pseudomonas alcaligenes

Lipid A fractions from Pseudomonas aeruginosa and Pseudomonas alcaligenes have similar compositions and structural features. By means of hydrazinolysis of the parent lipopolysaccharides and partial hydrolysis of the deacylation products, it was established that both lipids are derived from the β-(1→6)-linked disaccharide of glucosamine. Phosphorylated derivatives of the disaccharide from Ps. aeruginosa were also characterized. The lipids differ mainly in the absence of hexadecanoic acid and 2-hydroxydodecanoic acid from the lipid from Ps. alcaligenes. Evidence that in Ps. aeruginosa these acids are ester-linked to residues of 3-hydroxyalkanoic acids (including 3-hydroxydecanoic acid) was obtained. Heterogeneity of lipid A fractions was indicated by t.l.c., and by gel filtration of de-O-acylation products from mild alkaline methanolysis of the lipids.     

Monoclonal antibodies that distinguish inner core, outer core, and lipid A regions of Pseudomonas aeruginosa lipopolysaccharide.

In order to examine the immunochemistry of the core-lipid A region of Pseudomonas aeruginosa lipopolysaccharide (LPS), monoclonal antibodies (MAbs) specific for this region were produced in mice. Immunogen was prepared by coating a rough mutant of P. aeruginosa with column-purified core oligosaccharide fractions in order to enhance the immune response to the LPS core-lipid A region. Fourteen hybridoma clones were isolated, characterized, and further divided into three groups on the basis of their reactivities to rough LPS antigens in both enzyme-linked immunosorbent assays and Western immunoblots. In addition, another MAb, 18-19, designated group 1, was included in this study for defining core-lipid A epitopes. MAb 18-19 recognizes the LPS core-plus-one O-repeat unit of the serologically cross-reactive P. aeruginosa O2, O5, and O16. Group 2 MAbs are specific for the LPS outer core region and reacted with P. aeruginosa O2, O5, O7, O8, O10, O16, O18, O19, and O20, suggesting that these serotypes share a common outer core type. Group 3 MAbs recognize the inner core region and reacted with all 20 P. aeruginosa serotypes as well as with other Pseudomonas species, revealing the conserved nature of this region. Group 4 MAbs are specific for lipid A and reacted with all gram-negative organisms tested. Immunoassays using these MAbs and well-defined rough mutants, in addition to the recently determined P. aeruginosa core structures, have allowed us to precisely define immunodominant epitopes within the LPS core region.     

O-antigen conversion in Pseudomonas aeruginosa PAO1 by bacteriophage D3.

The lysogenization of Pseudomonas aeruginosa PAO by phage D3 results in derivatives which are resistant to superinfection by phage D3c by virtue of the fact that homologous phage cannot adsorb to these cells. The serologically and morphologically unrelated phage E79 showed a markedly decreased adsorption rate to the lysogen PAO(D3). Since both of these phages are lipopolysaccharide specific, these results suggested lysogenic conversion of the phage receptor. The lipopolysaccharide was extracted from strain PAO by the hot phenol-water technique, but this procedure was ineffective with PAO(D3). We developed a technique involving cold trichloroacetic acid extraction, followed by ultracentrifugation, digestion of the high-speed pellet with proteinase K, and ultimate purification on CsCl step gradients. The lipopolysaccharide from the wild type had inactivating activity against D3 and E79, whereas that from PAO(D3) inactivated neither. Chromatographic analysis indicated that the convertant lipopolysaccharide was smooth, and quantitative chemical analyses of the two preparations showed no differences in the level of the major fatty acids, amino compounds, or neutral sugars. On the other hand, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the side chains had a decreased migration rate through the gel matrix. The application of 1H and 13C nuclear magnetic resonance spectroscopic analysis revealed that the PAO side chain is chemically identical to that of serotype O:2a,d, containing 2,3-(1-acetyl-2-methyl-2-imidazolino-5,4)-2,3-dideoxy-D-mannuronic acid, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, and 2-acetamido-2,6-dideoxy-D-galactose (D-fucosamine). The molecular basis of the conversion event was (i) the introduction of an acetyl group into position 4 of the fucosamine residue and a change in the bonding between trisaccharide repeating units from alpha 1 leads to 4 to beta 1 leads to 4.     

Characterization of 2-amino-2,6-dideoxy-D-glucose as a constituent of the lipopolysaccharide antigen of Pseudomonas aeruginosa immunotype 4.

The title lipopolysaccharide was freed from its lipid A component by mild, acid hydrolysis, to give a polysaccharide fraction that was subsequently hydrolyzed completely to afford a mixture of neutral sugars and amino sugars. The amino sugars were separated, and identified as 2-amino-2-deoxy-D-galactose, 2-amino-2,6-dideoxy-galactose as a 2:1 mixture of the D and L enantiomers, and 2-amino-2,6-dideoxy-D-glucose. A reference sample of 2-amino-2,6-dideoxy-D-glucose was synthesized by an improved preparative route. Among the lipopolysaccharide antigens of the seven recognized immunotypes of Pseudomonas aeruginosa, 2-amino-2,6-dideoxyglucose is also characterized as a constituent of two others, types 3 and 5.     

Synthesis of lipopolysaccharide O side chains by Pseudomonas aeruginosa PAO1 requires the enzyme phosphomannomutase.

We have cloned a lipopolysaccharide (LPS) biosynthetic gene from Pseudomonas aeruginosa PAO1 that complements the defect in the production and incorporation of LPS O side chains in the LPS-rough strain AK1012. This gene was characterized by pulsed-field gel electrophoresis, deletion and restriction mapping of the cloned DNA, and biochemical analysis of the protein product. The cloned DNA was found to map to the 7-to-11-min region of the P. aeruginosa chromosome, and the gene needed for complementation of the LPS-rough phenotype was contained on a 2.6-kb HindIII-SacI fragment. This same size restriction fragment contains the alginate gene algC, which encodes the enzyme phosphomannomutase (PMM) and also maps to this region of the P. aeruginosa chromosome. The LPS-rough strain AK1012 was deficient in PMM activity, and this activity was restored to parental levels when the cloned gene was transferred to strain AK1012. In addition, the cloned gene could complement the PMM deficiency in the algC mutant strain 8858, and the cloned algC gene could restore the LPS-smooth phenotype to strain AK1012. These results indicate that the gene we have cloned is equivalent to the alginate gene algC. We designate this gene pmm to emphasize that it encodes the enzyme PMM, which has been shown to be essential for alginate production, and we demonstrate that PMM activity is required for the LPS-smooth phenotype in P. aeruginosa PAO1.     

The lipopolysaccharide from Pseudomonas aeruginosa NCTC 8505. Structure of the O-specific polysaccharide

Structural studies have been carried out on the O-specific fraction from the lipopolysaccharide of Pseudomonas aeruginosa NCTC 8505, Habs serotype 03. The O-specific polysaccharide has a tetrasaccharide repeating-unit containing residues of L-rhamnose (Rha), 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamido-2-deoxy-L-galacturonic acid (GalNAcA), and 2,4-diacetamido-2,4,6-trideoxy-D-glucose (BacNAc2). The following structure has been assigned to the repeating-unit: leads to 3)Rhap(beta 1 leads to 6)GlcpNAc(alpha 1 leads to 4)GalpNAcA(alpha 1 leads to 3)BacpNAc2(alpha 1 leads to. The parent lipopolysaccharide is a mixture of S, R, and SR species, and its high phosphorus content is partly due to the presence of triphosphate residues, as found for other lipopolysaccharides from P. aeruginosa. In addition to phosphorus, heptose, a 3-deoxyoctulosonic acid, and amide-bound alanine, the core oligosaccharide contains glucose, rhamnose, and galactosamine (molar proportions 3:1:1). The rhamnose and part of the glucose are present as unsubstituted pyranoside residues: other glucose residues are 6-substituted.     

Structural studies of lipid A from Pseudomonas aeruginosa PAO1: occurrence of 4-amino-4-deoxyarabinose.

Lipid A derived from Pseudomonas aeruginosa PAO1 contains a biphosphorylated 1-6-linked glucosamine disaccharide backbone. The reducing glucosamine has an unsubstituted glycosidically linked phosphate at C-1. The nonreducing glucosamine has an ester-bound phosphate at C-4' which is nonstoichiometrically substituted with 4-amino-4-deoxyarabinose. Induction of 4-amino-4-deoxyarabinose was dependent on cultural conditions. No pyrophosphate groups were detected. Acyloxyacyl diesters are formed by esterification of the amide-bound 3-hydroxydodecanoic acid with dodecanoic acid and 2-hydroxydodecanoic acids in an approximate molar ratio of 2:1. Dodecanoic and 3-hydroxydecanoic acids are esterified to positions C-3 and C-3' in the sugar backbone. All hydroxyl groups of the glucosamine disaccharide except C-4 and C-6' are substituted. Lipopolysaccharide chemical analyses measured glucose, rhamnose, heptose, galactosamine, alanine, phosphate, and glucosamine. The proposed lipid A structure differs from previous models. There are significant differences in acyloxyacyl diesters, and the proposed model includes an aminopentose substituent.     

Structural determination of lipid A of the lipopolysaccharide from Pseudomonas reactans. A pathogen of cultivated mushrooms.

The chemical structure of lipid A from the lipopolysaccharide of the mushroom-associated bacterium Pseudomonas reactans, a pathogen of cultivated mushroom, was elucidated by compositional analysis and spectroscopic methods (MALDI-TOF and two-dimensional NMR). The sugar backbone was composed of the beta-(1'-->6)-linked d-glucosamine disaccharide 1-phosphate. The lipid A fraction showed remarkable heterogeneity with respect to the fatty acid and phosphate composition. The major species are hexacylated and pentacylated lipid A, bearing the (R)-3-hydroxydodecanoic acid [C12:0 (3OH)] in amide linkage and a (R)-3-hydroxydecanoic [C10:0 (3OH)] in ester linkage while the secondary fatty acids are present as C12:0 and/or C12:0 (2-OH). A nonstoichiometric phosphate substitution at position C-4' of the distal 2-deoxy-2-amino-glucose was detected. Interestingly, the pentacyl lipid A is lacking a primary fatty acid, namely the C10:0 (3-OH) at position C-3'. The potential biological meaning of this peculiar lipid A is also discussed.     

Characterization of the 2-ketogluconate utilization operon in Pseudomonas aeruginosa PAO1

The Pseudomonas aeruginosa protein PtxS negatively regulates its own synthesis by binding to the upstream region of its gene. We have recently identified a 14 bp palindromic sequence within the ptxS upstream region as the PtxS operator site (OP1). In this study, we searched the P. aeruginosa genomic sequence to determine whether this 14 bp sequence exists in other regions of the P. aeruginosa chromosome. Another PtxS operator site (OP2) was located 47 bp downstream of ptxS. DNA gel shift experiments confirmed that PtxS specifically binds to a 520 bp fragment that carries OP2. The DNA segment 3' of OP2 contains four open reading frames (ORF1-ORF4), which code for 29, 32, 48 and 35 kDa proteins respectively. The molecular weight of the products of ORFs 2 and 3 were confirmed by T7 expression experiments. Computer analyses suggest that ORF2 encodes an ATP-dependent kinase; ORF3, a transporter; and ORF4, a dehydrogenase. The predicted product of ORF1 showed no homology to previously identified proteins and contains all the conserved amino acids within the aldose 1-epimerase protein motif. Examination of the ptxs-ORF1 intergenic region (using promoter fusion experiments) showed that no potential promoter exists. An isogenic mutant defective in ORF1 was constructed in the P. aeruginosa strain PAO1. In contrast to its parent strain, the mutant failed to grow on a minimal medium in which 2-ketogluconate was the sole carbon source. Similarly, a previously constructed ptxS isogenic mutant of PAO1 did not grow in a minimal medium containing 2-ketogluconate as the sole carbon source. Furthermore, a plasmid carrying a fragment that contains ptxS and ORFs 1-4 complemented the defect of the previously described P. aeruginosa 2-ketogluconate-negative mutant. In the presence of 10 mM 2-ketogluconate, the in vitro binding of PtxS to a DNA fragment that carries either OP1 or OP2 was inhibited. These results suggest that: (i) ptxS together with the other four ORFs constitute the 2-ketogluconate utilization operon (kgu) in P. aeruginosa. Therefore, ORFs 1-4 were designated kguE, kguK, kguT and kguD respectively. (ii) PtxS regulates the expression of the kgu operon by binding to two operators (OP1 and OP2) within the operon; and (iii) 2-ketogluconate is the molecular inducer of the kgu operon or the molecular effector of PtxS.     

Common antigen lipopolysaccharide from Pseudomonas aeruginosa AK1401 as a receptor for bacteriophage A7.

A-band, a D-rhamnose-containing common lipopolysaccharide antigen isolated from Pseudomonas aeruginosa AK1401, was found to be a receptor for bacteriophage A7. The phage-borne rhamnanase was capable of hydrolyzing the A-band to expose core-lipid A containing only two or three rhamnose repeats. Interaction of the hydrolyzed A-band with core- or lipid A-specific monoclonal antibodies revealed that common epitopes exist in the inner core and lipid A regions, while the outer core of A-band appears to be different from that of the serotype-specific (B-band) lipopolysaccharide.     

Production of Pyoluteorin and Its Derivatives from n-Paraffin by Pseudomonas aeruginosa S10B2

In investigations of biological active substances in metabolites of n-paraffin-utilizing microorganisms, Pseudomonas aeruginosa (Schroeter) S10B2 isolated from soil was found to produce pyoluteorin and its derivatives. One derivative was identified as 3′-nitropyoIuteorin, a new metabolite of microorganisms. Some of these products were found to have antimicrobial activity in vitro and in vivo. In addition, the herbicidal activity of these products was discovered.     

Characterization of the outer membrane protein OprF of Pseudomonas aeruginosa in a lipopolysaccharide membrane by computer simulation

The N-terminal domain of outer membrane protein OprF of Pseudomonas aeruginosa forms a membrane spanning eight-stranded antiparallel beta-barrel domain that folds into a membrane channel with low conductance. The structure of this protein has been modeled after the crystal structure of the homologous protein OmpA of Escherichia coli. A number of molecular dynamics simulations have been carried out for the homology modeled structure of OprF in an explicit molecular model for the rough lipopolysaccharide (LPS) outer membrane of P. aeruginosa. The structural stability of the outer membrane model as a result of the strong electrostatic interactions compared with simple lipid bilayers is restricting both the conformational flexibility and the lateral diffusion of the porin in the membrane. Constricting side-chain interactions within the pore are similar to those found in reported simulations of the protein in a solvated lipid bilayer membrane. Because of the strong interactions between the loop regions of OprF and functional groups in the saccharide core of the LPS, the entrance to the channel from the extracellular space is widened compared with the lipid bilayer simulations in which the loops are extruding in the solvent. The specific electrostatic signature of the LPS membrane, which results in a net intrinsic dipole across the membrane, is found to be altered by the presence of OprF, resulting in a small electrically positive patch at the position of the channel.