The chemical composition of the lipopolysaccharide of Pseudomonas aeruginosa

1. Lipopolysaccharide was isolated from both cell walls and acetone-dried whole cells of Pseudomonas aeruginosa (N.C.T.C. 1999). 2. Closely similar products are obtained, although that from whole cells cannot be completely freed from small amounts (2-7%) of residual nucleic acids. 3. The lipid moiety (23-33%) has a similar amino sugar backbone to that of lipids of enterobacterial lipopolysaccharides, but contains different hydroxy acids (2- and 3-hydroxydodecanoic acid and 3-hydroxydecanoic acid). 3-Hydroxytetradecanoic acid is absent, and 3-hydroxydodecanoic acid is the main N-acylating acid. No clear evidence permitting a distinction between the possibilities that phosphodiester or glycosidic linkages exist between the glucosamine residues was obtained. 4. Identifiable sugars (glucose, rhamnose, 3-deoxy-2-octulonic acid and heptose) account for less than 20% of the lipopolysaccharide, and alanine, galactosamine and fucosamine are apparently components of the polysaccharide moiety. 5. The polysaccharide moiety is unusual in that it is not readily obtained from the lipopolysaccharide by treatment with dilute acetic acid, which does, however, solubilize much of the phosphorus of the lipopolysaccharide. 6. The ;polysaccharide' fraction (approx. 21%) obtained by treatment with dilute acetic acid contains only a small proportion of the total polysaccharide components, and in one case only 45% of the fraction was accountable for in terms of identifiable components. 7. Evidence suggests that unidentified nitrogenous components are concentrated in the residual material after removal of both the lipid and the ;polysaccharide' fraction from the lipopolysaccharide.     

The effect of lipopolysaccharide composition on the ultrastructure of Pseudomonas aeruginosa.

The surface structure of Pseudomonas aeruginosa PACl and PAClR and of lipopolysaccharide-defective mutants derived from them was studied by negative-staining and thin-section electron microscopy and compared with that of a rough mutant with wild-type lipopolysaccharide. The rough mutant and the parent strains had fairly smooth outer layers. Negatively stained preparations of all the mutants lacking polymerized O-antigenic sidechains, including a semi-rough mutant, showed numerous blebs on the surface. In thin sections of these mutants occasional extrusions from the surface were seen. They appeared to consist of material extruded from the outer membrane, but there was no evidence to suggest they were complete unit membranes. Polymerized O-antigenic side-chains in the lipopolysaccharide appear to be required to produce the wild-type appearance of the outer membrane in P. aeruginosa.     

The chemical composition of the lipopolysaccharide from Pseudomonas aeruginosa strain PAO and a spontaneously derived rough mutant.

The chemical composition of the lipopolysaccharide (LPS) of the smooth strain Pseudomonas aeruginosa PAO 307 and a spontaneously derived rough mutant, obtained by selection for resistance to the LPS-specific phage E79, are compared. The rough LPS was shown to contain lipid A, heptose, 2-keto 3-deoxyoctonic acid, galactosamine, alanine and phosphate but lacked glucose, rhamnose and fucosamine which were important constituents, on a weight basis, of the smooth LPS. These results, and chromatographic analysis of the polysaccharide fraction indicate that the rough strain lacked side chain material and was defective in its inner core region. The chemical date obtained were consistent with a core in the PAO strain similar to that of strain NCTC 1999, enhancing the evidence for a common core polysaccharide in the LPS of P. aeruginosa strains.     

The purification and chemical composition of the lipopolysaccharide of Pseudomonas alcaligenes

1. A method for obtaining lipopolysaccharide free from glycosaminopeptide from isolated cell walls of Pseudomonas alcaligenes is discussed. 2. About 70-75% of the lipopolysaccharide and 86-90% of the isolated lipid A have been accounted for in terms of identifiable components. 3. Hydrolysates of lipid A contain mainly inorganic phosphate, glucosamine, O-phosphorylglucosamine and fatty acids (dodecanoic acid, dodec-2-enoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid), of which the last is the main N-acylating acid of the glucosamine backbone. 4. Material corresponding to the polysaccharide moiety of the lipopolysaccharide is extensively degraded. 5. Solubilization of the lipopolysaccharide by using sodium deoxycholate appreciably affects the chemical composition of the material.     

Further studies on Pseudomonas aeruginosa LasA: analysis of specificity

Full elastolytic activity in Pseudomonas aeruginosa is a result of the combined activities of elastase, alkaline proteinase, and the lasA gene product, LasA. The results of this study demonstrate that an active fragment of the LasA protein which is isolated from the culture supernatant fraction is capable of degrading elastin in the absence of elastase, thus showing that LasA is a second elastase produced by this organism. In addition, it is shown that LasA-mediated enhancement of elastolysis results from the separate activities of LasA and elastase upon elastin. The LasA protein does not affect the secretion or activation of a proelastase as previously proposed in other studies. Furthermore, LasA has specific proteolytic capability, as demonstrated by its ability to cleave beta-casein. Preliminary analysis of beta-casein cleavage in the presence of various protease inhibitors suggests that LasA may be classified as a modified serine protease.     

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.     

Structural studies on the core and the O-polysaccharide repeating unit of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide.

The structure of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa immunotype 1 was studied after mild acid and strong alkaline degradations by MS and NMR spectroscopy. Three types of LPS molecules were found, including those with an unsubstituted glycoform 1 core (A) or an isomeric glycoform 2 core substituted with one O-polysaccharide repeating unit (B) or with a long-chain O-polysaccharide. Therefore, of two core glycoforms, only glycoform 2 accepts the O-polysaccharide. In the structures A and B, Kdo, Hep, Hep7Cm, GalNAcAN3Ac, GalNFoAN, QuiNAc, GalNAla represent 3-deoxy-d-manno-octulosonic acid, l-glycero-d-manno-heptose, 7-O-carbamoyl-l-glycero-d-manno-heptose, 2-acetamido-3-O-acetyl-2-deoxygalacturonamide, 2-formamido-2-deoxygalacturonamide, 2-acetamido-2,6-dideoxyglucose and 2-(l-alanylamino)-2-deoxygalactose, respectively; all sugars are in the pyranose form and have the d configuration unless otherwise stated. One or more phosphorylation sites may be occupied by diphosphate groups. In a minority of the LPS molecules, an O-acetyl group is present in the outer core region at unknown position. The site and the configuration of the linkage between the O-polysaccharide and the core and the structure of the O-polysaccharide repeating unit were defined in P. aeruginosa immunotype 1. The QuiNAc residue linked to the Rha residue of the core was found to have the beta configuration, whereas in the interior repeating units of the O-polysaccharide this residue is in the alpha-configuration. The data obtained are in accordance with the initiation of biosynthesis of the O-polysaccharide of P. aeruginosa O6, which is closely related to immunotype 1, by transfer of d-QuiNAc-1-P to undecaprenyl phosphate followed by synthesis of the repeating O-antigen tetrasaccharide.     

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.     

Computer simulation of the rough lipopolysaccharide membrane of Pseudomonas aeruginosa.

Lipopolysaccharides (LPSs) form the major constituent of the outer membrane of Gram-negative bacteria, and are believed to play a key role in processes that govern microbial metal binding, microbial adsorption to mineral surfaces, and microbe-mediated oxidation/reduction reactions at the bacterial exterior surface. A computational modeling capability is being developed for the study of geochemical reactions at the outer bacterial envelope of Gram-negative bacteria. A molecular model for the rough LPS of Pseudomonas aeruginosa has been designed based on experimentally determined structural information. An electrostatic model was developed based on Hartree-Fock SCF calculations of the complete LPS molecule to obtain partial atomic charges. The exterior of the bacterial membrane was assembled by replication of a single LPS molecule and a single phospholipid molecule. Molecular dynamics simulations of the rough LPS membrane of P. aeruginosa were carried out and trajectories were analyzed for the energetic and structural factors that determine the role of LPS in processes at the cell surface.     

Antigenic epitope in Pseudomonas aeruginosa lipopolysaccharide immunologically cross-reactive with Escherichia coli 026 lipopolysaccharide

The human monoclonal antibody MH-4H7 recognizes the lipopolysaccharide outer core region of some Pseudomonas aeruginosa strains and in of some Pseudomonas aeruginosa strains and in particular strongly binds to strains of Lányi serotype 04. In this paper, we report that this monoclonal antibody also reacts with Escherichia coli O26 LPS. However, our results suggest that the previous reported immunological cross reaction between P. aeruginosa 04 and E. coli O26 strains (which was observed by using antisera against heat-stable antigens) is not due to the similarity of the O-polysaccharides.     

Influence of nutrient media on the chemical composition of the exopolysaccharide from mucoid and non-mucoid Pseudomonas aeruginosa

Two mucoid Pseudomonas aeruginosa strains and their non-mucoid revertants isolated from two different clinical origins (cystic fibrosis and bronchiectasis) were grown in various chemically defined media. The extracted exopolysaccharide was characterized by gas-liquid chromatography and 1H-NMR spectroscopy. The exopolysaccharide was always heterogeneous, with an alginate fraction and a neutral fraction essentially composed of glucose, galactose, rhamnose and hexosamines. The alginate composition (mannuronate/guluronate ratio and O-acetylation degree) changed according to the carbon source in nutrient media and whether the strains tested were responding differently to these environmental stimuli. In all cases, the best carbon source for the alginate production was glycerol: the two cystic fibrosis strains produced a predominantly O-acetylated alginate whereas only the mucoid bronchiectasis strain produced a polymannuronate exopolysaccharide.     

Monolayer film behavior of lipopolysaccharide from Pseudomonas aeruginosa at the air-water interface

Lipopolysaccharide (LPS) is an essential biomacromolecule making up approximately 50% of the outer membrane of gram-negative bacteria. LPS chemistry facilitates cellular barrier and permeability functions and mediates interactions between the cell and its environment. To better understand the local interactions within LPS membranes, the monolayer film behavior of LPS extracted from Pseudomonas aeruginosa, an opportunistic pathogen of medical importance, was investigated by Langmuir film balance. LPS formed stable monolayers at the air-water interface and the measured lateral stresses and modulus (rigidity) of the LPS film in the compressed monolayer region were found to be appreciable. Scaling theories for two-dimensional (2D) polymer chain conformations were used to describe the pi-A profile, in particular, the high lateral stress region suggested that the polysaccharide segments reside at the 2D air-water interface. Although the addition of monovalent and divalent salts caused LPS molecules to adopt a compact conformation at the air-water interface, they did not appear to have any influence on the modulus (rigidity) of the LPS monolayer film under biologically relevant stressed conditions. With increasing divalent salt (CaCl2) content in the subphase, however, there is a progressive reduction of the LPS monolayer's collapse pressure, signifying that, at high concentrations, divalent salts weaken the ability of the membrane to withstand elevated stress. Finally, based on the measured viscoelastic response of the LPS films, we hypothesize that this property of LPS-rich outer membranes of bacteria permits the deformation of the membrane and may consequently protect bacteria from catastrophic structural failure when under mechanical-stress.     

Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis

Natriuretic peptides of various forms are present in animals and plants, and display structural similarities to cyclic antibacterial peptides. Pretreatment of Pseudomonas aeruginosa PAO1 with brain natriuretic peptide (BNP) or C-type natriuretic peptide (CNP) increases bacterium-induced glial cell necrosis. In eukaryotes, natriuretic peptides act through receptors coupled to cyclases. We observed that stable analogs of cAMP (dibutyryl cAMP) and cGMP (8-bromo-cGMP) mimicked the effect of brain natriuretic peptide and CNP on bacteria. Further evidence for the involvement of bacterial cyclases in the regulation of P. aeruginosa PAO1 cytotoxicity by natriuretic peptides is provided by the observed doubling of intrabacterial cAMP concentration after exposure to CNP. Lipopolysaccharide (LPS) extracted from P. aeruginosa PAO1 treated with both dibutyryl cAMP and 8-bromo-cGMP induces higher levels of necrosis than LPS extracted from untreated bacteria. Capillary electrophoresis and MALDI-TOF MS analysis have shown that differences in LPS toxicity are due to specific differences in the structure of the macromolecule. Using a strain deleted in the vfr gene, we showed that the Vfr protein is essential for the effect of natriuretic peptides on P. aeruginosa PAO1 virulence. These data support the hypothesis that P. aeruginosa has a cyclic nucleotide-dependent natriuretic peptide sensor system that may affect virulence by activating the expression of Vfr and LPS biosynthesis.     

Variability in Pseudomonas aeruginosa lipopolysaccharide expression during crude oil degradation

Bacterial utilization of crude oil components, such as the n-alkanes, requires complex cell surface adaptation to allow adherence to oil. To better understand microbial cell surface adaptation to growth on crude oil, the cell surface characteristics of two Pseudomonas aeruginosa strains, U1 and U3, both isolated from the same crude oil-degrading microbial community enriched on Bonny Light crude oil (BLC), were compared. Analysis of growth rates demonstrated an increased lag time for U1 cells compared to U3 cells. Amendment with EDTA inhibited U1 and U3 growth and degradation of the n-alkane component of BLC, suggesting a link between cell surface structure and crude oil degradation. U1 cells demonstrated a smooth-to-rough colony morphology transition when grown on BLC, while U3 cells exhibited rough colony morphology at the outset. Combining high-resolution atomic force microscopy of the cell surface and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracted lipopolysaccharides (LPS), we demonstrate that isolates grown on BLC have reduced O-antigen expression compared with that of glucose-grown cells. The loss of O-antigen resulted in shorter LPS molecules, increased cell surface hydrophobicity, and increased n-alkane degradation.     

Promises and pitfalls of Pseudomonas aeruginosa lipopolysaccharide as a vaccine antigen

Antibodies directed to the Pseudomonas aeruginosa lipopolysaccharide (LPS) O-antigens have clearly shown to mediate the most effective immunity to infection caused by LPS-smooth strains. Such strains are major causes of disease in immunocompromised hosts such as burn or cancer patients, individuals in intensive care units, and those who utilize extended-wear contact lenses. Yet producing an effective vaccine composed of non-toxic, immunogenic polysaccharides has been challenging. The chemical diversity among the different O-antigens representative of the 20 major serotypes, plus additional diversity among some O-antigens representing variant subtype antigens, translates into a large degree of serologic variability that increases the complexity of O-antigen specific vaccines. Further complications come from the poor immunogenicity of the major protective epitope expressed by some O-antigens, and a large degree of diversity in animal responses that preclude predicting the optimal vaccine formulation from such studies. Nonetheless human trials over the years of vaccines eliciting O-antigen immunity have been encouraging, though no vaccine has yet been fully evaluated and found to be clinically efficacious. Newer vaccine approaches such as using polysaccharide-protein conjugates and passive therapy with monoclonal or polyclonal immune sera offer some additional means to try and produce an effective immunotherapeutic reagent for this problematic pathogen.     

Sedimentation-equilibrium studies of the polysaccharide components of Pseudomonas aeruginosa

Mild acid hydrolysis of lipopolysaccharide antigens from seven different serotype strains antigen immunotypes nos. 1–7 [in the classification of Fisher, M. W., Devlin, H. B. & Gnabasik, F. J. (1969) J. Bacteriol. 98 , 835–836] of Pseudomonas aeruginosa gave polysaccharide components of high molecular weight, which were isolated by gel filtration and dialysis. These components were examined by ultracentrifugation at equilibrium with the Rayleigh interferometric optical system. The partial specific volumes were calculated from densities obtained by using a mechanical oscillator. The average molecular weights (M _n, M _w, and M _z) were calculated and compared to evaluate the polydispersity of the polysaccharides. The nonideality was investigated by varying the rotor speed, the height of the solution column, and the concentrations of the polysaccharide fractions. The molar masses were found to range from 14,000 for the polysaccharide from immunotype two to 24,000 for that from immunotype one, when extrapolated to zero rotor speed and solution column height.