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PubMed1969

The chemical composition of the lipopolyacarideof 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.

Acetates↗

PubMed1972

Further studies of the chemical composition of the lipopolysaccharide of Pseudomonas aeruginosa.

1. Qualitative and quantitative analytical results for the lipopolysaccharide from acetone-dried cells of Pseudomonas aeruginosa (N.C.T.C. 1999) are presented and possible contamination of the material with nucleic acid was further examined. 2. Additional sugars detected (only in large-scale hydrolysates) were mannose and arabinose; traces of spermidine and putrescine were also found. 3. The heptose component is l-glycero-d-mannoheptose. 4. The thiobarbituric acid-positive component is a 3-deoxy-2-octulonic acid, of which only 35-40% links lipid A to the polysaccharide. This linkage is not broken by hydrolysis with acetic acid up to 0.08m. 5. Liberation of lipid A required hydrolysis with 0.1m-hydrochloric acid, which substantially degraded the polysaccharide moiety. 6. Fractions obtained from the degraded polysaccharide by high-voltage electrophoresis were examined; in these, the alanine/galactosamine molar ratio is approx. 1. 7. Hydrazinolysis of whole lipopolysaccharide showed that at least 40% of the alanine is in amide linkage, possibly with galactosamine. 8. Lipid A, solubilized by alkaline methanolysis was fractionated; most of the phosphorus of the higher-molecular-weight fractions was released as P(i) by a phosphomonoesterase. 9. Hydrazinolysis of lipid A destroyed approx. 80% of the glucosamine, and glycosidically linked glucosamine oligosaccharides could not be isolated.

Alanine↗

PubMed1976

Dehydrogenation of the phosphonate analogue of glucose 6-phosphate by glucose 6-phosphate dehydrogenase.

6,7 -Dideoxy-alpha-D-gluco-heptose 7-phosphonic acid, the isosteric phosphonate analogue of glucose 6-phosphate, was synthesized in six steps from the readily available precursor benzyl 4,6-O-benzylidene-alpha-D-glucopyranoside. The analogue is a substrate for yeast glucose 6-phosphate dehydrogenase, showing Michaelis-Menten kinetics at pH7.5 and 8.0. At both pH values the Km values of the analogue are 4-5 fold higher and the values approx. 50% lower than those of the natural substrate. The product of enzymic dehydrogenation of the phosphonate analogue at pH8.5 is itself a substrate for gluconate 6-phosphate dehydrogenase.

Deoxy Sugars↗

PubMed2000

Lipopolysaccharide core phosphates are required for viability and intrinsic drug resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that is notorious for its intrinsic drug resistance. We have used chemical and genetic techniques to characterize three putative kinase genes that are involved in the addition of phosphate to the inner core region of P. aeruginosa lipopolysaccharide. The first gene is a waaP homologue, whereas the other two (wapP and wapQ) are unique to P. aeruginosa. Repeated attempts using a variety of membrane-stabilizing conditions to generate waaP:Gm (Gm, gentamicin) or wapP:Gm mutants were unsuccessful. We were able to generate a chromosomal waaP mutant that had a wild-type copy of either waaPPa or waaPEc in trans, but were unable to cure this plasmid-borne copy of the gene. These results are consistent with the fact that P. aeruginosa mutants lacking inner core heptose (Hep) or phosphate have never been isolated and demonstrate the requirement of Hep-linked phosphate for P. aeruginosa viability. A wapQ:Gm mutant was isolated and it had an unaltered minimum inhibitory concentration (MIC) for novobiocin and only a small decrease in the MIC for sodium dodecyl sulphate (SDS), suggesting that the loss of a phosphate group transferred by WapQ may only be having a small impact on outer-membrane permeability. Nuclear magnetic resonance and methylation linkage analysis showed that WaaPPa could add one phosphate to O4 of HepI in a Salmonella typhimurium waaP mutant. The expression of WaaPPa increased the outer-membrane integrity of these complemented mutants, as evidenced by 35-fold and 75-fold increases in the MIC for novobiocin and SDS respectively. The S. typhimurium waaP mutant transformed with both waaP and wapP had over 250-fold and 1000-fold increases, respectively, in these MICs. The inner core phosphates of P. aeruginosa appear to be playing a key role in the intrinsic drug resistance of this bacterium.

Carbohydrate Sequence↗

PubMed2003

Structural characterization of a novel branching pattern in the lipopolysaccharide from nontypeable Haemophilus influenzae.

Structural analysis of the lipopolysaccharide (LPS) from nontypeable Haemophilus influenzae strain 981 has been achieved using NMR spectroscopy and ESI-MS on O-deacylated LPS and core oligosaccharide (OS) material as well as by ESI-MSn on permethylated dephosphorylated OS. A heterogeneous glycoform population was identified, resulting from the variable length of the OS branches attached to the glucose residue in the common structural element of H. influenzae LPS, l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-[beta-d-Glcxp-(1-->4)]-l-alpha-d-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A. Notably, the O-6 position of the beta-d-Glcp residue was either substituted by PCho or the disaccharide branch beta-d-Galp-(1-->4)-d-alpha-d-Hepp, while the O-4 position was substituted by the globotetraose unit, beta-d-GalpNAc-(1-->3)-alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp, or sequentially truncated versions thereof. This is the first time a branching sugar residue has been reported in the outer-core region of H. influenzae LPS. Additionally, a PEtn group was identified at O-3 of the distal heptose residue in the inner-core.

Acetylation↗

PubMed2005

The role of galacturonic acid in outer membrane stability in Klebsiella pneumoniae.

In most members of the Enterobacteriaceae, including Escherichia coli and Salmonella, the lipopolysaccharide core oligosaccharide backbone is modified by phosphoryl groups. The negative charges provided by these residues are important in maintaining the barrier function of the outer membrane. Mutants lacking the core heptose region and the phosphate residues display pleiotrophic defects collectively known as the deep-rough phenotype, characterized by changes in outer membrane structure and function. Klebsiella pneumoniae lacks phosphoryl residues in its core, but instead contains galacturonic acid. The goal of this study was to determine the contribution of galacturonic acid as a critical source of negative charge. A mutant was created lacking all galacturonic acid by targeting UDP-galacturonic acid precursor synthesis through a mutation in gla(KP). Gla(KP) is a K. pneumoniae UDP-galacturonic acid C4 epimerase providing UDP-galacturonic acid for core synthesis. The gla(KP) gene was inactivated and the structure of the mutant lipopolysaccharide was determined by mass spectrometry. The mutant displayed characteristics of a deep-rough phenotype, exhibiting a hypersensitivity to hydrophobic compounds and polymyxin B, an altered outer membrane profile, and the release of the periplasmic enzyme beta-lactamase. These results indicate that the negative charge provided by the carboxyl groups of galacturonic acid do play an equivalent role to the core oligosaccharide phosphate residues in establishing outer membrane integrity in E. coli and Salmonella.

Carbohydrate Sequence↗

PubMed1976

Immunochemical characterization of the "native" type III polysaccharide of group B Streptococcus.

The type III polysaccharide of -roup B Streptococcus has been isolated and purified by a method that employs washing of intact cells at neutral pH. That the polysaccharide prepared by this procedure is the "native" type III antigen is suggested by its molecular size in excess of 10(6) daltons, its degradation by acid and heat treatment to a fragment with immunologic characteristics of the classical HCl antigen, and its type-specific serologic activity. The type III polysaccharide in native form contains sialic acid, galactose, glucose, glucosamine, heptose, and mannose. It is acidic in nature, is resistant to neuramindiase degradation, contains no O-acetyl groups, and does not share antigenic determinants with capsular type K1 antigen of Escherichia coli or Group B polysaccharide antigen of Neiserria meningitidis.

Epitopes↗

PubMed1976

Chemical and biological characterization of the lipopolysaccharide of Bacteroides fragilis subspecies fragilis.

Bacterial lipopolysaccharides extracted from Bacteroides fragilis subspecies fragilis lacked 2-keto-3-deoxyoctonate and heptose, sugars which make up part of the inner core of most bacterial endotoxins. Over 98% of the lipid portion of this material could be removed easily with chloroform-methanol and alcohol, a finding which indicates a loose association between the polysaccharide and lipid moieties. The lipopolysaccharides caused gelation of limulus lysate at a concentration significantly higher than that for the endotoxin of Salmonella typhi. None of the extracts was lethal in 10-day-old chick embryos at doses of greater than 200 mug per egg, whereas the endotoxin of Neisseria meningitidis was lethal at a dose of 1.2 mug per egg. The local Shwartzman reaction could not be induced by levels of B. fragilis endotoxin of up to 1,000 mug per rabbit, whereas a (control) endotoxin of S. typhi induced this phenomenon at a level of 3 mug per rabbit. Intact oxygen-killed B. fragilis failed to provoke the local Shwartzman reaction at doses of 2,500 mug. These results indicate that B. fragilis has a lipopolysaccharide different from that of most gram-negative bacteria. Although it retains some of the chemical and biologic properties of classical endotoxin, it seems to lack others. This observation may have significant clinical implications.

Animals↗

PubMed1978

Characterization of lipopolysaccharide of Haemophilus influenzae.

Lipopolysaccharide from strains of Haemophilus influenzae was extracted and isolated by the hot phenol-water procedure. The preparations were relatively insoluble in water but could be solubilized with surface-active agents. The preparations contained carbohydrate (30%), fatty acid (29%), and phosphate (4.7%); protein content was less than 1%. Thin-layer chromatography, gas-liquid chromatography, and colorimetric assays detected glucose, galactose, glucosamine, heptose, and a 2-keto-3-deoxy-octonate-like molecule (less than 1%). Neither methylpentose nor dideoxyhexose was detected. The lipid portion was composed of fatty acids common to lipopolysaccharide of Salmonella. The preparations provoked positive dermal Shwartzman reactions and biphasic febrile responses in rabbits, responses typical of endotoxic activity. The 50% lethal dose for mice was decreased from 16.5 microgram/g to 0.015 microgram/g by concomitant administration of actinomycin D. The preparations were shown to be polyclonal activators of bone marrow-derived (B) cells. Limulus lysate gelation was seen with 8.0 ng of lipopolysaccharide. Preliminary hemagglutination data suggested at least three different antigenic factors associated with the lipopolysaccharide of H. influenzae type b. The H. influenzae lipopolysaccharide appeared biologically similar to that of enterobacteria but chemically different.

Animals↗

PubMed1980

Immunochemical and biological properties of the outer membrane-associated lipopolysaccharide and protein of Rochalimaea quintana.

The outer membrane complex of Rochalimaea quintana was isolated by use of ethylene-eiaminetetraacetate and was compared biochemically and biologically both with lipopolysaccharide (LPS) isolated by phenol-water extraction of whole organisms and with lipids isolated by chloroform-methanol extractions of the phenol-water insoluble residues. The outer membrane consisted of protein and LPS components, as distinguished by precipitin tests with sera from patients with trench fever or tests with hyperimmune animal sera. The outer membrane protein component, but not LPS, also reacted with sera from infections with Rickettsia tsutsugamushi. The LPS contained 2-keto-3-deoxy-octonate and heptose. The outer membrane and phenol-extracted LPS were reactive in the chick embryo lethality test, limulus assay, and complement activation. Outer-membrane activity was confined to the LPS component. The lipid extracts were reactive in chick embryo lethality and limulus assays, but did not activate complement.

Animals↗

PubMed1975

Studies on lipopolysaccharides isolated from strains of Neisseria gonorrhoeae.

Lipopolysaccharides, extracted by phenol-water from five strains fo Neisseria gonorrhoeae, were purified by treatment with ribonuclease followed by multiple washes. These preparations were fatal to mice when administered in submicrogram amounts with actinomycin D, the LD50 values varying from 4 to 16 mug/kg. Analyses showed that all preparations contained glucose, galactose, glucosamine, heptose, 2-keto-3-deoxyoctonic acid and phosphate. All the lipopolysaccharides contained the same fatty acids, namely beta-OH-10:0, beta-OH-12:0, beta-OH-14:0, 12:0, 14:0,16:0, 16:1, 18:0 and 18:1. We were unable to detect significant differences between the lipopolysaccharides of virulent and avirulent gonococci or between penicillin-sensitive and resistant strains. Gonococcal lipopolysaccharides appeared to lack O-antigen side chains.

Animals↗

PubMed1996

The O polysaccharide chain of the lipopolysaccharide from Vibrio cholerae O76 is a homopolymer of N-[(S)-(+)-2-hydroxypropionyl]-alpha-L-perosamine.

Chemical and serological studies of LPS from Vibrio cholerae O76 (O76) were performed. The LPS of O76 contained D-glucose, D-galactose, L-glycero-D-manno-heptose, D-fructose, D-glucosamine, D-quinovosamine (2-amino-2,6-dideoxy-D-glucose) and L-perosamine (4-amino-4,6-dideoxy-L-mannopyranose). The sugar composition of the LPS from O76 was quite similar to that of LPS from V. cholerae O1 with the exception of the presence of a small amount of D-galactose in the LPS of O76. However, perosamine, a major sugar component of the LPS from O76, was in the L configuration in contrast to the D configuration of the perosamine in the LPS of V. cholerae O1. The L-perosamine was N-acylated with an (S)-(+)-2-hydroxypropionyl group in the LPS from O76. Structural analysis by NMR spectroscopy, as well as GC/MS, revealed that the O polysaccharide chain of the LPS from O76 was an alpha(1-->2)-linked homopolymer of N-[(S)-(+)-2-hydroxypropionyl]-L-perosamine. The serological cross-reactivity between the LPS of O76 and the LPS from other strains, such as V. cholerae O1 (Ogawa and Inaba O forms), Vibrio bio-serogroup 1875 (Original and Variant strains), V. cholerae O140 (Hakata) and Yersinia enterocolitica O9, was examined in passive haemolysis tests with sheep red blood cells that had been sensitized with LPS and antisera raised against whole cells of these bacteria. The latter six strains have in common the O antigen that includes Inaba antigen factor C, in addition to their own O-antigenic factors. Thus, they crossreact serologically. The O polysaccharide chains of the LPS of these six trains are known to consist exclusively of alpha(1-->2)-linked D-perosamine homopolymers and differences are found only among the N-acyl substituents. In passive haemolysis tests, the LPS of O76 did not cross-react serologically with any of the other LPS examined. Thus, the results obtained in this study support the hypothesis that Inaba antigen factor C, associated with the O antigens of these six strains, which include V. cholerae O1, is related substantially and exclusively to their alpha(1-->2)-linked homopolymers of N-acylated D-perosamine, and not to such homopolymers of N-acylated L-perosamine.

Acetylation↗

PubMed1993

Chemical analysis of lipopolysaccharides of Shigella sonnei form II strains expressed by cloned form I antigen genes.

A compositional sugar analysis was carried out on lipopolysaccharide (LPS) from Shigella sonnei form II in which a plasmid with cloned form I antigen genes had been introduced. The recipient form II strains contained galactose, glucose, heptose, glucosamine, and 2-keto-3-deoxyoctonic acid (KDO) (2: 3: 1: 2: 2) in its LPS, while the transformant form I LPS contained, besides these sugars, N-acetyl-L-altrosaminouronic acid as an additional sugar constituent, which is known to be one of the antigenic determinants of form I antigen.

Cloning, Molecular↗

PubMed2005

Novel lipopolysaccharide biosynthetic genes containing tetranucleotide repeats in Haemophilus influenzae, identification of a gene for adding O-acetyl groups.

Many of the genes for lipopolysaccharide (LPS) biosynthesis in Haemophilus influenzae are phase variable. The mechanism of this variable expression involves slippage of tetranucleotide repeats located within the reading frame of these genes. Based on this, we hypothesized that tetranucleotide repeat sequences might be used to identify as yet unrecognized LPS biosynthetic genes. Synthetic oligonucleotides (20 bases), representing all previously reported LPS-related tetranucleotide repeat sequences in H. influenzae, were used to probe a collection of 25 genetically and epidemiologically diverse strains of non-typeable H. influenzae. A novel gene identified through this strategy was a homologue of oafA, a putative O-antigen LPS acetylase of Salmonella typhimurium, that was present in all 25 non-typeable H. influenzae, 19 of which contained multiple copies of the tetranucleotide 5'-GCAA. Using lacZ fusions, we showed that these tetranucleotide repeats could mediate phase variation of this gene. Structural analysis of LPS showed that a major site of acetylation was the distal heptose (HepIII) of the LPS inner-core. An oafA deletion mutant showed absence of O-acetylation of HepIII. When compared with wild type, oafA mutants displayed increased susceptibility to complement-mediated killing by human serum, evidence that O-acetylation of LPS facilitates resistance to host immune clearance mechanisms. These results provide genetic and structural evidence that H. influenzae oafA is required for phase variable O-acetylation of LPS and functional evidence to support the role of O-acetylation of LPS in pathogenesis.

Acetyltransferases↗

PubMed1975

Heterogeneity of the lipopolysaccharide from Pseudomonas aeruginosa.

Lipopolysaccharide isolated from pseudomonas aeruginosa PAC1 and its phage-resistant mutant was degraded by mild acid hydrolysis into lipid A and three major polysaccharide-containing fractions which were separated on Sephadex G-75. The low-molecular-weight fraction contained glucose, rhamnose, heptose, galactosamine, alanine and phosphate. The higher-molecular-weight fractions consisted mainly of glucose, rhamnose and glucosamine together with amino compounds. Alkaline degradation of the lipopolysaccharide produced at least four different species each of which contained a low-molecular-weight polysaccharide similar if not identical to that produced by acid hydrolysis. Under certain growth conditions an abnormal lipopolysaccharide was produced which was defective in the low-molecular-weight polysaccharide and contained mainly high-molecular-weight material. Strains of different serotype yielded lipopolysaccharides which also exhibited heterogeneity but contained a low-molecular-weight polysaccharide similar to that obtained from strain PAC1 and PAC1R. It is suggested that each strain of P. aeruginosa may produce several lipopolysaccharides each containing a polysaccharide common to all. The relative proportions of the various lipopolysaccharides may be changed by growth conditions.

Alanine↗

PubMed2001

Separation of 6-deoxy-heptan [correction of 6-deoxy-heptane] from a smooth-type lipopolysaccharide preparation of Burkholderia pseudomallei.

Smooth-type lipopolysaccharide (LPS) of Burkholderia pseudomallei has been reported to contain two kinds of O-antigenic polysaccharides, a 1,3-linked homopolymer of 6-deoxy-heptose and a polymer with a repeating unit of -->3)-glucose-(1-->3)-6-deoxy-talose-(1--> with O-acetyl or O-methyl modifications. A LPS preparation containing these two polysaccharides was separated by gel-permeation chromatography in this study. Chemical analysis of the separated fractions revealed the 6-deoxy-heptan [corrected] to be a polysaccharide without a lipid portion and the polymer of glucose and 6-deoxy-talose to be an O-antigenic polysaccharide of the LPS. This result was further supported by the assay of these polysaccharide molecules for macrophage activation activity. The 6-deoxy-heptan [corrected] showed no macrophage activation, indicating that this polysaccharide was not the LPS, but one of the capsular polysaccharides of B. pseudomallei.

Animals↗

PubMed1976

Cellular monosaccharide patterns of Neisseriaceae.

Sixty-four strains of Neisseria, Moraxella, and Acinetobacter were screened for cellular monosaccharides by gas-liquid chromatography and other chromatographic techniques. The four sugars ribose, glucose, glucosamine, and 2-keto-3-deoxyoctonate (KDO) were detected in all strains. Heptose was detected only in "true neisseriae" (Neisseria gonorrhoeae, N. meningitidis, N. sicca, N. cinerea, N. flavescens, and N. elongata) and in the tentaively named species Moraxella urethralis. Some marked interspecies dissimilarities within groups were revealed. Thus, N. ovis and M. atlantae were characterized by the presence of mannose. Intraspecies differences were also encountered. N. meningitidis strains of serogroups B and C were distinguished from strains of serogroup A by their sialic acid content. This sugar was also detected in two out of three examined strains of M. nonliquefaciens. In Acinetobacter, heterogeneity of monosaccharide patterns was rather pronounced. The results show the applicability of gas chromatographic "monosaccharide" profiles fo whole cells or extracted carbohydrate in bacterial classification and identification, including differentiation at the subspecies level. In addition, such profiles may be useful for monitoring during purification of cellular polysaccharides.

Acinetobacter↗

PubMed1980

Fatty acids and neutral sugars present in lipopolysaccharides isolated from Fusobacterium species.

Lipopolysaccharides (LPS) extracted from cells of Fusobacterium necrophorum, F. mortiferum, F. Gonidiaformans. varium and single strains of F. naviforme and F. russi were analysed for sugars and fatty acids. All preparations contained a monosaccharide tentatively identified as L-glycero-D-mannoheptose, but showed variation with respect to the presence of glucose, galactose, rhamnose and another heptose isomer, tentatively identified as L-glycero-D-mannoheptose. L-glycero-D-mannoheptose was a monosaccharide of great abundance in all LPS examined. 2-keto-3-deoxy-octonate and glucosamine were present in all LPS preparations. The fatty acids present were 3-hydroxy-tetradecanoic acid as the main component, and n-tetradecanoic acid. Some LPS contained n-hexadecanoic acid. All the strains of F. nucleatum contained 3-hydroxy-hexadecanoic acid as a group-specific LPS constituent.

Calorimetry↗