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Background Proteus mirabilis is a Gram-negative bacteria most noted for its involvement with catheter-associated urinary tract infections. It is also known for its multicellular migration over solid surfaces, referred to as ‘swarming motility’. Here we analyzed the genomic sequences of two P. mirabilis isolates, designated K38 and K39, which exhibit varied swarming ability. Methods and results The isolates genomes were sequenced using Illumina NextSeq sequencer, resulting in about 3.94 Mbp, with a GC content of 38.6%, genomes. Genomes were subjected for in silico comparative investigation. We revealed that, despite a difference in swarming motility, the isolates showed high genomic relatedness (up to 100% ANI similarity), suggesting that one of the isolates probably originated from the other. Conclusions The genomic sequences will allow us to investigate the mechanism driving this intriguing phenotypic heterogeneity between closely related P. mirabilis isolates. Phenotypic heterogeneity is an adaptive strategy of bacterial cells to several environmental pressures. It is also an important factor related to their pathogenesis. Therefore, the availability of these genomic sequences will facilitate studies that focus on the host–pathogen interactions during catheter-associated urinary tract infections.

One of the most dangerous pests of cereals is Zabrus tenebrioides and, in Poland, it is becoming a serious pest. Entomopathogenic nematodes (EPNs) seem to be a very promising, biological control agent for this pest. Native EPN populations are well adapted to local environmental conditions. The current study characterized three Polish isolates of the EPN Steinernema feltiae, which differed in their effectiveness against Z. tenebrioides. In the field, isolate iso1Lon reduced the pest population by 37%, compared with 30% by isolate iso1Dan and 0% by the iso1Obl isolate; the number of plants damaged by Z. tenebrioides in the presence of the different isolates reflected the results in terms of the decrease in pest population size. After incubation in the soil for 60 days, recovered EPN juveniles of all three isolates were able to infect 93–100% of the test insects, with isolate iso1Obl again showing the lowest effectiveness. The juveniles of isolate iso1Obl were also morphometrically distinct from the other two isolates, as revealed by principal component analysis (PCA), which helped to distinguish the EPN isolates. These findings showed the value of using locally adapted isolates of EPNs; two of the three isolates randomly selected from Polish soil outperformed a commercial population of S. feltiae.

The pathogenicity of entomopathogenic nematodes (EPNs) depends directly on the presence of bacteria in the nematode digestive tracts. Based on 16S rRNA and MALDI-TOF analyses 20 isolated bacteria were assigned to 10 species with 10 isolates classified as Pseudomonas ssp. Six strains (30%) show ureolytic activity on Christensen medium. Spectroscopic analysis of the strains showed that the ureolytic activity is strongly correlated with the following wavenumbers: 935 cm(-1) in window W4, which carries information about the bacterial cell wall construction and 1158 cm(-1) in window W3 which corresponds to proteins in bacterial cell. A logistic regression model designed on the basis of the selected wavenumbers differentiates ureolytic from non-ureolytic bacterial strains with an accuracy of 100%. Spectroscopic studies and mathematical analyses made it possible to differentiate EPN-associated Pseudomonas sp. strains from clinical Pseudomonas aeruginosa PAO1. These results suggest, that infrared spectra of EPN-associated Pseudomonas sp. strains may reflect its adaptation to the host.

Purpose: Proteus mirabilis is a common pathogen associated mainly with complicated urinary tract infections and sometimes with septicemia. There is great serological diversity of the microorganism. While P. mirabilis O3 is commonly found in patients with infections, the serotype O18 rarely occurs. The O18 lipopolysaccharide contains a phosphocholine substitute, which makes it unique among Proteus strains. To explain different clinical significance of the strains we evaluated the biological activity of the lipopolysaccharides of P. mirabilis O3 and O18, as measured by interleukin-8 (IL-8) production. Materials and Methods: Three cell lines were used, namely uroepithelial cells, renal epithelial cells and monocytes. IL-8 production was determined on the protein and mRNA levels using enzyme-linked immunosorbent assay and real-time polymerase chain reaction, respectively, and CD14 expression on the cell surface was studied using flow cytometry. Results: Uroepithelial cells and monocytes reacted to lipopolysaccharides by higher IL-8 production compared with renal epithelial cells. Cell specific IL-8 response corresponded to the cell expression of CD14. Renal epithelial cells produced more IL-8 after stimulation with the phosphocholine rich lipopolysaccharide O18, although adding phosphocholine alone did not induce or increase IL-8 production. Conclusions: Our data suggest that different cells within the human urinary tract have different roles during urinary tract infection. The biological activity and pathogenicity of P. mirabilis lipopolysaccharides might be determined by their specific chemical structure.

Mild acid degradation of the lipopolysaccharide (LPS) of Proteus mirabilis O20 resulted in depolymerisation of the O-polysaccharide to give a repeating-unit pentasaccharide. A polysaccharide was obtained by O-deacylation of the LPS followed by nitrous acid deamination. The derived pentasaccharide and polysaccharide were studied by NMR spectroscopy, including 2D 1 H, 1 H COSY, TOCSY, ROESY, 1 H, 13 C HMQC and HMQC–TOSCY experiments, along with chemical methods, and the following structure of the repeating unit of the O-polysaccharide was established: As opposite to most other P. mirabilis O-polysaccharides studied, that of P. mirabilis O20 is neutral. A week serological cross-reactivity was observed between anti- P. mirabilis O20 serum and LPS of a number of Proteus serogroups with known O-polysaccharide structure. The ability of LPS of P. mirabilis O20 to activate the serine protease cascade was tested in Limulus amoebocyte lysate and in human blood plasma and compared with that of P. mirabilis O14a,14c having an acidic O-polysaccharide. The LPS of P. mirabilis O20 was found to be less active in both assays than the LPS of P. mirabilis O14a,14c and, therefore, the structurally variable O-polysaccharide may influenced the biological activity of the conserved lipid A moiety of the LPS.

A phosphorylated, choline-containing polysaccharide was obtained by O-deacylation of the lipopolysaccharide (LPS) of Proteus mirabilis O18 by treatment with aqueous 12% ammonia, whereas hydrolysis with dilute acetic acid resulted in depolymerisation of the polysaccharide chain by the glycosyl phosphate linkage. Treatment of the O-deacylated LPS with aqueous 48% hydrofluoric acid cleaved the glycosyl phosphate group but, unexpectedly, did not affect the choline phosphate group. The polysaccharide and the derived oligosaccharides were studied by NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HMQC and HMQC-TOSCY experiments, along with chemical methods, and the following structure of the pentasaccharide phosphate repeating unit was established: [carbohydrate structure in text] Where ChoP=Phosphocoline Immunochemical studies of the LPS, O-deacylated LPS and partially dephosphorylated pentasaccharide using rabbit polyclonal anti-P. mirabilis O18 serum showed the importance of the glycosyl phosphate group in manifesting the serological specificity of the O18-antigen.

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria may activate blood platelets. The aim of our study was to evaluate the effects of different forms of Proteus mirabilis LPS and isolated lipid A and polysaccharide part on the production of superoxide radicals in blood platelets and to estimate the role staurosporin, wortmannin and indomethacin on this process. We compared the generation of superoxide radicals in platelets treated with LPS after preincubation with inhibitors of the signal transduction pathways, namely staurosporin (inhibitor of protein kinase C), wortmannin (inhibitor of phosphoinositide 3-kinase), and indomethacin (inhibitor of cycloxygenase). Our results demonstrate that all LPS molecules and their fragments caused a stimulation of O2- generation in platelets (P.5). LPSS1959 had the strongest stimulatory effect. Straurosporin and wortmannin, but not indomethacin inhibited O2- production in LPS-stimulated platelets. Staurosporin (8 nM) and wortmannin (50 nM) caused about 50% inhibition of thrombin-induced O2- generation in platelets, while indomethacin (10 microM) had only a slight inhibitory effect on this process. Our results provide support that in LPS- and thrombin-activated platelets, at least part of O2- generation in platelets, while indomethacin (10 microM) had only a slight inhibitory effect on this process. Our results provide support that in LPS- and thrombin-activated platelets, at least part of O2- is generated due to the activation of the enzymes (protein kinase C and phosphoinositide 3-kinase) involved in signal transduction pathway. Cycloxygenase seems to be not involved in this process.

The structure of a neutral polysaccharide isolated by degradation with dilute acetic acid of the lipopolysaccharide (LPS) of P. mirabilis O24 has been determined recently [E. Literacka et al., FEBS Lett., 456 (1999) 227–231]. Further studies of this LPS using alkaline degradation and hydrolysis at pH 4.5 showed that the polysaccharide chain includes an acetal-linked pyruvic acid residue, which is removed completely during delipidation with acetic acid. A revision using 1H and 13C NMR spectroscopy and methylation analysis resulted in determination of the following full structure of the repeating unit of the O-specific polysaccharide: Download : Download full-size image where d -Gal3,4(S-Pyr) is 3,4-O-[(S)-1-carboxyethylidene]- d -galactose.

An O-specific polysaccharide was obtained by mild acid degradation of Proteus mirabilis O14 lipopolysaccharide (LPS) and found to contain D-galactose, 2-acetamido-2-deoxy-D-glalactose, phosphate, N-(2-hydroxyethyl)-D-alanine (D-AlaEtn), and O-acetyl groups. Studies of the initial and O-deacetylated polysaccharides using one- and two-dimensional 1H- and 13C-NMR spectroscopy, including COSY, TOCSY, NOESY, H-detected 1H,13C heteronuclear multiple-quantum coherence, and heteronuclear multiple-bond correlation experiments, demonstrated the following structure of the repeating unit: [equation: see text] This is the second bacterial polysaccharide reported to contain alpha-D-Galp6PAlaEtn, whereas the first one was the O-antigen of P. mirabilis EU313 taken erroneously as strain PrK 6/57 from the O3 serogroup [Vinogradov, E. V., Kaca, W., Shashkov, A.S., Krajewska-Pietrasik, D., Rozalski, A., Knirel, Y.A.Kochetkov, N.K. (1990) Eur. J. Biochem., 188, 645-651]. Anti-(P. mirabilis O14) serum cross-reacted with LPS of P. mirabilis EU313 and vice versa in passive hemolysis and ELISA. Absorption of both O-antisera with the heterologous LPS decreased markedly but did not abolish the reaction with the homologous LPS. These and chemical data indicated that both strains have similar but not identical O-antigens. Therefore, we propose that P. mirabilis EU313 should belong to a new subgroup of the O14 serogroup.

The O-specific polysaccharide of Proteus mirabilis O11 was studied by sugar analysis, Smith degradation, 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, and 1H-detected 1H, 13C HMQC experiments. The following structure of a pentasaccharide repeating unit of the polysaccharide was established: [formual: see text] where D-GalA6LThr is N-(D-galacturonoyl)-L-threonine. ELISA with anti-P. mirabilis O11 serum showed that D-GalA6LThr is of minor importance for manifesting the O11 immunospecificity.

The O-specific polysaccharide of Proteus vulgaris O25 was studied by acid hydrolysis and by 1H-NMR and 13C-NMR spectroscopies, including one-dimensional NOE and two-dimensional COSY and heteronuclear 13C,1H correlation (HETCOR) spectroscopy. The polysaccharide was found to contain 3-O-[(R)-1-carboxyethyl]-D-glucose (D-RGlc), and the following structure of the pentasaccharide repeating unit was established: [structure in text]

0-specific polysaccharide was isolated from Proteus penneri strain 12 (ATCC 33519) lipopolysaccharide (LPS) and studied using NMR spectroscopy, including selective spin-decoupling, one-dimensional NOE, two-dimensional homonuclear correlation spectroscopy, I3C,’H heteronuclear correlation spectroscopy and chemical methods (0-deacetylation, Smith degradation, partial acid hydrolysis followed by borohydride reduction and methylation). The amide of D-gdaCtUrOniC acid with L-threonine [D-GalA(LThr)] was identified as a constituent of the polysaccharide and the following structure of the tetrasaccharide repeating unit was established

Administration of alpha alpha-crosslinked stroma-free hemoglobin (SFH) as a cell-free resuscitation fluid is associated with multiple organ toxicities. Many of these toxicities are characteristic of the pathophysiological effects of bacterial endotoxins (lipopolysaccharide, LPS). To better understand the potential role of LPS in the observed in vivo toxicities of SFH, we examined mixtures of SFH and E. coli LPS for evidence of LPS-SFH complex formation. LPS-SFH complexes were demonstrated by three techniques: ultrafiltration through 300 kDa cut-off membranes, which distinguished LPS in complexes (87-89%300 kDa) from LPS alone (90%300 kDa); density centrifugation through 5% sucrose, which distinguished denser LPS alone from LPS-SFH complexes; and precipitation by 67% ethanol, which demonstrated 2-3 fold increased precipitability of complexes compared to SFH alone. Interaction of LPS with SFH was also associated with markedly increased biological activity of LPS, as manifested by enhancement of LPS activation of Limulus amebocyte lysate (LAL), increased release of human mononuclear cell tissue factor, and enhanced production of cultured human endothelial cell tissue factor. These results demonstrated that hemoglobin can serve as an endotoxin binding protein, and that this interaction results in the alteration of several LPS physical characteristics and enhancement of LPS biological activities.

Proteus bacteria are important human opportunistic pathogens which frequently cause urinary tract infections. According to Bergey's Manual of Systematic Bacteriology,1 this genus includes three species: P. mirabilis, P. vulgaris, and P. myxofaciens. A novel species of P. penneri has been recently proposed2,3 for strains formerly called P. vulgaris biogroup I. Proteus is an antigenically heterogeneous group of bacteria, and this is mainly associated with diverse composition and structures of O-specific polysaccharide chains of outer-membrane lipopolysaccharides (O-antigens). The Kauffman-Perch serological classification4 of P. mirabilis and P. vulgaris includes 49 O-serogroups. However, a number of S-strains remain unclassified,5 including strains of P. penneri.

Proteus mirabilis bacilli play an important role in human urinary tract infections, bacteremia, and rheumatoid arthritis. The authors previously studied human complement C3 conversion by smooth-form P. mirabilis O10, O23, O30, and O43 lipopolysaccharides (LPSs) and showed that smooth Proteus LPSs fragmented C3 in a dose- and time-dependent manner. In the present study, one smooth P. mirabilis S1959 and its two polysaccharide-truncated LPSs isolated from an R mutant strain were used to study the C3 conversion. The conversion of C3 to C3c by smooth and rough P. mirabilis LPSs was studied by capture ELISA and crossed immunoelectrophoresis. Proteins isolated from the outer membrane were analyzed by discontinuous sodium dodecyl sulfate gel electrophoresis. The smooth P. mirabilis S1959 (O3) strain was resistant to the bactericidal activity of human serum, in contrast to the Ra and Re mutant strains. The presence of an exposed core oligosaccharide in R110 LPS was not sufficient to protect the strain from serum-dependent killing. In addition to LPS structure, the outer-membrane proteins may also play roles in protecting the smooth P. mirabilis S1959 (O3) strain from the bactericidal action of serum. It was shown that the Ra P. mirabilis R110 and the Re P. mirabilis R45 mutants possess very different OMP compositions from that of the P. mirabilis S 1959 strain. Regardless of the complement resistance of the P. mirabilis strains, the S1959, R110, and R45 LPSs fragmented C3 and induced C3c neo-antigen exposure. The use of complement-deficient human serum allows the conclusion that the Re-type P. mirabilis R45 LPS fragmented C3 by the antibody-independent classical pathway.

Measurement of the main Proteus mirabilis virulence factors would increase our understanding of how the organism infects and colonizes the urinary tract. The purpose of this study was to quantify the virulence factors of twelve P. mirabilis laboratory strains and to determine whether expression of virulence factors of P. mirabilis depends on the presence of homoserine-lactone derivatives.Twelve P. mirabilis strains with defined lipopolysaccharide structures were used. The activity levels of urease, proteases, and hemolysins and the swarming abilities of P. mirabilis rods were tested by qualitative and quantitative methods. The effect of addition of acylated homoserine lactones (acyl-HSLs) was evaluated in order to determine their influence on the pathogenic features of the P. mirabilis strains.The ureolytic, proteolytic, and hemolytic abilities and the swarming motility of P. mirabilis rods were strain-specific. The P. mirabilis strains which possessed a negatively charged O-polysaccharide demonstrated strong ureolytic and proteolytic properties and faster migration speed on solid media. There was no influence of acyl-HSLs on the process of urea decomposition. The acyl-HSLs inhibited the protease activity of five P. mirabilis strains. N-butanoyl-L-homoserine lactone accelerated the migration speed of the tested P. mirabilis strains.The levels of tested virulence factors were strain-specific. The acetylated homoserine lactone derivatives modified the expression of some virulence factors of the P. mirabilis strains.

The structures of the O-polysaccharides of the lipopolysaccharides of Proteus mirabilis O7 and O49 were determined by chemical methods, mass spectrometry, including MS/MS, and NMR spectroscopy, including experiments run in an H2O/D2O mixture to reveal correlations for NH protons. The O-polysaccharides were found to contain N-carboxyacetyl (malonyl) and N-(3-carboxypropanoyl) (succinyl) derivatives of 4-amino-4,6-dideoxyglucose (4-amino-4-deoxyquinovose, Qui4N), respectively. The behavior of Qui4N derivatives with the dicarboxylic acids under conditions of acid hydrolysis and methanolysis was studied using GLC-MS.

A highly phosphorylated O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis O41 followed by GPC. The initial and dephosphorylated polysaccharides and phosphorylated products from two sequential Smith degradations were studied by 1H, 13C and 31P NMR spectroscopy and ESI-MS. The O-polysaccharide was found to have a tetrasaccharide repeating unit containing one ribitol phosphate (presumably d -Rib-ol-5-P) and two ethanolamine phosphate (Etn-P) groups, one of which is present in the stoichiometric amount and the other in a nonstoichiometric amount. The following structure of the O-polysaccharide was established

The O-antigen of Proteus mirabilis O38 was found to be unique among bacterial polysaccharides and to have the following structure: Download : Download full-size image where d -Qui4N(Ac- d -Asp) is 4-(N-acetyl- d -aspart-4-ylamino)-4,6-dideoxy- d -glucose and AcEtnP is 2-acetamidoethyl phosphate. Neither of these entities have been hitherto found in natural polysaccharides. Structural studies were performed using 1D and 2D NMR spectroscopy, including experiments run in an H2O/D2O mixture to reveal correlations for NH protons. In addition, dephosphorylation, carboxyl reduction and selective cleavages were applied. Solvolysis of the polysaccharide with anhydrous HF gave an α- d -GlcNAc-(1→3)- d -Qui4N(Ac- d -Asp) disaccharide. Solvolysis with trifluoromethanesulfonic (triflic) acid afforded d -GlcNAc6(AcEtnP), thus showing the suitability of this reagent for the preparation of phosphorylated sugar derivatives.

The effect of lipopolysaccharide (LPS, endotoxin), isolated from Proteus mirabilis S1959 strain, on red blood cell (RBC) membranes in whole cells as well as on isolated membranes was studied. Lipid membrane fluidity, conformational state of membrane proteins and the osmotic fragility of RBCs were examined using electron paramagnetic resonance spectroscopy and spectrophotometric method. Lipid membrane fluidity was determined using three spin-labeled fatty acids: 5-, 12- and 16-doxylstearic acid (5-, 12- and 16-DS). The addition of LPS S1959 to RBC suspension resulted in an increase in membrane fluidity, as indicated by 12-DS. At the concentrations of 0.5 and 1 mg/ml, LPS treatment led to a significant (P0.05) increase in lipid membrane fluidity in the deeper region of lipid bilayer (determined by 12-DS). The conformational changes in membrane proteins were determined using two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The highest concentration of endotoxin significantly (P0.05) decreased the relative rotational correlation time of ISL and significantly (P0.05) increased the osmotic fragility of RBCs. The effect of endotoxin was much more profound in isolated membranes than in intact cells treated with LPS. At the concentrations 0.5 and 1 mg/ml, LPS led to a significant increase in h(w)/h(s) ratio. These results indicated increased membrane protein mobility, mainly in the spectrin-actin complex in membrane cytoskeleton. These data suggest that LPS-induced alterations in membrane lipids and cytoskeleton proteins of RBCs lead to loss of membrane integrity.

The following structure of the O-polysaccharide (O-antigen) of the lipopolysaccharide of Proteus mirabilis O-9 was determined by NMR spectroscopy, including 2D 1 H, 1 H COSY, TOCSY, ROESY, and 1 H, 13 C HMQC experiments, along with chemical methods: Download full-size image where the degree of O-acetylation is ∼70%. Immunochemical studies using rabbit polyclonal anti- Proteus mirabilis O-9 serum showed the importance of the O -acetyl groups in manifesting the serological specificity of the O-9 antigen. Anti- P. mirabilis O-9 cross-reacted with the lipopolysaccharides (LPS) of P. vulgaris O-25 and Proteus penneri 14, which could be accounted for by a structural similarity of their O-polysaccharides.

The following structure of the O-specific polysaccharide of Proteus mirabilis O5 was established by 1H and 13C NMR spectroscopy at 500 MHz, including two-dimensional COSY, TOCSY, NOESY, and H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC) experiments: Download : Download full-size image where O-acetylation of α- d -GlcNAc at both positions is nonstoichiometric.

Strains of Proteus mirabilis belonging to serogroups O24 and O29 are frequent in clinical specimens. Anti-P. mirabilis O24 serum cross-reacted with the lipopolysaccharide (LPS) of P. mirabilis O29 and vice versa. The structures of the O-specific polysaccharides (OPSs, O-antigens) of both LPSs were established using sugar analysis and one- and two-dimensional 1H- and 13C-NMR spectroscopy and found to be different. SDS-PAGE and Western immunoblotting suggested that the serological cross-reactivity of the LPSs is due to a common epitope(s) on the core-lipid A moiety, rather than on the OPS. Therefore, the epitope specificity and the structures of the O-antigens studied are unique among Proteus serogroups.

A marked serological cross-reactivity was observed by ELISA and a precipitation test between anti-Proteus mirabilis O23 serum and the lipopolysaccharide as well as the O-specific polysaccharide from the Proteus mirabilis strain belonging to serogroup O6. The structures of the O-specific polysaccharides were elucidated using chemical and NMR spectroscopic analyses, and the only common component, 2-acetamido-2-deoxy-beta-D-glucopyranose (beta-D-GlcNAc), was revealed, which was suggested to be responsible for the cross-reactivity observed. Both anti-O23 and anti-O6 sera were shown to react with 1, 3-Linked beta-D-GlcNAc-containing O-antigen from Salmonella enterica ssp. arizonae O59 also. The lack of reactivity of Smith-degraded P. mirabilis O6 O-specific polysaccharide with homologous antiserum indicated the crucial role of alpha-D-glucuronic acid in specific antibody binding.

Toxicities of hemoglobin (Hb) solutions, which have been demonstrated in numerous animal resuscitation models, prominently include fever, hypertension, thrombocytopenia, activation of the complement and coagulation cascades, disseminated intravascular coagulation with parenchymal organ damage, reduced tolerance to sepsis, susceptibility to bacterial infections, reticuloendothelial cell blockade and lethal toxicity (Bolin et al. 1983, Bornside, Bouis, and Cohn 1970, Brandt, Frank, and Lichtman 1951, Feola et al. 1988a and 1988b, Feola et al. 1990, Marks et al. 1989, Savitsky et al. 1978, Smith et al. 1990, White et al. 1986a). In addition, recent clinical trials of cross-linked Hb have been associated with production of hypertension and gastrointestinal dysmotility. Of particularly great current interest is the recent demonstration that injection of non-lethal doses of gram-negative bacteria into animals produced 50% and 100% mortality when the animals had been pre-infused with either native or cross-linked preparations of cell-free Hb, respectively (Griffiths et al. 1995). In vitro Hb has been shown to stimulate tissue factor production by mononuclear cells (Smith and Winslow 1992), cause endothelial cell injury (Feola et al. 1989) and to activate complement (Smith and Winslow 1992). These in vivo and in vitro effects are characteristic of bacterial endotoxins (lipopolysaccharide, LPS). Investigations of the possibility that LPS may contribute to the observed side effects of Hb infusions have been a major focus of our laboratory during the past several years, and a significant role for LPS in Hb toxicity has been suggested by our studies.

Previous investigations have demonstrated that hemoglobin (Hb) is a binding protein for bacterial endotoxin (lipopolysaccharide, LPS) and that the structure and biological activity of LPS are altered in the presence of Hb. In the present study, the influence of LPS on the structure of native human HbA0 and covalently cross-linked Hb (alpha alpha Hb) was studied by analyzing the absorption and circular dichroic spectra of Hb in the wavelength region of 200-650 nm. Incubation of oxyHb with each of several LPSs resulted in a decrease in the intensity of the major Soret band at 414 nm with a shift in the maximum peak to 410 nm, decreases in the intensities of the major visible region peaks at 541 and 577 nm, and the appearance of increased absorbance in the visible region in the range of 630 nm. The resultant spectra are characteristic of methemoglobin formation. These spectral changes were time-dependent and LPS-concentration-dependent. Production of methemoglobin was prominent with chemically modified, partially deacetylated rough LPS, and was observed to a lesser extent both with native, complete rough and with native smooth LPSs. The influence of LPS on the absorption spectrum of methemoglobin also was directly tested. The conversion of methemoglobin to hemichrome in the presence of LPS was demonstrated and was shown to be reversible. Analysis of circular dichroic spectra of Hb demonstrated LPS-induced spectral changes in the visible and Soret regions consistent with the production of a substantial quantity of metHb, but did not demonstrate any alteration in the far-UV region (210-240 nm). Moreover, Hb oxygen affinity was only slightly altered after incubation with any of several LPSs. In conclusion, analyses of absorption and circular dichroic spectra reveal the potential of LPS to produce a facilitated oxidation of both alpha alpha-cross-linked human Hb and native human HbA0, without substantial changes in the secondary structure of the globin.

Purified human hemoglobin is being developed as an alternative to transfusions of homologous erythrocytes. However, toxicity associated with infusion of hemoglobin has limited the development of this resuscitation fluid. Some observed toxicities, including activation of the complement cascade, have been associated with contamination of hemoglobin solutions by bacterial endotoxin. Recent studies have demonstrated complex formation between hemoglobin and endotoxin, and have documented a resultant increase in the ability of endotoxin to activate coagulation, stimulate tissue factor production by human peripheral blood mononuclear cells, and stimulate tissue factor activity and protein synthesis in cultured human endothelial cells. The process of hemoglobin enhancement of endotoxin toxicity suggests a possible mechanism by which the consequences of endotoxin contamination of hemoglobin solutions, including complement activation, could be magnified. Therefore, we studied the potential of hemoglobin to either fix complement directly, or modify the ability of endotoxin to fix complement. Human crosslinked and native hemoglobins, at concentrations between 0.2 mg/ml and 3 mg/ml, were shown to fix complement. Complement fixation by hemoglobin was identical in normal human serum or in factor B-depleted serum, suggesting that fixation occurred via the classical pathway of complement activation. Complement fixation then was examined with a battery of smooth and rough endotoxins tested in the absence and presence of hemoglobin. Addition of hemoglobin to a solution of a rough Salmonella endotoxin partial structure, from which a single fatty acid had been hydrolyzed from the lipid A portion of the macromolecule, resulted in decreased efficiency of complement fixation. However, addition of hemoglobin had little or no effect on the intrinsic complement fixing abilities of eight other smooth endotoxins, rough endotoxins, or endotoxin partial structures. Our results demonstrated the ability of hemoglobin to fix complement at hemoglobin concentrations which would be achieved during infusion for resuscitation, but failed to demonstrate a reproducible effect of hemoglobin on the activation of complement by endotoxin.

The O-specific polysaccharide of Proteus mirabilis O28 was found to contain D-galactose, D-galacturonic acid (GalA), 2-acetamido-2-deoxy-D-glucose, L-serine, L-lysine, and O-acetyl groups in molar ratios 1:2:1:1:1:1, the amino acids being linked via their alpha-amino group to the carboxyl group of GalA. The polysaccharide was studied using 1H- and 13C-NMR spectroscopy, including selective spin-decoupling, one-dimensional total correlation spectroscopy, two-dimensional homonuclear correlation spectroscopy (COSY), heteronuclear 13C,1H COSY, one-dimensional NOE, and two-dimensional rotating-frame NOE spectroscopy and partial acid hydrolysis followed by borohydride reduction, methylation, and GLC/MS analysis of the derived glycosyl alditols. The following structure of the repeating unit was established: [formula: see text] Epitope specificity of the P. mirabilis O28 polysaccharide was analysed using a homologous rabbit polyclonal antiserum in quantitative precipitation, passive immunohemolysis, and inhibition of passive immunohemolysis. Study with related synthetic glycopolymers (2-acrylamidoethyl glycosides of amides of alpha-D-GalA with amino acids copolymerised with acrylamide) showed the importance of D-GalA(L-Lys) for manifesting serological specificity of the O-antigen. Serological cross-reactions between P. mirabilis O28, S1959, and R14/S1959 (a transient-like form) are discussed.

Cell-free hemoglobin (Hb) is a purified preparation of human hemoglobin that is being developed as a resuscitation fluid. In vivo administration of hemoglobin has resulted in significant toxicity, due in part to contamination with bacterial endotoxin (lipopolysaccharide (LPS)). To better understand this toxicity, we have studied the interaction between Hb and LPS. Mixtures of each of three different Hb preparations (cross-linked alpha alpha Hb, cross-linked carbon monoxy-alpha alpha HbCO, and non-cross-linked (native) HbAo) and LPS (Escherichia coli O26:B6 or Proteus mirabilis S1959) were examined by several independent methods for evidence of Hb.LPS complex formation. Binding assays in microtiter plates demonstrated saturable binding of LPS to immobilized Hb, with a kD of 3.1 x 10(-8) M. Binding of LPS to Hb also was demonstrated wiht a radiolabeled LPS photoaffinity probe. Ultrafiltration of Hb/LPS mixtures by 300- and 100-kDa cut-off membranes showed that the majority of LPS in these mixtures (87-97 and 64-72%, respectively) was detected in the filtrates, in contrast to the lack of filterability of LPS in the absence of Hb. Density centrifugation demonstrated that LPS co-migrated with each of the three Hbs, whereas unbound LPS had a distinctly greater sedimentation velocity than Hb or Hb.LPS complexes. Nondenaturing polyacrylamide gel electrophoresis demonstrated that in the presence of Hb, LPS migrated into the gel and co-electrophoresed with Hb, whereas LPS alone did not appreciably enter the gel. Finally, precipitation by ethanol of each of the three Hb preparations was increased in the presence of LPS compared with precipitation in the absence of LPS. Interaction of LPS with each of the three Hb preparations was also associated with altered biological activity of LPS, as shown by enhancement of LPS activation of Limulus amebocyte lysate. Therefore, our data provide several lines of independent evidence for Hb-LPS complex formation and indicated that LPS exhibited altered physical characteristics and enhanced biological activity in the presence of Hb.

The chemical composition of lipopolysaccharides isolated from respective Proteus vulgaris and P. mirabilis strains belonging to 49 different serotypes (Kauffmann and Perch classification) allowed us to divide the investigated LPS’s into 16 chemotypes. The major part of LPS’s is clustered in two chemotypes — IX and XV (4). The structure of the 0 specific part of LPS, isolated from P. vulgaris serotype 019 strain, from the chemotype IX, together with some serological data, is presented in this paper.

The mutant Proteus mirabilis R4 (R4/028) was obtained from the wild-type strain P. mirabilis 028 (F87) by ultraviolet irradiation. Isolation of R4/028 lipopolysaccharide (LPS) and the partial elucidation of the glucose-heptose region as a trisaccharide: β-glucosyl-(1→ 3/4)-L-glycero-α -D-manno-heptosyl-(1 → 4/3)-L-glycero-α-D-manno-heptosyl-7-phosphate has already been described (11). The linkage region between d0clA and heptose, the terminal and side chain-linked d0clA, the substituents of the phosphate groups and the lipid A structure were the aim of this study. In addition, we examined the effect of polymyxin B on P. mirabilis R4/028 mutant, after finding that its LPS is lacking 4-amino-4-deoxy-L-arabinose (Ara4N). The presence of that unusual aminopentose has been suggested by Vaara et al., (15, 16) to be the reason for the resistance of P. mirabilis strains towards the action of polymyxin.

The lipopolysaccharide (LPS) of a rough mutant (95R) of Vibrio cholerae Ogawa has been investigated chemically and serologically. d -Fructose was released from LPS under conditions (10m m trifluoroacetic acid, 60°, 1 h) that liberated no other sugar constituent of the LPS (2-amino-2-deoxy- d -glucose, d -glucose, l - glycero - d - manno -heptose). Upon periodate oxidation, d -fructose and d -glucose were oxidised quantitatively, whereas ∼50% of heptose was periodate-resistant. The data indicate that d -fructose does not link the polysaccharide and lipid A portion as proposed earlier, and suggest that d -fructose is present as a branch. By passive hemolysis inhibition, it was shown that the release of d -fructose paralleled the exposure of an antigenic determinant cryptic in LPS.

Three strains of B. thetaiotaomicron of different origin were investigated. Lipopolysaccharides were extracted from the studied strains using a phenol-water method. The best purification of LPS was achieved by digestion with nuclease and subsequent ultracentrifugation. Capsular material (CPS) was obtained from the most heavily encapsulated strain. The preparation were analyzed chemically, and their serological activity was determined. All antigens were active with homologous antibacterial sera in immunodiffusion, crossed immunoelectrophoresis, and passive hemagglutination tests. In the CPS equal amounts of saccharides and proteins were detected. All antigens were analyzed by polyacrylamide gel electrophoresis with SDS. Capsular antigen slowly migrated in the gel in the form of a single band. Migration pattern of lipopolysaccharides of the studied B. thetaiotaomicron strain was characteristic for S-type LPS.