The structure of the carbohydrate backbone of core-lipid A region ofthe lipopolysaccharides from Proteus mirabilis wild-type strain S1959 (serotype O3) and its Ra mutant R110/1959.

The following structure of core-lipid A region of the lipopolysaccharide (LPS) from Proteus mirabilis strain 1959 (serotype O3) and its rough mutant R110/1959 (Proteus type II core) was determined using NMR and chemical analysis of the core oligosaccharide, obtained by mild acid hydrolysis of LPS, and of the products of alkaline deacylation of the LPS: Incomplete substitutions are indicated by italics. All sugars are in pyranose form, alpha-Hep is the residue Lglycero-alpha-Dmanno-Hep, alpha-DD-Hep is the residue Dglycero-alpha-Dmanno-Hep. The differences with the previously reported structures are discussed.     

Structural and immunochemical studies on the lipopolysaccharide of the ‘T-antigen’-containing mutant Proteus mirabilis R14/1959

Abstract In DOC-PAGE, lipopolysaccharide (LPS) of Proteus mirabilis R14/1959 (Rb-type) mutant showed a ladder-like migration pattern indicating the presence of a high molecular weight polysaccharide chain. The isolated polysaccharide, called T-antigen because of similarity with the T1 chain of Salmonella friedenau LPS, contained d -glucose, d -galacturonic acid ( d -GalA), and d -GlcNAc in molar ratios 2:1:1 and was structurally different from the O-antigen of the parental S-strain P. mirabilis S1959 but identical to the O-antigen of another S-strain Proteus penneri 42. The importance of a d -GalA( l -Lys)-containing epitope, most likely present in the core region of LPS, and of GalA present in the T-antigen chain in manifesting the serological specificity of P. mirabilis R14/1959 were revealed using rabbit polyclonal homologous and heterologous R- and O-specific antisera and the appropriate antigens, including synthetic antigens which represent partial structures of various Proteus LPS.     

Interaction of lipopolysaccharide with detergents and its possible role in the detergent resistance of the outer membrane of Gram-negative bacteria.

In the presence of MgCl2, amounts of detergents which disrupted phospholipid vesicles caused lipopolysaccharide I from Proteus mirabilis to aggregate and form vesicular, membrane-like structures. Vesicle formation with P. mirabilis lipopolysaccharide II containing longer O-polysaccharide chains was extremely poor. Lipopolysaccharides of Salmonella minnesota R mutants (chemotypes Ra, Rc and Re) displayed a growing tendency for vesicle formation with increasing deficiency of the R core polysaccharide. Lipopolysaccharides of chemotypes Rc and Re produced vesicles even in the absence of MgCl2 and detergent. Spherical aggregates consisting of P. mirabilis lipopolysaccharide I MgCl2 and detergent were unable to either entrap or retain [14C]-sucrose, [3H=inulin or [3H]dextran. On the other hand, S. minnesota R mutant lipopolysaccharides of chemotypes Rc and Re could entrap all three saccharides and retain them for at least short periods of time. Leakage of [3H]-inulin out of re-lipopolysaccharide vesicles was greatly retarded by addition of MgCl2 to the vesicle system. Incorporation of P. mirabilis lipopolysaccharide I or S. minnesota Rc lipopolysaccharide into phospholipid vesicles protected these model membranes from disruption by detergent. This suggested a similar protective function of lipopolysaccharide in the outer membrane of enteric bacteria against the action of surfactants occurring in their normal intestinal habitat.     

Structural and serological studies of the related O-specific polysaccharides of Proteus vulgaris O21 and Proteus mirabilis O48 having oligosaccharide-phosphate repeating units.

The O-specific polysaccharide chains (O-antigens) of the lipopolysaccharides (LPSs) of Proteus mirabilis O48 and Proteus vulgaris O21 were found to have tetrasaccharide and pentasaccharide repeating units, respectively, interlinked by a glycosidic phosphate. Polysaccharides and an oligosaccharide were derived from the LPSs by various degradation procedures and studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, H-detected 1H,13C and 1H,31P HMQC experiments. The following related structures of the repeating units of the O-antigens were established (top: Proteus mirabilis O48; bottom: Proteus vulgaris O21) The O-specific polysaccharide of P. vulgaris O21 has the same structure as that of Hafnia allvei 744 and PCM 1194 [Petersson C., Jachymek, W., Klonowska, A., Lugowski, C., Niedziela, T. & Kenne, L. (1997) Eur. J. Biochem., 245, 668-675], except that the GlcN residue carries the N-acetyl rather than the N-[(R)-3-hydroxybutyryl] group. Serological investigations confirmed the close relatedness of the Proteus and Hafnia O-antigens studied.     

Effect of polymyxins on the lipopolysaccharide-defective mutants of Proteus mirabilis

The effects of polymyxins (Pmx) B and E on smooth and rough Proteus mirabilis strains were investigated. P. mirabilis mutant R4/028 which completely lacked 4-amino-4-deoxy-L-arabinose was sensitive towards both polymyxins, and the other P. mirabilis strains investigated were resistant. Lipopolysaccharide (LPS) from Pmx-sensitive R4/028 strain, binds 50% more Pmx B than LPS derived from resistant P. mirabilis strains. The presence of iodoacetamide, N-ethylmaleimide and chloramphenicol rendered the Pmx-resistant P. mirabilis strains sensitive towards both polymyxins.     

Structural studies on the lipopolysaccharide core of Proteus OX strains used in Weil-Felix test: a mass spectrometric approach

The core region of the lipopolysaccharides of Proteus group OX bacteria, which are used as antigens in Weil-Felix test for serodiagnosis of rickettsiosis, were studied by chemical degradations in combination with ESI FTMS, including infrared multi-photon dissociation (IRMPD) MS/MS and capillary skimmer dissociation. Structural variants of the inner core region were found to be the same as in Proteus non-OX strains that have been studied earlier. The outer core region has essentially the same structure in Proteus vulgaris OX19 (serogroup O1) and OX2 (serogroup O2) and a different structure in Proteus mirabilis OXK (serogroup O3). A fragmentation due to the rupture of the linkage between GlcN or GalN and GalA was observed in IRMPD-MS/MS of core oligosaccharides and found to be useful for screening of Proteus strains to assign structures of the relatively conserved inner core region and to select for further studies strains with distinct structures of a more variable outer core region.     

Structure and serological properties of the O-antigen of two clinical Proteus mirabilis strains classified into a new Proteus O77 serogroup

Two Proteus mirabilis strains, 3 B-m and 3 B-k, were isolated from urine and faeces of a hospitalized patient from Lodz, Poland. It was suggested that one strain originated from the other, and the presence of the bacilli in the patient's urinary tract was most probably a consequence of autoinfection. The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of P. mirabilis 3 B-m and studied by sugar analysis and nuclear magnetic resonance spectroscopy, including two-dimensional rotating frame Overhause effect spectroscopy (ROESY) and 1H,13C heteronuclear single quantum coherence (HSQC) experiments. The following structure of the linear trisaccharide-repeating unit of the O-polysaccharide was established:-->2)-beta-D-Glcp-(1-->3)-alpha-L-6dTalp2Ac-(1-->3)-beta-D-GlcpNAc-(1-->where 6dTal2Ac stands for 2-O-acetyl-6-deoxy-L-talose. It resembles the structure of the O-polysaccharide of Proteus penneri O66, which includes additional lateral residues of 2,3-diacetamido-2,3,6-trideoxy-L-mannose. The lipopolysaccharides from two P. mirabilis strains studied were serologically identical to each other but not to that from any of the existing 76 Proteus O-serogroups. Therefore, the strains were classified into a new O77 serogroup specially created in the genus Proteus. Serological studies using Western blot and enzyme-linked immunosorbent assay with intact and adsorbed O-antisera showed that the P. mirabilis O77 antigen is related to Proteus vulgaris O2 and P. penneri O68 antigens, and a putative disaccharide epitope responsible for the cross-reactivity was revealed.     

Structure of the glycerol phosphate-containing O-polysaccharides and serological studies of the lipopolysaccharides of Proteus mirabilis CCUG 10704 (OE) and Proteus vulgaris TG 103 classified into a new Proteus serogroup, O54

O-Polysaccharides were obtained from the lipopolysaccharides of Proteus mirabilis CCUG 10704 (OE) and Proteus vulgaris TG 103 and studied by chemical analyses and one- and two-dimensional (1)H and (13)C nuclear magnetic resonance spectroscopy, including rotating-frame nuclear Overhauser effect spectroscopy, H-detected (1)H,(13)C heteronuclear single-quantum spectroscopy and (1)H,(31)P heteronuclear multiple-quantum spectroscopy experiments. The Proteus mirabilis OE polysaccharide was found to have a trisaccharide repeating unit with a lateral glycerol phosphate group. The Proteus vulgaris TG 103 produces a similar O-polysaccharide, which differs in incomplete substitution with glycerol phosphate (c. 50% of the stoichiometric amount) and the presence of an O-acetyl group at position 6 of the 2-acetamido-2-deoxygalactose (GalNAc) residue. These structures are unique among the known bacterial polysaccharide structures. Based on the structural and serological data of the lipopolysaccharides, it is proposed to classify both strains studied into a new Proteus serogroup, O54, as two subgroups, O54a,54b and O54a,54c. The serological relatedness of the Proteus O54 and some other Proteus lipopolysaccharides is discussed.     

Structure of the O-specific polysaccharide of Proteus mirabilis D52 and typing of this strain to Proteus serogroup O33.

The acidic O-specific polysaccharide chain (O-antigen) of the lipopolysaccharide (LPS) of Proteus mirabilis strain D52 was studied using chemical analyses along with 1H-NMR and 13C-NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H,13C and 1H,31P HMQC experiments. The polysaccharide was found to contain D-ribitol 5-phosphate (D-Rib-ol-5-P) and ethanolamine phosphate (Etn-P) and has the following structure: D-Rib-ol-5-P (3) approximately 75% EtnP(6)-->2)-beta-D-Galp-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-GlcpNAc-(1-->). This structure is identical with that of the O-polysaccharide of P. mirabilis O33 strain 59/57, and, hence, P. mirabilis D52 belongs to the same Proteus serogroup O33. Serological studies with O-antiserum against P. mirabilis D52 confirmed this but showed that the LPS species of P. mirabilis 59/57 and D52 are not identical, having different epitopes in the core region. A serological cross-reactivity of P. mirabilis D52 O-antiserum was observed with LPS of two other Proteus strains, P. mirabilis O16 and P. penneri 103, which have structurally different O-polysaccharides. The role of charged groups, Rib-ol-5-P and Etn-P in the immunospecificity is discussed.     

Structure of the O-polysaccharide of a serologically separate strain of Proteus mirabilis, TG 332, from a new proposed Proteus serogroup O50

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis TG 332 strain. The following structure of the O-polysaccharide was determined by chemical methods along with NMR spectroscopy, including 2D COSY, TOCSY, ROESY and 1H, 13C HMQC experiments: [see equation in text]. The O-polysaccharide studied has a unique structure among Proteus O-antigens. Accordingly, P. mirabilis TG 332 is serologically separate, and we propose to classify this strain into a new Proteus serogroup, O50. The nature of minor epitopes that provide a cross-reactivity of P. mirabilis TG 332 O-antiserum with the LPS of P. mirabilis O30 and Proteus penneri 34 (O60) is discussed.     

Three enzymatic steps required for the galactosamine incorporation into core lipopolysaccharide

The core lipopolysaccharides (LPS) of Proteus mirabilis as well as those of Klebsiella pneumoniae and Serratia marcescens are characterized by the presence of a hexosamine-galacturonic acid disaccharide (αHexN-(1,4)-αGalA) attached by an α1,3 linkage to L-glycero-D-manno-heptopyranose II (L-glycero-α-D-manno-heptosepyranose II). In K. pneumoniae, S. marcescens, and some P. mirabilis strains, HexN is D-glucosamine, whereas in other P. mirabilis strains, it corresponds to D-galactosamine. Previously, we have shown that two enzymes are required for the incorporation of D-glucosamine into the core LPS of K. pneumoniae; the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS, and WabN catalyzes the deacetylation of the incorporated GlcNAc. Here we report the presence of two different HexNAc transferases depending on the nature of the HexN in P. mirabilis core LPS. In vivo and in vitro assays using LPS truncated at the level of galacturonic acid as acceptor show that these two enzymes differ in their specificity for the transfer of GlcNAc or GalNAc. By contrast, only one WabN homologue was found in the studied P. mirabilis strains. Similar assays suggest that the P. mirabilis WabN homologue is able to deacetylate both GlcNAc and GalNAc. We conclude that incorporation of d-galactosamine requires three enzymes: Gne epimerase for the generation of UDP-GalNAc from UDP-GlcNAc, N-acetylgalactosaminyltransferase (WabP), and LPS:HexNAc deacetylase.     

Structure of the O-specific polysaccharide of the bacterium Proteus vulgaris O23

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the bacterium Proteus vulgaris O23 (strain PrK 44/57) and found to contain 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, and D-galacturonic acid. Based on 1H- and 13C-NMR spectroscopic studies, including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments, the following structure of the branched tetrasaccharide repeating unit of the polysaccharide was established: [figure], where the degree of O-acetylation of the terminal GalA residue at position 4 is about 80%. A structural similarity of the O-specific polysaccharides of P. vulgaris O23 and P. mirabilis O23 is discussed.     

Structure of the O-polysaccharide of Proteus mirabilis OC (CCUG 10702) from a new proposed Proteus serogroup O75

A neutral O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis OC (CCUG 10702) and studied by sugar and methylation analyses and (1)H and (13)C NMR spectroscopy. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: [structure: see text]. Based on the unique structure of the O-polysaccharide and serological data, we propose classifying P. mirabilis OC (CCUG 10702) into a new separate Proteus serogroup O75. A weak cross-reaction of O-antiserum against P. mirabilis OC with the lipopolysaccharide of P. mirabilis O49 was accounted for by a similarity in the O-polysaccharide structures.     

Structure of the O-polysaccharide of Proteus mirabilis CCUG 10705 (OF) containing an amide of D-galacturonic acid with L-alanine

The structure of the O-polysaccharide of Proteus mirabilis CCUG 10705 (OF) was determined by chemical analyses along with one- and two-dimensional (1)H and (13)C NMR spectroscopy. The polysaccharide was found to contain an amide of D-galacturonic acid with L-alanine and based on the uniqueness of the O-polysaccharide structure and serological data, it was suggested to classify P. mirabilis OF into a new separate Proteus serogroup, O74. A weak cross-reactivity of P. mirabilis OF and P. mirabilis O5 was observed and accounted for by a similarity of their O-repeating units. The following structure of the polysaccharide of P. mirabilis OF was established: [chemical structure: see text]     

Serotyping of clinical isolates belonging to Proteus mirabilis serogroup O36 and structural elucidation of the O36-antigen polysaccharide

The O-specific polysaccharide (OPS) isolated from the lipopolysaccharide of Proteus mirabilis O36 was found to have a pentasaccharide repeating unit of the following structure: -->2)-beta-D-Ribf-(1-->4)-beta-D-Galp-(1-->4)-alpha-D-GlcpNAc6Ac-(1-->4)-beta-D-Galp-(1-->3)-alpha-D-GlcpNAc-(1-->. The structure is unique among Proteus OPS, which is in agreement with the classification of this strain into a separate Proteus O-serogroup. Remarkably, the P. mirabilis O36-polysaccharide has the same structure as the OPS of Escherichia coli O153, except that the latter is devoid of O-acetyl groups. The cross-reaction of anti-O36 antibodies with the O-part of E. coli O153 lipopolysaccharide is observed. In the present study, two steps of serotyping Proteus strains are proposed: screening of dry mass with enzyme-linked immunosorbent assay and immunoblot with the crude lipopolysaccharides. This method allowed serotyping of 99 P. mirabilis strains infecting the human urinary tract. Three strains were classified into serogroup O36. The migration pattern of these lipopolysaccharides fraction with long O-specific PSs was similar to the standard laboratory P. mirabilis O36 (Prk 62/57) lipopolysaccharide. The relatively low number of clinical strains belonging to serogroup O36 did not correspond to the presence of anti-P. mirabilis O36 antibodies in the blood donors' sera. Twenty-five percent of tested sera contained a statistically significant elevated level of antibodies reacting with thermostable surface antigens of P. mirabilis O36. The presence and amount of antibodies correlated with Thr399Ile TLR4 polymorphism types (P=0.044).     

Structural elucidation of the lipopolysaccharide core region of the O-chain-deficient mutant strain A28 from Pseudomonas aeruginosa serotype 06 (International Antigenic Typing Scheme).

The lipopolysaccharide (LPS) of the Pseudomonas aeruginosa serotype 06 rough-type mutant A28 was isolated by a modified phenol-chloroform-petroleum ether extraction method. Deoxycholate-polyacrylamide gel electrophoresis indicated a single band with mobility similar to that of the complete core region of the wild-type parent serotype 06 (International Antigenic Typing Scheme) strain. Compositional analysis of the LPS indicated that the core oligosaccharide was composed of D-glucose (three units), L-rhamnose (one unit), 2-amino-2-deoxy-D-galactose (one unit), L-glycero-D-manno-heptose (two units), 3-deoxy-D-manno-octulosonic acid (two units), L-alanine (one unit), and phosphate (two units). Under the mild conditions of hydrolysis with methanolic hydrogen chloride, a 7-O-carbamoyl substituent was observed on the second heptose residue. The glycan structure of the LPS was determined by employing one- and two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry-based methods with a backbone oligosaccharide that was obtained from the LPS by deacylation, dephosphorylation, and reduction of the terminal glucosamine. On the basis of the results of the present study and our earlier work with the P. aeruginosa 06-derived core-defective mutant R5 (H. Masoud, E. Altman, J. C. Richards, and J. S. Lam, Biochemistry, 33:10568-10578, 1994), a structural model for the complete core oligosaccharide is proposed.     

Properties of an hydrogen peroxide resistant Proteus mirabilis mutant]

A peroxide-resistant mutant (PR) was isolated from Proteus mirabilis using the hydrogen peroxide mutagenic property. Under the same conditions, resistance of mutant PR bacteria to H2O2 was 50 to 100 times greater than that of the wild type. The total amount of catalase produced by P. mirabilis PR was on the average 10 times greater than that of the wild type. When PR bacteria were subjected to high doses of H2O2 (150mM), the determination of catalasic activity in vivo increased; paradoxically, there was a net decrease in the activity of the solubilized catalase after the breakdown of the cells. The hypothesis of an enzyme transfer from the inside towards the periphery of the cells is discussed. The behavior of a membrane enzyme (L-phenylalanine oxidase) of the PR mutant shows that H2O2 may cause lesions way up to the internal membrane of bacteria.     

Structure of the neutral O-polysaccharide and biological activities of the lipopolysaccharide of Proteus mirabilis O20

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 1H,1H COSY, TOCSY, ROESY, 1H,13C HMQC and HMQC-TOSCY experiments, along with chemical methods, and the following structure of the repeating unit of the O-polysaccharide was established: [Carbohydrate structure: see text]. 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.     

Structure of the O-polysaccharide of Proteus mirabilis CCUG 10701 (OB) classified into a new Proteus serogroup, O74

An acidic O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Proteus mirabilis CCUG 10701 (OB) and studied by chemical analyses and (1)H and (13)C NMR spectroscopy. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: --> 3)-beta-D-GlcpNAc6Ac-(1 --> 2)-beta-D-GalpA4Ac-(1--> 3)-alpha-D-GalpNAc-(1 --> 4)-alpha-D-GalpA-(1 -->, where the degree of O-acetylation at position 6 of GlcNAc is approximately 50% and at position 4 of beta-GalA approximately 60%. Based on the unique structure of the O-polysaccharide and serological data, it is proposed to classify P. mirabilis CCUG 10701 (OB) into a new Proteus serogroup, O74.