Properties of a deep Proteus R mutant isolated from clinical material.

Some biological features of a deep P. mirabilis 17301 R mutant isolated from the urine of a patient with chronic UTI were studied and compared with similar features of P. mirabilis S forms and five induced Proteus R mutants of different chemotypes. There were no differences in lethal toxicity and adhesion to human uroepithelial cells. Of all the R mutants tested, two of them, 17301 and R4, exhibited strong cell-bound hemolytic activity. The P. mirabilis R 17301 was characterized as the most invasive (tested in L929 mouse fibroblasts) compared to the other Proteus S and R forms. The structure of PS from a clinical R mutant investigated and the results of serological studies prove that this mutant belongs to the Rc chemotype.

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

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.

Immunochemical studies on R mutants of Yersinia enterocolitica O:3.

Three mutants of Yersinia enterocolitica O:3, namely: YeO3-R1, YeO3-RfbR7 and YeO3-c-trs8-R were classified on the basis of sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) profile of isolated lipopolysaccharides (LPS) as belonging to the Ra- (the first) and the Rc-type (the other two mutants). Methylation analysis, in addition to 13C and 1H NMR studies of purified core oligosaccharides revealed structures similar to those established previously for the full core of Y. enterocolitica O:3 in the case of the Ra mutant, and identical to that reported for the Rc mutant Ye75R, in the case of the two other mutants. The O-specific sugar, 6d-L-altrose, which forms a homopolymeric O-chain, was present in small amounts in all three LPS preparations, as well as in the core oligosaccha ride preparations along with the Ra and the Rc sugars, characteristic of the Y. enterocolitica O:3 core. This result is in line with genetic data, indicating that it is the inner core region which is the receptor for the O-specific chain in Y. enterocolitica O:3. This region seems likewise to be the anchoring region for the enterobacterial common antigen (ECA), as shown by SDS/PAGE/Western blot analysis with monoclonal antibodies against ECA. In addition, we also demonstrated that the Ye75R mutant Rc and its parental strain Ye75S, both were ECA-immunogenic strains. So far, ECA-immunogenic strains, i.e. those with LPS-linked ECA, were only identified in E. coli mutants of the R1, R4 and K-12 serotype.

Structure of a new acidic O-antigen of Proteus vulgaris O22 containing O-acetylated 3-acetamido-3,6-dideoxy-D-glucose.

The acidic O-specific polysaccharide of Proteus vulgaris O22 was studied using 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC) experiments, and the following structure for the branched pentasaccharide repeating unit was established: [sequence: see text] where Qui3NAc is 3-acetamido-3,6-dideoxyglucose, O-acetylation of QuiNAc at position 4 is stoichiometric and at position 2 nonstoichiometric. Serological relationships of P. vulgaris O22 with some other Proteus strains were substantiated on the level of the O-antigen structures.

Structure of the acidic O-specific polysaccharide from Proteus vulgaris O39 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid.

The O-specific polysaccharide of Proteus vulgaris O39 was found to contain a new acidic component of Proteus lipopolysaccharides, 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid (di-N-acetylpseudaminic acid, Pse5Ac7Ac). The following structure of the polysaccharide was determined by NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, ROESY, and 1H,(13)C HMQC experiments, along with selective cleavage of the polysaccharide by solvolysis with anhydrous trifluoromethanesulfonic (triflic) acid: -->8)-beta-Psep5Ac7Ac-(2-->3)-alpha-L-FucpNAc-(1-->3)-alpha-D-GlcpNAc-(1--> The structure established is unique among the O-specific polysaccharides, which is in accordance with classification of the strain studied into a separate Proteus serogroup.

Structure of an O-acetylated acidic O-specific polysaccharide of Proteus vulgaris O46.

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O46 and studied by chemical methods (O-deacetylation, sugar and methylation analyses, partial solvolysis) and 1H and 13C NMR spectroscopy. Solvolysis of the O-deacetylated polysaccharide with trifluoromethanesulfonic acid resulted in a alpha-D-GlcpNAc-(1 --> 3)-D-GlcA disaccharide that demonstrated the usefulness of this reagent for selective cleavage of heteropolysaccharides. The following structure for the polysaccharide was established: --> 4)-alpha-D-Glcp6Ac(1 --> 3)-beta-D-GlcpA4Ac-(1 --> 3)-alpha-D-GlcpNAc-(1 --> 3)-beta-D-GlcpA4Ac-(1 --> where the degree of O-acetylation is approximately 65% at position 6 of Glc and 80-95% at position 4 of GlcA residues.

[Tests for studying invasive properties of selected Proteus mirabilis strains]. / Badanie wlasciwosci inwazyjnych wybranych szczepów Proteus mirabilis.

Proteus mirabilis is an important pathogen of the urinary tract infections (UTI). Lipopolysaccharide (LPS, endotoxin) is one of the pathogenic factors of pathogenicity of these bacteria. In this paper we described the invasion of L929 mouse fibroblasts by P. mirabilis strains, classified into the O10, O23, O30, O43 serogroups. The maximal invasiveness was observed between 4-6 hours of incubation of the tested cells with bacteria. The cytotoxic effect slightly increased with the incubation time, probably as a result of the production of HpmA hemolysin. Incubation of L929 fibroblasts with LPS led to decrease of bacterial invasiveness. We observed that with the time of incubation of L929 cells with LPS (2-22 h), the invasiveness decreased (longer incubation time with LPS--weaker penetration).

[Studies of epitope specificity of polyclonal antibodies against Proteus mirabilis R mutants]. / Badania swoistosci epitopowej przeciwcial poliklonalnych przeciwko mutantom R Proteus mirabilis.

The chemical structure of the following P. mirabilis R mutants lipopolysaccharide (LPS) were already established: R110/1959 (Ra), R4/028 (Rc) and R45/1959 (Re). In this report we focus on P. mirabilis R5/O28, R13/1959 and R14/1959 and R14/1959. The last one corresponds to Salmonella transient forms, and synthesis truncated core oligosaccharide lacking terminal DD-Hep and nevertheless substituted by T polysaccharide whose structure occurred to be similar to P. penneri 42 O-repeating unit. The knowledge of chemical structure of P. mirabilis R mutants lipopolysaccharides led us to the study of the epitope specificity of rabbit polyclonal R specific antisera. The results show strong structural and serological relatedness of LPS from P. mirabilis R110 and R13. Antibodies against P. mirabilis R4 recognize in homologous LPS an epitope sharing oligosaccharide Glc-Hep. The serological studies revealed also close similarities of LPS from P. mirabilis R14 and P. mirabilis S1959, O28 as well as P. penneri 42. These data indicate that polyclonal antibodies against P. mirabilis R14 are directed against four epitopes: two in T-polysaccharide (D-Glc-(beta 1,4)-D-Glc and terminal GalA residue) and two in core oligosaccharide (D-Glc-(alfa 1,6)-D-Glc and terminal GlcNAc residue) of lipopolysaccharide molecule.

[Characterization of hemolytic activity of Proteus penneri]. / Charakterystyka aktywnosci hemolitycznej Proteus penneri.

Bacteria belonging to the genus Proteus synthesise two kinds of hemolysins HpmA and HlyA which represent "RTX proteins". In previous papers we described the production of an extracellular HlyA hemolysin by some P. penneri strains. Now we are reporting on the synthesis by P. penneri, typical for P. mirabilis HpmA hemolysin. There were identified two P. penneri strains 5 and 37 in which both hpmA and hlyA regions are present. In two other strains P. penneri 13 and 44 only hlyA region was found, whereas in strain P. penneri 42 operon hpmA was identified. The production of HpmA hemolysin was revealed in the cases of P. penneri 5, 42 and P. mirabilis 03 and 1959. The dynamics of HlyA hemolysin synthesis by P. penneri 44 was also investigated and its highest activity was observed during logarithmic phase of growth of bacterial culture. HlyA hemolysin was isolated from culture filtrate by precipitation with polyethylene glycol 4000. The invasiveness of HpmA+ and/or HlyA+ P. penneri strains was also checked by use of mouse L929 fibroblasts. Both kinds of strains were able to penetrate tested cells. The invasion of L929 fibroblasts by strains producing HlyA hemolysin is accompanied by cytotoxic effect.

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 and serological specificity of a new acidic O-specific polysaccharide of Proteus vulgaris O45.

The following structure of the O-specific polysaccharide (OPS) of Proteus vulgaris O45 lipopolysaccharide (LPS) was established using 1H- and 13C-NMR spectroscopy, including two-dimensional NOESY and H-detected 1H, 13C heteronuclear multiple-quantum coherence ( HMQC) experiments: [structure: see text text] Immunochemical studies, using rabbit polyclonal anti-P. vulgaris O45 serum and LPS, OPS and Smith-degraded OPS of P. vulgaris O45, showed the importance of beta-D-GlcA in manifesting the serological specificity of the O-antigen studied.

Structure and epitope characterisation of the O-specific polysaccharide of Proteus mirabilis O28 containing amides of D-galacturonic acid with L-serine and L-lysine.

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.

Structural and serological studies on a new acidic O-specific polysaccharide of Proteus vulgaris O32.

The following structure of the O-specific polysaccharide chain (O-antigen) of the Proteus vulgaris 032 lipopolysaccharide (LPS) was established by 1H-NMR and 13C-NMR spectroscopy, including two-dimensional NOESY and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments: -->2)-alpha-L-RhapI-(1-->2)-alpha-L-RhapII-(1-->4)-beta-D-++ +GalpA(I)-(1-->3)-beta-D-GlcpNAc-(1-->4)-alpha-D-GalpA(II)-(1-- >. In addition, an O-acetyl group was detected, which, most probably, is located at position 3 of a part of RhapI residues. Serological studies, using rabbit polyclonal anti-(P. vulgaris 032) serum, homologous and heterologous Proteus O-antigens and related artificial antigens, revealed the importance of an a-D-GalA-associated epitope in manifesting the immunospecificity of P. vulgaris 032 and substantiated serological relationships between the O-antigen studied and those of some other Proteus strains.

New structures of the O-specific polysaccharides of Proteus. 3. Polysaccharides containing non-carbohydrate organic acids.

Four new Proteus O-specific polysaccharides were isolated by mild acid degradation from the lipopolysaccharides of P. penneri 28 (1), P. vulgaris O44 (2), P. mirabilis G1 (O3) (3), and P. myxofaciens (4), and their structures were elucidated using NMR spectroscopy and chemical methods. They were found to contain non-carbohydrate organic acids, including ether-linked lactic acid and amide-linked amino acids, and the following structures of the repeating units were established: [Figure: see text], where (S)-Lac and (R)-aLys stand for (S)-1-carboxyethyl (residue of lactic acid) and N(epsilon)-[(R)-1-carboxyethyl]-L-lysine ("alaninolysine"), respectively. The data obtained in this work and earlier serve as the chemical basis for classification of the bacteria Proteus.

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.