Characterization of anti-ECA antibodies in rabbit antiserum against rough Yersinia enterocolitica O:3.

Enterobacterial common antigen (ECA) is a characteristic surface component in bacteria belonging to the Enterobacteriaceae family. It is generally integrated in the outer membrane via a linkage to phosphatidylglycerol (ECA(PG)) and at the same time in some special cases via a linkage to lipopolysaccharide (ECA(LPS)); the latter form is immunogenic. Yersinia enterocolitica O:3 expresses both ECA(PG) and ECA(LPS). To study whether ECA-immunogenicity of Y. enterocolitica O:3 is temperature-regulated, rabbits were immunized with ECA-expressing Y. enterocolitica O:3 bacteria grown at 22 and 37°C. To induce minimal amount of anti-LPS antibodies, immunization was performed with YeO3-c-trs8-R, an LPS mutant missing both O-polysaccharide and the outer core hexasaccharide. However, abundant antibodies specific for LPS core were still present in the obtained antisera such that the reactivity of ECA-specific antibodies could not be detected. To obtain "monovalent" anti-ECA antisera, the sera were absorbed with ECA-negative bacteria. Absorption with live bacteria removed efficiently the anti-LPS antibodies, whereas this was not the case with boiled bacteria. Western blotting revealed that the specificity of the monovalent anti-ECA antiserum was different from that of a monoclonal anti-ECA antibody (mAb 898) as it did not react with ECA(PG), and this suggested that in Y. enterocolitica O:3 ECA(LPS) only one or two ECA repeat unit(s) is/are linked to LPS. Both ECA(PG) and ECA(LPS) expression were found to be regulated by temperature and repressed at 37°C.

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.

4-Amino-4-deoxy-L-arabinose in LPS of enterobacterial R-mutants and its possible role for their polymyxin reactivity.

The content of 4-amino-4-deoxy-L-arabinopyranose (L-Arap4N) and the phosphate substitution pattern of the LPS of various strains from Salmonella minnesota, Yersinia enterocolitica and Proteus mirabilis was determined by GC/MS, HPLC and 31P-NMR. These data allowed us to examine the possible role of these components for the polymyxin B-binding capacity of LPS and for the minimal inhibiting concentration (MIC) and the minimal bactericidal concentration (MBC) of polymyxins B and E towards the respective R-mutants. Contrary to other investigated Re-, Rd- and Rc-mutants of S. minnesota, strain R595 (Re-mutant) showed about a 90% substitution of the ester-linked phosphate-group with L-Arap4N, whereas the L-Arap4N content of the other S. minnesota strains amounted to 17-25%. Neither the binding capacity of LPS to polymyxin B, determined by a bioassay, nor the MIC- and MBC-values of the R-mutants were significantly affected by this alteration. Similar results were obtained after using the temperature-dependent changes in the L-Arap4N-content and phosphate substitution pattern of Y. enterocolitica 75R. In order to explore the relevant polymyxin B binding site, lipid A samples with or without substitution of their ester-linked phosphate group were prepared and subjected to the polymyxin-binding assay. The results obtained so far indicated that the inner core bound L-Arap4N, detected in all resistant strains investigated, may play a decisive role in the decreased binding of polymyxin B, responsible for the bacterial resistance towards polymyxin(s).

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.

Galacturonic acid as the terminal constituent in the R core polysaccharide of proteus R110 (Ra) mutant.

Terminal non-reducing position of GalUA residue in the R core region of P. mirabilis R110 (Ra)mutant was established by GLC/MS analysis of methylated degraded polysaccharide. This position of GalUA is exceptional as compared with the terminal constituents present in the known structures of R core polysaccharides in Enterobacteriaceae. As it was shown in serological study, GalUA does not play a role of immunodominant in the examined Proteus R core region.

The structure of the carbohydrate backbone of the core-lipid A region of the lipopolysaccharide from Proteus mirabilis serotype O28.

The following structure of the lipid A-core region of the lipopolysaccharide (LPS) from Proteus mirabilis serotype O28 was determined using NMR, MS, and chemical analysis of the core oligosaccharide, obtained by mild acid hydrolysis of LPS, and of the products of alkaline deacylation of the LPS: carbohydrate sequence [see text] where S = beta-GalALys (amide of beta-D-galactopyranosyluronic acid with the alpha-amino group of L-lysine) or beta-GalALys4PEtN are present in the ratio of approximately 1:1. beta-GalA and Ara4N (indicated by bold italics) are present in non stoichometric amount. All sugars are present in the pyranose form and all except L-Ara4N have the D configuration.

Chemical and immunochemical studies on lipopolysaccharides of Coxiella burnetii phase I and phase II.

Lipopolysaccharides of Coxiella burnetii phase I and II were comparatively investigated by chemical and immunochemical methods. LPS of phase I (LPS I) and phase II cells (LPS II) show no serological cross reaction, indicating that the serological determinants of LPS II are masked in LPS I. Chemical analysis of LPS I and II show that phase I and II cells can be considered as S and R forms of Coxiella burnetii. The structure of LPS II has recently been elucidated and shows a dimannosylated core of an alpha(1,3)-linked heptose-disaccharide which is attached to a "KDO-like" substance. In enterobacterial core-types, alpha(1,3)-linked heptose-disaccharide is also part of the inner core structure, although the heptose occurring in enterobacterial R cores is the L-glycero-D-manno-heptose. In Coxiella burnetii we have only the rare D-glycero-D-manno-heptose which is the biosynthetic precursor of the former and is in many enteric LPS, present only in addition to L-glycero-D-mannoheptose. In these R-cores, it is occupying mostly terminal positions (Radziejewska-Lebrecht et al., 1981) and is absent from the main chain. The complete structure of LPS I is not yet available, but some important points could recently be clarified. The immunodominant sugars in LPS I are C-3-branched sugars, 6-deoxy-3-C-methyl-L-gulose (L-virenose) and 3-C-(hydroxymethyl)-L-lyxose (dihydro-hydroxy-L-streptose). These two sugars have not been found so far in other lipopolysaccharides and the latter one not previously in any other natural product. Their identification is based on GLC-MS comparison with authentic and synthetic compounds. Both branched sugars (and in addition part of the mannose) are the terminal sugars in LPS I. Sites of attachment of phase I-specific sugars to the LPS II-core are: the 3-position of a branched heptose and, presumably, the 4-position of a terminal D-mannose. The extreme acid-lability of the linkages of both branched sugars was investigated in detail and is caused by the nature of the branched sugars (deoxyhexose with bulky axial substituents; pentofuranose with axial OH-groups). No information is so far available on the (penultimate) sugars to which the branched sugars are linked, but methylation analyses with LPS I, and with the recently described I/CR mutant, which is selectively lacking the virenopyranose, are presently performed.

[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.

[Further types of core lipopolysaccharides in Proteus]. / Dalsze typy polisacharydu rdzeniowego (core) rodzaju Proteus.

Comparative analysis of the chemical composition of 11 core oligosaccharides isolated from lipopolysaccharides of the wild (S) and phenotypically rough (R) strains Proteus mirabilis (nine) and Proteus vulgaris (two) allowed to recognize three new types Proteus core, classified as IV, V, VI. All of them contained D-galactose and D-galactosamine in addition to common core constituents: D-glucose, D-galacturonic acid, L-glycero-D-manno-heptose, KDO, EtN described for Proteus core types I, II, III (6, 7, 8). D-glucosamine was characteristic for Proteus core type VI whereas D-glycero-D-manno-heptose for types V and VI.

The core region of Proteus mirabilis R110/1959 lipopolysaccharide.

The complete core structure present in the lipopolysaccharide of the R mutant R110/1959 from Proteus mirabilis (Proteus type II core) was investigated using methylation analysis and a number of degradation methods such as Smith degradation and beta-elimination. These studies combined with earlier work on a Rc-type mutant of P. mirabilis O28 (R4/O28) which shares the same inner core region, allowed formulation of the complete core structure of the Proteus type II core as shown in Scheme 1. (formula; see text) A characteristic feature of the Proteus core of type II is the presence of two units of D-galacturonic acid (DGalA); one in terminal, the other one in a chain-linked position. In addition, the presence of the two isomers of glycero-D-manno-heptose (LDHep and DDHep) and the lack of galactose are conspicuous. DDHep occupies a terminal position in the external core region, whereas the three units of LDHep in addition to dOclA form, as in other enterobacterial core types, the internal core region. The taxonomic significance of the presence of DGalA in the Proteus type II core, but also in all R cores of other Proteeae investigated so far, will be discussed.

Phosphate localization in carbohydrates - a study on enterobacterial lipopolysaccharides.

The localization of the phosphate substituents in the core oligosaccharide of the lipopolysaccharides of Enterobacteriaceae has been reported for Salmonella minnesota and Escherichia coli B only. In these cases the localizations were done by a beta-elimination reaction in mild alkaline solution after periodate oxidation. We report now on a method generally applicable on carbohydrates. The localization of phosphate groups and the extent of substitution with phosphate residues in carbohydrates can be determined by the following reaction sequence: methylation, dephosphorylation, and reetherification (labelling) with C2H3J or C2H5J followed by derivatizing to partially methylated alditol acetates and analysis by combined gas liquid chromatography/mass spectrometry. The results presented here are obtained by application of this method to isolated core oligosaccharides of lipopolysaccharides from E. coli C23.1, E. coli C71, E. coli F2515, and P. mirabilis R4/O 28. Phosphate is localized at C-4 of the chain heptoses in the lipopolysaccharides of E. coli C and E. coli R4, and at C-7 of the branching heptose in the lipopolysaccharide of P. mirabilis R4/O 28.

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.

Structure of the heptose region of lipopolysaccharies from Rhodospirillum tenue.

There is a common structure (core region) in the lipopolysaccharides of Rhodospirillum tenue. It is composed of a branched trisaccharide of L-glycero-D-mannoheptose (and of 2-keto-3-deoxyoctonate), as revealed by methylation analyses of degraded polysaccharides of four different R. tenue strains. The structure is similar or might even be identical to the inner core of enterobacterial O antigens. In addition, each of the four R. tenue lipopolysaccharides contains a strain-specific region that consists of heptose(s) (L-glycero-D-mannoheptose or D-glycero-D-mannoheptose or both) or hexoses. There is a partial substitution of the core region and the strain-specific region by phosphorus, showing microheterogeneity.

3-C-branched aldoses in lipopolysaccharide of phase I Coxiella burnetii and their role as immunodominant factors.

Mild acid hydrolysis with 1% acetic acid (100 degrees C, 15-60 min) of lipopolysaccharide (LPS) isolated from Coxiella burnetii phase I cells leads to a drastic decrease in its serological reactivity as shown by the passive hemolysis test. This decrease in reactivity occurs parallel or even prior to the cleavage of LPS into free lipid A and the polysaccharide moiety. During this mild hydrolysis two unusual sugars (X and Y) are released from the LPS, which were obtained in pure state by thin-layer chromatography. Analysis of their alditol acetate derivatives by gas chromatography/mass spectrometry revealed that sugar X is a 6-deoxy-3-C-methyl-hexose and sugar Y a 3-C-(hydroxymethyl)-pentose. Using a range of authentic standards and different thin-layer and gas chromatographic conditions, X could be recognized as 6-deoxy-3-C-methyl-gulose (virenose), very probably as the L form of this sugar (L-virenose). Y has been identified as 3-C-(hydroxymethyl)-lyxose (dihydrohydroxystreptose) by comparing it with newly synthesized 3-C-(hydroxymethyl)-pentoses (Dahlman, O., Garegg, P. J., Mayer, H., Schramek, S., unpublished results). Both branched sugars are (at least partially) in terminal positions since methylation analysis of LPS afforded (mainly) their permethylated derivatives. This analysis further showed virenose to be linked in C. burnetii phase I LPS as pyranose and dihydro-hydroxystreptose as furanose. The terminal linkage and the chemical nature of X and Y are in accordance with the observed acid-lability of the serological determinants.

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.

Lipopolysaccharide of Providencia rettgeri. Chemical studies and taxonomical implications.

The chemical constitutional analysis of the lipopolysaccharide (LPS) isolated from Providencia rettgeri was carried out. Polyacrylamide gel electrophoresis using sodium dodecylsulfate or sodium deoxycholate showed that the lipopolysaccharide mostly consisted of short sugar chains. The lipid A was precipitated out after mild acid hydrolysis of LPS. From the supernatant degraded polysaccharide and unsubstituted core fractions were isolated. Compositional analysis of the core material revealed the presence of galacturonic acid, galactose, glucose, glucosamine, L-glycero-D-manno-heptose, 3-deoxy-D-manno-octulosonic acid, alanine and phosphorus. Methylation analysis of the core material indicated the presence of terminal units of glucose, galacturonic acid and glucosamine. The chemical structure of the lipid A was elucidated. It constitutes a beta-1,6-glucosamine disaccharide substituted on either side by ester and glycosidically-bond phosphate residues. The ester-bound phosphate was found to be substituted by a 4-amino-4-deoxy-L-arabinosyl residue. The amino groups of the backbone disaccharide are N-acylated by 3-O-(14:0)14:0 and 3-O-14:0. Two hydroxyl groups of the disaccharide are esterified by 3-O-(14:0)14:0 and 3-O-14:0. The taxonomical importance of these structural details will be discussed.

Structural studies on the core and lipid A region of a 4-amino-L-arabinose-lacking Rc-type mutant of Proteus mirabilis.

The structure of the 4-amino-L-arabinose-lacking lipopolysaccharide of the Proteus mirabilis Rc-type mutant R4, derived from wild-type O28, was elucidated. The lipopolysaccharide core structure has previously been partially characterized. The linkage between heptose and deoxyoctulosonic acid(dOclA) is now reported, as well as the structure of the lipid A moiety of this mutant strain. Besides the tentative identification of an alpha-linked glucosamine disaccharide in the lipid A backbone accompanying the usual beta 1----6-linked glucosamine-disaccharide, the only significant structural variation to previous studies was the lack of substitution of the C-4' phosphate by 4-amino-L-arabinose. In addition, the substitution at C-8 of one dOclA unit by 4-amino-L-arabinose, previously reported for the R45 mutant of P. mirabilis 1959, is lacking in this R mutant. Also in addition to previous findings, the terminal unit of heptose was found to be substituted at C-7 with phosphorylethanolamine (PEtN) and not only with phosphate, although this substitution is not complete as demonstrated by the relevant signals in 31P-NMR. Additional studies with the wild-type strain P. mirabilis O28 revealed the presence of 4-amino-L-arabinose in both the core and the lipid A regions suggesting that the R4 mutant is defective in the biosynthesis of this amino sugar rather than in its transfer. Otherwise the lipid A regions of the mutant and the wild-type strain show no structural differences. The following formula is proposed for the lipopolysaccharide of 4-amino-L-arabinose-lacking mutant R4/O28 P. mirabilis: (Formula; see text)

Structural studies on the glucose-heptose region of the Proteus mirabilis R core.

Methylation analysis of the core oligosaccharide of the Proteus mirabilis mutant R4 (derived from serotype 028 was carried out in order to obtain information on the internal (glucose-heptose) region of the P. mirabilis R core. The isolated core oligosaccharide was composed of glucose, L-glycero-D-manno-heptose, 3-deoxy-D-manno-octulosonic acid (dOclA) and phosphorus in a molar ratio of about 1:2:1:1.4. It was methylated either directly or after dephosphorylation. To localize the position of the phosphate substituents, the permethylated product was dephosphorylated with hydrogen fluoride and the oligosaccharide obtained was remethylated using C2H3I. Location of phosphate at C-7 of the terminal heptose was shown by isolation of the sugar phosphate from partial hydrolysates and gas-liquid chromatography/mass spectrometry of the permethylated product. Combining the data of the methylation analysis with the data of an NMR study allows one to formulate the structure of the core oligosaccharide as folllows: (formula: see text).