Structural elucidation of the O-antigen of the Shigella flexneri provisional serotype 88-893: structural and serological similarities with S. flexneri provisional serotype Y394 (1c).

The structure of the repeating unit of the O-antigen polysaccharide from Shigella flexneri provisional serotype 88-893 has been determined. (1)H and (13)C NMR spectroscopy as well as 2D NMR experiments were employed to elucidate the structure. The carbohydrate part of the hexasaccharide repeating unit is identical to the previously elucidated structure of the O-polysaccharide from S. flexneri prov. serotype Y394. The O-antigen of S. flexneri prov. serotype 88-893 carries 0.7 mol O-acetyl group per repeating unit located at O-2 of the 3-substituted rhamnosyl residue, as identified by H2BC and BS-CT-HMBC NMR experiments. The O-antigen polysaccharide is composed of hexasaccharide repeating units with the following structure: →2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap2Ac-(1→3)[α-D-Glcp-(1→2)-α-D-Glcp-(1→4)]-ß-D-GlcpNAc-(1→. Serological studies showed that type antigens for the two provisional serotypes are identical; in addition 88-893 expresses S. flexneri group factor 6 antigen. We propose that provisional serotypes Y394 and 88-893 be designated as two new serotypes 7a and 7b, respectively, in the S. flexneri typing scheme.

Altering trends in the dominance of Shigella flexneri serotypes and emergence of serologically atypical S. flexneri strains in Dhaka, Bangladesh.

Of 469 recently isolated Shigella flexneri strains, 452 agglutinated with Shigella flexneri-specific monoclonal antibodies. Of these, 396 could be assigned to 10 of the currently recognized 15 serotypes, with S. flexneri 2b dominating (23.2%). Of the 56 untypeable strains which showed invasive properties, 17 were serologically atypical and the remaining 39 belonged to a new serotype.

Identification of Shigella flexneri subserotype 1c in rural Egypt.

In a population-based study of diarrhea in rural, northern Egypt, 60 Shigella flexneri strains were identified, of which 10 could not be definitively serotyped. Serological analysis with commercial reagents suggested that they were serotype 1, but the strains failed to react with subserotype 1a- or 1b-specific antibodies. All 10 strains reacted with MASF 1c, a monoclonal antibody specific for a provisional S. flexneri subserotype, 1c, first identified in Bangladesh and not previously detected outside of that region. Our results show that S. flexneri subserotype 1c is not unique to Bangladesh and that the inability to detect it may reflect both the limited use of suitable screening methods and the rarity of this subserotype.

Lipopolysaccharide with an altered O-antigen produced in Escherichia coli K-12 harbouring mutated, cloned Shigella flexneri rfb genes.

Cloning of the rfb genes of Shigella flexneri 2a into Escherichia coli K-12 strain DH1 results in the synthesis of lipopolysaccharides (LPS) with an O-antigen chain having type antigen IV and group antigens 3,4. During genetic studies of these rfb genes in E. coli K-12, we observed that strains harbouring plasmids with certain mutations (inversion and transposon insertions) which should have blocked O-antigen synthesis nevertheless still produced LPS with O-antigen chains. These LPS migrated differently on silver-stained SDS-polyacrylamide gels, compared with the LPS produced by wild-type rfb genes, and the group 3,4 antigens were barely detectable, suggesting that the O-antigen was altered. Investigation of the genetic determinants for production of the altered O-antigen/LPS indicated that: (i) these LPS are produced as a result of mutations which are either polar on rfbF or inactivate rfbF; (ii) the rfbX gene product (or a similar protein in the E. coli K-12 rfb region) is needed for production of the altered O-antigen in the form of LPS; (iii) the rfbG gene product is required for the production of both the parental and altered LPS; (iv) the dTDP-rhamnose biosynthesis genes are required. Additionally, an E. coli K-12 gene product(s) encoded outside the rfb region also contributes to production of the O-antigen of the altered LPS. An antiserum raised to the altered LPS from strain DH1(pPM2217 (rfbX::Tn1725)) was found to cross-react with nearly all S. flexneri serotypes, and with the altered LPS produced by other DH1 strains harbouring plasmids with different rfb mutations, as described above. The reactivity of the altered LPS with a panel of monoclonal antibodies specific for various S. flexneri O-antigen type and group antigens demonstrated that their O-antigen components were closely related to that of S. flexneri serotype 4. The RfbF and RfbG proteins were shown to have similarity to rhamnose transferases, and we identified a motif common to the N-termini of 6-deoxy-hexose nucleotide sugar transferases. We propose that the E. coli K-12 strains harbouring the mutated S. flexneri rfb genes produce LPS with a hybrid O-antigen as a consequence of inactivation of RfbF and complementation by an E. coli K-12 gene product. Analysis of the genetic and immunochemical data suggested a possible structure for the O-antigen component of the altered LPS.

Monoclonal antibodies specific for elongation factor Tu and complete nucleotide sequence of the tuf gene in Mycobacterium tuberculosis.

Monoclonal antibodies against mycobacterial antigens were produced by immunizing LOU/C rats with live Mycobacterium bovis BCG. The antibodies were characterized by an enzyme-linked immunosorbent assay and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting (immunoblotting). One antibody, MAMB 2, reactive with a 47-kDa protein was used to screen a lambda gt11 M. tuberculosis gene library (R. A. Young, B. R. Bloom, C. M. Grosskinsky, J. Ivanji, D. Thomas, and R. W. Davis, Proc. Natl. Acad. Sci. USA 82:2583-2587, 1985). Three recombinant phages reactive with MAMB 2 in plaque lysates were isolated, and part of the insert was sequenced. The mycobacterial inserts were all expressed as proteins fused with beta-galactosidase when the phages were induced as lysogens in Escherichia coli. The entire M. tuberculosis tuf gene was obtained by screening the lambda gt11 library with a DNA probe specific for the primary clones. A phage isolated from this screening was able to express the native protein in E. coli when introduced as a lysogen. A comparison of the entire gene sequence and the deduced protein sequence with the EMBL DNA and Swiss-Prot protein data libraries revealed strong homologies with elongation factors of bacteria, yeast mitochondria, and a plant chloroplast.

Use of monoclonal antibodies to type Shigella flexneri in Bangladesh.

A panel of 10 mouse and rat monoclonal antibodies specific for different type- and group-specific O-antigenic determinants of Shigella flexneri lipopolysaccharide was used to serotype 240 isolates of S. flexneri from Bangladesh. Three immunoglobulin M antibodies were used in a direct slide agglutination test; seven immunoglobulin G antibodies were absorbed to Staphylococcus aureus and used in a coagglutination assay. All but 13 of the isolates could be serotyped by using the monclonal antibodies. The six most common serotypes were (in descending order) 2a, 2b, Y (E1037), 1a, 3a, and 1b and accounted for more than 80% of all isolates. Two of the nontypable strains were found to be of a new provisional serotype of S. flexneri (T. Wehler and N.I.A. Carlin, Eur. J. Biochem. 176:471-476, 1988). The 11 remaining strains were found to be rough and therefore nontypable. The serotyping scheme based on the panel of monoclonal antibodies is specific and holds the potential to be developed into a useful tool for epidemiological investigation. The study also demonstrates that the recently described E1037 antigen is commonly found among at least four serotypes (4a, 6, X, and Y) of S. flexneri.

Structural and immunochemical studies of the lipopolysaccharide from a new provisional serotype of Shigella flexneri.

The chemical structure of the O-antigen of a proposed new provisional serotype of Shigella flexneri has been determined. Methylation analysis, GLC-MS, 1H-NMR and 13C-NMR showed that the linear O-antigenic polysaccharide is the same as for all S. flexneri [Kenne, L., Lindberg, B., Petersson, K. & Romanowska, E. (1977) Carbohydr. Res. 56, 363-370]. A novel structural feature is that the disaccharide alpha-D-Glcp-(1----2)-alpha-D-Glcp is linked to O4 of the N-acetyl-glucosamine residue. (Formula: see text) Western blotting of the lipopolysaccharide with an E. coli R3 core-specific monoclonal antibody, suggested the presence of an E. coli R3 core.

Characterization of five Shigella flexneri variant Y-specific monoclonal antibodies using defined saccharides and glycoconjugate antigens.

The structural domains of the Shigella flexneri variant Y O-antigen epitopes 3,4 have defied definition, despite knowledge of the structure of the linear polysaccharide chain of the LPS molecule. The dual epitope designation of group antigen 3,4 is based on absorption data using polyvalent rabbit antisera. Five monoclonal antibodies specific for the Y antigen, generated after immunization of BALB/c mice or LOU/C rats, were selected on the basis of ELISA by using well-characterized S. flexneri Y LPS and chemically defined glycoconjugates. Chemically defined LPS from all S. flexneri serogroups, synthetic oligosaccharides, and saccharides obtained by phage Sf6-mediated hydrolysis of the O-polysaccharide were used either as free haptens or glycoconjugates in Farr assays and ELISA titrations. Two different patterns of antibody specificities were seen: two monoclonal antibodies had combining sites recognizing the terminal nonreducing end of the O-polysaccharide complementary to the tetrasaccharide repeating unit; and three antibodies bound to intrachain determinants and had larger combining sites, possibly accommodating at least an octasaccharide. The precise specificity of these two general types of antibodies indicate that variant Y polysaccharide generates more than two O-factors.

Monoclonal antibodies specific for Shigella flexneri lipopolysaccharides: clones binding to type IV, V, and VI antigens, group 3,4 antigen, and an epitope common to all Shigella flexneri and Shigella dysenteriae type 1 stains.

Monoclonal antibodies reactive with Shigella flexneri O antigens were generated in both mouse and rat systems. Antibody-producing hybridomas were screened in an enzyme-linked immunosorbent assay using chemically defined lipopolysaccharides as antigens, and the epitope specificities were determined with a panel of lipopolysaccharides and synthetic O-antigen-specific glycoconjugates as antigens. To verify the specificity seen in the enzyme-linked immunosorbent assay, the antibodies were used in agglutination against a large number of S. flexneri strains. Monoclonal antibodies with the following specificities were identified: type, antigen IV (reactive with serotype 4a and 4b bacteria); type antigen V (reactive with serotype 5a and 5b bacteria); type antigen VI (reactive with serotype 6 bacteria); group antigen 3,4(reactive with serotype 1a, 2a, 3b, 4a, 5a, and Y bacteria); and group antigen 1 (reactive with an epitope present on all S. flexneri and Shigella dysenteriae type 1 bacteria). Furthermore, a monoclonal antibody defining a new O-antigenic epitope present on some S. flexneri strains of serotypes 4a, X, and Y was characterized (4X). The monoclonal antibodies analyzed in this study define epitopes described by polyclonal antisera (type antigens IV, V, and VI), define a hitherto uncharacterized epitope (group antigen 1), and finally identify new epitopes in what has previously been considered as one epitope (group antigen 3,4 and type antigen IV). These immunochemically characterized monoclonal antibodies may have a powerful potential in studies of the importance of humoral immunity in shigellosis.

Role of monoclonal O-antigen antibody epitope specificity and isotype in protection against experimental mouse typhoid.

A panel of 14 monoclonal antibodies with specificity for the O-antigenic polysaccharide chain of the lipopolysaccharide of the cell envelope of Salmonella typhimurium was established. The specificity of each antibody clone was determined against a set of Salmonella saccharide antigens, natural and synthetic, in passive hemagglutination and enzyme immunoassays. The monoclonal antibodies could be classified into at least five different groups: (i) O4 epitope specific, (ii) O4,12 specific, (iii) O4,12(2) specific, (iv) O5 specific, and (v) O12 specific. These specificities correspond to different structural and conformational domains of the polysaccharide chain, and often extend over more than one repeating unit (tetrasaccharide) of the polymer. The passive protection afforded by these antibodies was estimated in an experimental mouse typhoid model using S. typhimurium SH2201 for intraperitoneal challenge. Monoclonal antibodies of the IgG3 isotype were available for four of the epitope groups and were protective in the following order of activity O4 greater than O4,12 greater than O4,12(2) greater than or equal to O12. The difference between O4 and 012 antibodies was greater than 2500 fold in protective activity. Antibodies of the IgM class were highly protective irrespective of being of the O4,12 or O12 epitope specificity. Two IgA antibodies with O5 epitope specificity were not protective. The results show that both isotype and epitope specificity can be of importance for the protective ability of antibodies generated by the host.

Characterization of Shigella flexneri-specific murine monoclonal antibodies by chemically defined glycoconjugates.

Chemically defined glycoconjugates are demonstrated to have considerable potential for selecting hybridoma antibodies directed toward O-antigenic determinants, especially when used in combination with a panel of well-characterized LPS molecules. Monoclonal antibodies specific for the Shigella flexneri O-antigens of serogroup 5b, variants X and Y, were generated after immunization of BALB/c mice with killed bacterial cells, and active hybrids were selected on the basis of ELISA performed with the purified serotype-specific LPS antigen. Subsequent screening with a variety of glycoconjugates, derived from synthetic oligosaccharides and larger structures obtained by phage Sf6/endo-rhamnosidase hydrolysis of purified LPS established a detailed profile of binding characteristics for Shigella flexneri variant Y-specific antibodies. Together with the results of precipitin analysis and heavy chain isotyping experiments, a limited number of antibodies were selected as candidates for detailed studies of the antibody combining site.

Monoclonal antibodies specific for Shigella flexneri lipopolysaccharides: clones binding to type I and type III:6,7,8 antigens, group 6 antigen, and a core epitope.

Monoclonal antibodies against the Shigella flexneri lipopolysaccharide (LPS) were generated in two fusions by using the myeloma cell line Sp2/0 as a fusion partner with spleen cells from BALB/c mice immunized with S. flexneri serotypes 1b and 3a bacteria. The antibodies were characterized by immunoblotting, enzyme-linked immunosorbent assay (ELISA), hemagglutination, and coagglutination. Four different types of monoclonal antibodies were isolated: antibodies specific for the core antigen of the LPS, antibodies specific for the type I O antigen, antibodies specific for the group 6 O antigen, and antibodies specific for the type III:6,7,8 O antigen. The core-specific antibodies were shown to be specific for the Escherichia coli R3 core, which all S. flexneri LPSs tested, except for S. flexneri serotype 6 LPS, have. The type I O antigen-specific antibodies were shown to bind exclusively to S. flexneri serotypes 1a and 1b in ELISA. The type III:6,7,8 O-antigen-specific antibodies were specific for S. flexneri serotype 3a in ELISA and hemagglutination. Two different group 6 O-antigen-specific antibodies were bound. One was bound in both ELISA and hemagglutination to LPSs of S. flexneri serotypes 1b, 3a, 3b, and 4b, whereas the second was bound only to LPSs of serotypes 3a, 3b, and 4b in ELISA but to LPSs of all four serotypes in hemagglutination. The specificity of the isolated I, III:6,7,8, and group 6 monoclonal antibodies was verified by coagglutination of 363 S. flexneri clinical isolates.

Shigella flexneri O-antigen epitopes: chemical and immunochemical analyses reveal that epitopes of type III and group 6 antigens are identical.

In the present investigation we studied the nature of a Shigella flexneri O antigen, the type III antigen. No structural entity has yet been attributed to this antigen. Phenol-water-extracted lipopolysaccharides (LPSs) from all S. flexneri serotypes possessing either group 6 antigen or both type III antigen and group 6 antigen were subjected to de-O-acetylation by weak alkali treatment. Two-dimensional nuclear magnetic resonance spectra of serotype 3a native LPS and de-O-acetylated LPS were completely resolved, confirming the established structure (L. Kenne, B. Lindberg, K. Petersson, E. Katzenellenbogen, and E. Romanowska, Eur. J. Biochem. 91:279-284, 1978) and proving that no other structural alteration was introduced by the treatment. Both native and de-O-acetylated LPSs of serotypes 1b, 3a, 3b, and 4b were tested in an enzyme-linked immunosorbent assay against four absorbed rabbit anti-S. flexneri antisera and three monoclonal antibodies with specificity for different parts of the S. flexneri type III antigen and group 6 antigen. Upon de-O-acetylation, all binding capacity for the tested antibodies was lost. The chemical and immunological results indicate that the type III and group 6 antigens of S. flexneri are one and the same, a result of the presence of the O-acetyl group in rhamnose III of the repeating unit of the S. flexneri O-antigen chain.

The Shigella flexneri O-antigenic polysaccharide chain. Nature of the biological repeating unit.

The sequence of monosaccharides in the biological repeating tetrasaccharide unit of Shigella flexneri variant Y O-antigenic polysaccharide chain was determined by subjecting three oligosaccharides of the polysaccharide, obtained by phage-Sf6-mediated enzymatic hydrolysis, to methylation analysis and proton nuclear magnetic resonance spectroscopy. The smallest saccharide was shown to be a tetrasaccharide with the structure alpha-L-Rhap-(1-2)-L-Rha. The next saccharide, an octasaccharide, was shown to be a dimer of the tetrasaccharide with the L-Rha residues linked alpha 1.3. The longest saccharide was shown to be a decasaccharide with the following structure: alpha-L-Rhap-(1-2)-alpha-L-Rhap-(1-3)-alpha-L-Rhap-(1- 3)-beta-D-GlcpNAc-(1-2)-alpha-L-Rhap-(1-2)-alpha-L-Rhap++ + +-(1-3)-alpha-L-Rhap-(1-3)-beta-D-GlcpNAc-(1-2)-alpha-L-R hap-(1-2)-L-Rha. Thus the decasaccharide differed from the octasaccharide and tetrasaccharide by having the alpha-L-Rhap-(1-2)-L-Rhap disaccharide added in the terminal non-reducing end of the saccharide chain. This shows that the alpha-L-Rhap-(1-2)-alpha-L-Rhap-(1-3)-alpha-L-Rhap-(1- 3)-D-GlcpNAc tetrasaccharide is the biological repeating unit of the O chain and that the repeating units are joined through a beta-D-GlcpNAc-(1-2)-L-Rhap linkage. Inhibition experiments utilizing the enzyme-linked immunosorbent assay (ELISA) with S. flexneri Y lipopolysaccharide/S. flexneri Y rabbit antiserum showed that the decasaccharide was the best inhibitor (threefold as active as the octasaccharide and sixtyfold as active as the tetrasaccharide); this supports the postulated structure of the biological repeating unit.

Monoclonal antibodies specific for O-antigenic polysaccharides of Shigella flexneri: clones binding to II, II:3,4, and 7,8 epitopes.

Hybrid cells producing monoclonal antibodies against the O-antigens of Shigella flexneri were obtained by polyethylene glycol-mediated fusion of myeloma cells and lymphocytes from BALB/c mice immunized with whole heat-killed S. flexneri bacteria of serotypes 2a and 2b. Clones were selected for their binding specificity to structurally defined S. flexneri lipopolysaccharides (LPS). The following three groups were identified as recognizing three different epitopes: monoclonal antibodies binding to (i) S. flexneri LPS with the II:3,4 antigens, (ii) S. flexneri LPS with the II:3,4 antigens and the II:7,8 antigens, and (iii) S. flexneri LPS with the 7,8 group antigen only. Of cloned and characterized antibodies, more than 90% had either the mu or gamma 3 heavy chain and 98% had the kappa light chain. The exquisite specificity of each monoclonal antibody preparation was in complete contrast to the polyclonal specificities seen in sera from immunized rabbits. Even absorbed rabbit S. flexneri typing sera contained antibodies reacting with several different LPS, i.e., they were not type antigen specific. Ascites from immunoglobulin G monoclonal antibody preparations representing the three different specificities were used for sensitizing Staphylococcus aureus Cowan 1 bacteria and were used in coagglutination. In testing 211 clinical isolates of all different serotypes of S. flexneri, the reagents were shown to be sensitive and specific in correctly identifying all S. flexneri II and 7,8 antigen-containing strains with no false positives. Two isolated immunoglobulin M antibody clones specific for the II:3,4 and 7,8 antigens were used as successfully for identification by direct slide agglutination. These results suggest that the monoclonal reagents are superior to conventional typing antisera.