Genus-specific Epitope Research Articles

TargeTron Inactivation of Chlamydia trachomatis gseA Results in a Lipopolysaccharide 3-Deoxy-d-Manno-Oct-2-Ulosonic Acid-Deficient Strain That Is Cytotoxic for Cells.

All members of the family Chlamydiaceae have lipopolysaccharides (LPS) that possess a shared carbohydrate trisaccharide antigen, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) that is functionally uncharacterized. A single gene, genus-specific epitope (gseA), is responsible for attaching the tri-Kdo to lipid IVA. To investigate the function of Kdo in chlamydial host cell interactions, we made a gseA-null strain (L2ΔgseA) by using TargeTron mutagenesis. Immunofluorescence microscopy and immunoblotting with a Kdo-specific monoclonal antibody demonstrated that L2ΔgseA lacked Kdo. L2ΔgseA reacted by immunoblotting with a monoclonal antibody specific for a conserved LPS glucosamine-PO4 epitope, indicating that core lipid A was retained by the mutant. The mutant strain produced a similar number of inclusions as the parental strain but yielded lower numbers of infectious elementary bodies. Transmission electron microscopy of L2ΔgseA-infected cells showed atypical developmental forms and a reduction in the number of elementary bodies. Immunoblotting of dithiothreitol-treated L2ΔgseA-infected cells lysates revealed a marked reduction in outer membrane OmcB disulfide cross-linking, suggesting that the elementary body outer membrane structure was affected by the lack of Kdo. Notably, lactic acid dehydrogenase release by infected cells demonstrated that L2ΔgseA was significantly more cytotoxic to host cells than the wild type. The cytotoxic phenotype may result from an altered outer membrane biogenesis structure and/or function or, conversely, from a direct pathobiological effect of Kdo on an unknown host cell target. These findings implicate a previously unrecognized role for Kdo in host cell interactions that facilitates postinfection host cell survival.

A monoclonal antibody recognizing the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) trisaccharide αKdo(2→4)αKdo(2→4)αKdo of Chlamydophila spacepsittaci 6BC lipopolysaccharide

A monoclonal antibody (mAb) S45-18 was generated against a synthetic neoglycoconjugate containing the trisaccharide alphaKdo(2-->4)alphaKdo(2-->4)alphaKdo (Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid) which represents a structure of the lipopolysaccharide (LPS) from Chlamydophila psittaci 6BC. The antibody was characterized by binding and inhibition assays in ELISA using: (i) the immunizing antigen and chemically synthesized derivatives thereof; (ii) chlamydial elementary bodies (EB); and (iii) LPS of Chl. psittaci 6BC and Chlamydia trachomatis L2. The specificity was determined in comparison to that of mAb S25-23 recognizing the alphaKdo(2-->8)alphaKdo(2-->4)alphaKdo trisaccharide which represents an epitope shared by all species of the family. MAb S45-18 bound to an epitope of the structure alphaKdo(2-->4)alphaKdo(2-->4)alphaKdo, with lower reactivity with the (2-->8)-(2-->4)-linked analog. Using chlamydial EB or LPS, mAb S45-18 bound preferentially to LPS and EB of Chl. psittaci. Therefore, Chl. psittaci LPS contains, in addition to the known genus-specific epitope, a species-specific epitope.

Far‐eastern strains of bean common mosaic virus and methods of their immunodiagnostics

The paper presents data of investigation on the physico‐chemical and antigenic properties of capsid proteins of the Bean common mosaic virus isolated from Phaseolus plants in the Russian Far East (BCMV‐R) and from China (BCMV‐C). A method for isolation of the virus preparation was selected. The purified preparations of two isolates BCMV have been obtained. The presence of one polypeptide in structural proteins of virions was established and their molecular masses determined (BCMV‐R ‐ 31,6 kD; BCMV‐C ‐ 32,1 kD). Polyclonal antiserum was obtained with titre 1:12800 and the indirect and “sandwich"‐variants of ELISA were developed to detect this virus. The allied relationships were established with the bean yellow mosaic virus and with the type representative of the genus Potyvirus ‐ PVY. Based on the data of physico‐chemical and antigenic properties it was concluded that isolates BCMV‐R and BCMV‐C are two independent strains of this virus. The presence of strain‐, virus‐ and genusspecific epitopes of capsid proteine was revealed as a result of comparison of antigenic characteristics of the Russian Far Eastern and Chinese strains of BCMV. A high antigenic activity of capsid protein of the Russian Far Eastern strain was observed.

Characterization of intertype specific epitopes on adenovirus hexons.

The specification and differentiation of eighteen intertype specific (IT) epitopes of adenovirus hexons defined by monoclonal antibodies are given by two groups of hexon types: on which they are and on which they are not present. The close specificity relationship among some groups of epitopes determined by pairwise analysis according to their presence on the hexon types indicate that they could be continuous (sequential), overlapping or discontinuous (topographic) epitopes. Based on the identification of the hydrophilic regions and the localization of beta-turns sixteen IT epitope sites were predicted on human adenovirus (HAdV) type 2 and nineteen on HAdV-41 hexon's amino acid (aa) sequences beside the type and genus specific ones. The 16 predicted IT epitopes on HAdV-2 hexon show good coincidency with the 14 IT epitopes demonstrated with monoclonal antibodies (MAbs) on this hexon type. The predicted number of common epitopes between HAdV types 2 and 41 also corresponds well with the eight IT epitopes determined by MAbs, but the 17 predicted non-common epitopes indicate the possibility of the existence of more epitopes on these hexon types. The location of the predicted epitopes of HAdV-2 were determined by alignment of their sequence numbers on the three dimensional ribbon representation of the hexon subunit. Most of the predicted IT epitopes were found between the type and genus specific epitopes i.e. in the "upper" regions of pedestal domains P1 and P2 orientated toward the virion surface and in the "lower" part of loop 1 region orientated inside the virion. Two peptides representing potential IT epitopes were synthesized corresponding to residues 309-320 from the "lower" part of loop 1 and 399-408 from the "upper" part of P1 region of HAdV-2 hexon. Antibodies raised against the peptide-carrier conjugates recognized different purified native hexon types in ELISA.

Reactivities of Genus-Specific Monoclonal Antibody EM-6E11 against Listeria Species and Serotypes of Listeria monocytogenes Grown in Nonselective and Selective Enrichment Broth Media

Depending on the growth medium used for enrichment of bacterial cells prior to assay, the monoclonal antibody (MAb) EM-6E11 recognizing Listeria genus-specific epitope on 43 and 94 to 97 kDa cell-surface antigens (A. K. Bhunia and M. G. Johnson, Appl. Environ. Microbiol. 58:1924–1929–1992) exhibited extensive variability in the detection of Listeria species. MAb EM-6E11 strongly detected live cells of all Listeria species and all serotypes of L. monocytogenes by ELISA when cells were grown in nonselective brain heart infusion (BHI) broth, in selective Listeria enrichment broth (LEB), or in Listeria repair broth (LRB). In contrast, EM-6E11 detected only four of the thirteen serotypes of L. monocytogenes (serotypes ½c, 3b, 4ab, and 7) when cells were grown in the UVM1 formulation of Listeria enrichment broth (UVM1) or Fraser broth (FRB). This MAb failed to react with live cells of four other Listeria species, including L. ivanovii, L. welshimeri, L. grayi, and L. murrayi cells grown in UVM1 or FRB. Heating of Listeria cells at 100°C for 20 min, irrespective of the enrichment media used, led to large losses of MAb EM-6E11 reactivity in ELISA, suggesting that the specific cell-surface epitopes involved may not be heat stable. Our results confirm that MAb EM-6E11 is suitable for detection of live cells but not heat-killed cells of Listeria spp. and can be used in conjunction with an enrichment step in BHI, LEB, or LRB but not in UVM1 or FRB.

Conformational analysis of a Chlamydia-specific disaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl in aqueous solution and bound to a monoclonal antibody: observation of intermolecular transfer NOEs.

The disaccharide alpha-Kdo-(2-->8)-alpha-Kdo (Kdo: 3-deoxy-D-manno-oct-2-ulosonic acid) represents a genus-specific epitope of the lipopolysaccharide of the obligate intracellular human pathogen Chlamydia. The conformation of the synthetically derived disaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl was studied in aqueous solution, and complexed to a monoclonal antibody S25-2. Various NMR experiments based on the detection of NOEs (or transfer NOEs) and ROEs (or transfer ROEs) were performed. A major problem was the extensive overlap of almost all 1H NMR signals of alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl. To overcome this difficulty, HMQC-NOESY and HMQC-trNOESY experiments were employed. Spin diffusion effects were identified using trROESY experiments, QUIET-trNOESY experiments and MINSY experiments. It was found that protein protons contribute to the observed spin diffusion effects. At 800 MHz, intermolecular trNOEs were observed between ligand protons and aromatic protons in the antibody binding site. From NMR experiments and Metropolis Monte Carlo simulations, it was concluded that alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl in aqueous solution exists as a complex conformational mixture. Upon binding to the monoclonal antibody S25-2, only a limited range of conformations is available to alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl. These possible bound conformations were derived from a distance geometry analysis using transfer NOEs as experimental constraints. It is clear that a conformation is selected which lies within a part of the conformational space that is highly populated in solution. This conformational space also includes the conformation found in the crystal structure. Our results provide a basis for modeling studies of the antibody-disaccharide complex.

Temperature-sensitive lesions in the Francisella novicida valA gene cloned into an Escherichia coli msbA lpxK mutant affecting deoxycholate resistance and lipopolysaccharide assembly at the restrictive temperature

The valAB locus of Francisella novicida has previously been found to be highly similar at the deduced amino acid level to msbA lpxK of Escherichia coli. Both ValA and MsbA are members of the superfamily of ABC transporters, and they appear to have similar functions. In this study we describe the isolation of a temperature-sensitive valAB locus. DNA sequence analysis indicates that the only changes to the ValAB deduced amino acid sequence are changes of S453 to an F and T458 to an I in ValA. E. coli strains defective in msbA and expressing temperature-sensitive ValA rapidly ceased growth when shifted from a permissive temperature to a restrictive temperature. After 1 h at the restrictive temperature, cells were much more sensitive to deoxycholate treatment. To test the hypothesis that ValA is responsible for the transport or assembly of lipopolysaccharide, we introduced gseA, a Kdo (3-deoxy-D-manno-octulosonic acid) transferase from Chlamydia trachomatis, into a strain with a temperature-sensitive valA allele and a nonfunctional msbA locus. These recombinants were defective in cell surface expression of the chlamydial genus-specific epitope within 15 min of a shift to the nonpermissive temperature. Also, there was enhanced association of the epitope with the inner membrane after a shift to the nonpermissive temperature. Thus, we propose that ValA is involved in the transport of lipopolysaccharide to the outer membrane.

Comparison of an LPS-specific competitive ELISA with a motility enrichment culture method (MSRV) for detection of Salmonella typhimurium and S. enteritidis in chickens

We report here evaluation of a competitive enzyme-linked immunosorbent assay (c-ELISA) for detection of Salmonella spp. in chicken organs and faeces. The c-ELISA used a monoclonal antibody (MAb), specific for a genus-specific epitope of the outer core oligosaccharide of salmonellae. Salmonella lipopolysaccharide (LPS) in samples competed with Salmonella LPS coated on microtitre plates, for binding to the MAb. Competition reduced binding of the MAb to the LPS on the plate and of the secondary antibody to the MAb hence reducing the chromogenic signal. Stable coating and minimal false positives were achieved by conjugating LPS to poly- l-lysine. The c-ELISA was compared with motility enrichment culture using modified semisolid Rappaport Vassiliadis (MSRV) medium, which detected less than 10 2 CFU/g, and did not allow migration of non-salmonella species. The c-ELISA detected 10 6 CFU of enriched culture or 10 2–10 3 CFU of Salmonella/g of faeces. Its limit of detection was thus higher than that of MSRV culture and it had a sensitivity of 92.9% and a specificity of 96.7%.

Structural requirements of synthetic oligosaccharides to bind monoclonal antibodies against Chlamydia lipopolysaccharide.

Monoclonal antibodies were generated against a synthetic glycoconjugate containing the trisaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo (Kdo, 3-deoxy-D-manno-2-octulopyranosonic acid) which represents the genus-specific epitope of the lipopolysaccharide from the obligatory intracellular human pathogen Chlamydia. Antibodies of all immunoglobulin G isotypes were obtained and characterized by enzyme immunobinding and inhibition assays using the immunizing antigen as well as chemically synthesized derivatives of the Kdo trisaccharide. The latter contained (1) one of the three residues in beta- instead of alpha-linkage, (2) a Kdo residue the carboxyl group of which had been reduced to a CH2OH group (Kdo(C1-red)), or (3) changing the linkage of the terminal Kdo from 2-->8 to 2-->4. Only one compound, namely, alpha-Kdo-(2-->8)-alpha-Kdo(C1-red)-(2-->4)-alpha-Kdo exhibited binding to and inhibition of Kdo trisaccharide-specific antibodies, whereas all other compounds were not active. Structural and conformational investigations using NMR spectroscopy at high field on the allyl glycosides of the oligosaccharides 6-12 confirmed the conformational similarities between those structures 4, 5, and 10 which were able to bind to the antibodies investigated.

Characterization of an outer membrane protein of Pasteurella multocida belonging to the OmpA family

The outer membrane vesicle and N-lauroylsarcosine-insoluble protein preparations of Pasteurella multocida 656 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A major outer membrane protein (OMP) was found to be heat-modifiable, having a molecular mass of 28 kDa when the OMP preparation was solubilized at 60°C and a molecular mass of 37 kDa when it was solubilized at 100°C. A monoclonal antibody, designated mAb MT4.1, was generated against heat-modifiable OMP of P. multocida. This mAb reacted with the heat-modifiable OMP irrespective of the temperature at which it was solubilized, as demonstrated by immunoblot results. The heat-modifiable OMP of P. multocida showed a significant N-terminal amino acid sequence homology with OmpA family. Immunoelectron microscopic study revealed that the mAb MT4.1 epitope was not surface exposed on the intact bacterium. The mAb MT4.1 reacted with all the reference strains of 5 capsular and 16 somatic serotypes, as well as with 75 field strains of P. multocida in immunoblot assay. This mAb MT4.1 also reacted with strains of various other Pasteurella species such as P. stomatis, P. aerogenes, P. gallinarum, P. betti, P. sp. B, P. SP-g and P. canis, but not with strains of 12 other Gram-negative bacteria. These results indicated that this protein carried a genus-specific epitope and mAb MT4.1 may be useful for identification of Pasteurella species. This is the first report in which a major heat-modifiable OMP has been identified and characterized using a mAb, and has been shown belonging to the OmpA family.

Rapid identification of porcine Serpulina species by colony blot assay using a genus-specific monoclonal antibody.

An immunoglobulin G monoclonal antibody (mAb C9E8) recognising a genus-specific epitope on the 26 kDa protein of porcine Serpulina species organisms was used in a simple colony blot assay to detect Serpulina in cultures grown directly on blood agar plates from pig faeces and tissues. The mAb detected even a few colonies of the organism in the presence of an abundant growth of non-Serpulina organisms. The whole procedure was completed in less than three hours. A total of 123 strains of S hyodysenteriae and S innocens were correctly identified by the colony blot assay whereas all the 26 non-Serpulina Gram-negative organisms commonly isolated from faecal material or tissues of pigs remained negative. The assay was rapid, highly specific and sufficiently reliable to be used with confidence for identifying porcine Serpulina colonies directly on blood agar plates.

Identification and characterization of genus-specific epitopes of Serpulina species using monoclonal antibodies

Four murine monoclonal antibodies (mAbs) designated as C9E8, A10, G12, and G8 which recognized both Serpulina hyodysenteriae and S. innocens were produced and characterized. The mAbs reacted with whole cell antigens in ELISA, indirect immunofluorescence and immunoblot assays. The mAbs did not show any cross reactivity in rapid dot ELISA or immunoblot assay with Leptospira icterohemorrhagiae, Campylobacter jejuni and Escherichia coli. Treatment of whole cell suspension with proteinase K and sodium periodate indicated that the reacting epitopes of the mAbs were protein in nature. The genus-specific antigens were identified as heat-stable proteins with molecular weight in the range of 26 to 45 kDa. Immunofluorescence and immunogold labelling studies showed that the antibody-binding epitopes were exposed on the outer-surface of the spirochaetal cell wall. The mAbs inhibited growth of reference strains of both S. hyodysenteriae and S. innocens in vitro but failed to cause agglutination. The detection of spirochaetal forms directly in fecal smears or paraffin-embeded tissue sections from experimentally infected pigs indicated that such mAbs were potentially useful for the diagnosis of swine spirochaetosis. This is the first report of mAbs identifying and characterizing common antigens of porcine Serpulina.

Molecular cloning, sequence analysis, and functional characterization of the lipopolysaccharide biosynthetic gene kdtA encoding 3-deoxy-alpha-D-manno-octulosonic acid transferase of Chlamydia pneumoniae strain TW-183.

The gene kdtA of Chlamydia pneumoniae strain TW-183, encoding the enzyme 3-deoxy-alpha-D-manno-octulosonic acid (Kdo) transferase of lipopolysaccharide biosynthesis, was cloned and sequenced. A single open reading frame of 1314 bp was identified, the deduced amino acid sequence of which revealed 69% similarity and 43% identity with KdtA of Chlamydia trachomatis and Chlamydia psittaci. The gene was expressed in the Gram-positive host Corynebacterium glutamicum and the primary gene product was characterized as a multifunctional glycosyltransferase. Cell-free extracts generated in vitro the genus-specific epitope of Chlamydia composed of the trisaccharide alphaKdo(2-8)alphaKdo(2-4)alphaKdo. The results show that a single polypeptide affords three different glycosidic bonds, which is in contradiction to the dogma of glycobiology: 'one enzyme - one glycosidic bond'.

Inhibition of Lipopolysaccharide Biosynthesis and Cell Growth following Inactivation of the kdtA Gene in Escherichia coli

The enzyme 3-deoxy-D-manno-octulosonic acid (Kdo) transferase is encoded by the kdtA gene in Escherichia coli. The enzyme is a single polypeptide that catalyzes the transfer of two Kdo residues to a tetraacyldisaccharide-1,4'-bisphosphate precursor of lipid A, designated lipid IVA (Belunis, C.J., and Raetz, C.R.H. (1992) J. Biol. Chem. 267, 9988-9997). To determine if Kdo transfer to lipid IVA is required for growth, we constructed a strain of E. coli with a chromosomal kdtA::kan insertion mutation. In mutants carrying the kdtA::kan allele on the chromosome, cell growth and Kdo transferase activity were dependent upon a copy of the intact kdtA gene on a plasmid. When the kdtA-bearing plasmid was itself temperature sensitive for replication, the growth of these strains was inhibited after several hours at 44 degrees C, and Kdo transferase activity in extracts became undetectable. Concomitantly, the cells accumulated massive amounts of lipid IVA, the precursor of (Kdo)2-lipid IVA. The kdtA::kan mutation could also be complemented by hybrid plasmids bearing the gseA gene of Chlamydia trachomatis. gseA specifies a distinct Kdo transferase that adds three Kdo moieties to lipid IVA. Lipopolysaccharide from E. coli kdtA::kan constructs complemented by gseA reacts strongly with antibodies directed against the genus-specific epitope of Chlamydia, whereas lipopolysaccharide from parental E. coli K-12 does not. Our studies prove that Kdo attachment during lipid A biosynthesis is essential for cell growth and accounts for the conditional lethality associated with mutations in Kdo biosynthesis.

Enzyme immunoassay for detection of antibodies against isolated flagella of Legionella pneumophila.

Flagella of Legionella pneumophila serogroup 1, strain Philadelphia (ATCC 33152), were isolated and used as antigen to detect antibodies by enzyme immunoassay (EIA) in sera of patients with significant titers against Legionella as determined by the indirect immunofluorescence assay (IFA). Healthy blood donors served as a control group. Whereas the sera of blood donors were negative in both assays, sera of patients showed two patterns of reactivity. Sera with elevated titers against Legionella pneumophila serogroup 1 or Legionella gormanii in IFA usually demonstrated high titers in EIA. However, high IFA titers against Legionella bozemanii were associated with low titers in EIA. The data show that flagella protein of Legionella pneumophila serogroup 1 strain Philadelphia is of limited value for the detection of antibodies against Legionella other than Legionella pneumophila serogroup 1, although in vitro studies revealed genus-specific epitopes. This might be due to unpredictable variabilities in the expression of flagella in vivo.

Evidence of Common and Genus-Specific Epitopes on Ornithodoros spp. Tick (Acari: Argasidae) Salivary Proteins

New Zealand White rabbits were repeatedly infested with Ornithodoros turicata (Duges), Ornithodoros talaje (Guérin-Méneville), and Ornithodoros coriaceus (Koch) at 2-wk intervals. Blood samples were taken from each animal 10 d after each infestation and the titer of anti-tick antibody was determined by enzyme-linked immunosorbent assay. Subsequent cross-reactivity studies demonstrated that the antitick antisera nonspecifically bound to salivary gland extract proteins prepared from several other tick genera and species' Amblyomma maculatum (Koch), Dermacentor andersoni (Stiles), Dermacentor variabilis (Say), and Ornithodoros moubata (Murray). Absorption of the antisera against an immobilized extract of A. maculatum substantially increased specificity at the genus level. Western blots of electrophoretically separated Ornithodoros salivary gland extract samples were used to further compare the specificity of absorbed and nonabsorbed antitick antisera. The blots demonstrated that many of the Ornithodoros salivary gland extract proteins bear genus specific epitopes. Some differences were noted among the Ornithodoros species examined with respect to the degree of antigenic relatedness with the ixodid ticks.

Chlamydia1 lipopolysaccharide: Chemical and antigenic structure, biosynthesis and biomedical application

Abstract

Nucleotide sequence variations within the lipopolysaccharide biosynthesis gene gseA (Kdo transferase) among the Chlamydia trachomatis serovars

The gene gseA, involved in the expression of the genus-specific epitope of chlamydial lipopolysaccharide (LPS), was analyzed by temperature gradient gel electrophoresis (TGGE) to visualize nucleotide sequence variations among the 15 serovars of Chlamydia trachomatis. Sequence analysis showed that the TGGE melting-profile patterns were able to detect single nucleotide variations within gseA and allowed the arrangement of the serovars in groups of both identical nucleotide sequences and sequences containing identical sites of nucleotide substitutions. Compared to serovar L2, four types of patterns were obtained: (i) serotypes A and Ba; (ii) B and C (causing endemic trachoma); (iii) D through K (causing sexually transmitted oculo-genital infections); (iv) L1 through L3 (the causative agents of lymphogranuloma venereum). A total of 58 isolates of C. trachomatis of genital or conjunctival origin were tested by this method in comparison to reference strains. Forty-eight isolates (13 of type E, 16 of type F, nine of type G, and ten of type K) yielded the same melting profile as the corresponding type strain, independent of whether they were isolated from genital or ocular infections. However, ten B serotype strains of genital origin behaved in TGGE like a typical genital strain and not a trachoma strain. Thus, although gseA was found to be highly conserved among C. trachomatis, the obtained TGGE profiles of the tested strains tended to correlate with their specific site of infection.

Preparation and structural analysis of oligosaccharide monophosphates obtained from the lipopolysaccharide of recombinant strains of Salmonella minnesota and Escherichia coli expressing the genus-specific epitope of Chlamydia lipopolysaccharide.

The lipopolysaccharide of the recombinant strain Salmonella minnesota r595-207 expressing the genus-specific epitope of Chlamydia lipopolysaccharide [Holst, O., Brade, L., Kosma, P. and Brade, H. (1991) J. Bacteriol, 173, 1862-1866] was sequentially de-O- and de-N-acylated by mild hydrazinolysis and treatment with 4 M KOH, respectively. The resulting mixture of compounds was separated by high-performance anion-exchange chromatography and gel-permeation chromatography, yielding four oligosaccharide phosphates two of which were readily identified by their 1H-NMR- and 13C-NMR spectra as alpha-Kdo-(2-4)-alpha-Kdo-(2-6)-beta-D-GlcpN-(1-6)-alpha-D-Glcp N 1,4'-bisphosphate (tetrasaccharide bisphosphate; Kdo = 3-deoxy-D-manno-octulopyranosonic acid) and alpha-Kdo-(2-8)-alpha-Kdo-(2-4)-alpha-Kdo-(2-6)-beta-D-GlcpN-(1-6) -alpha-D- GlcpN 1,4'-bisphosphate (pentasaccharide bisphosphate) [Holst, O., Broer, W., Thomas-Oates, J.E., Mamat, U. and Brade, H. (1993) Eur. J. Biochem. 214, 703-710]. The structures of the other two compounds were established by chemical analysis, NMR spectroscopy, and fast-atom-bombardment mass spectrometry as alpha-Kdo- (2-4)-alpha-Kdo-(2-6)-beta-D-GlcpN-(1-6)-alpha-D-GlcpN 1-phosphate (tetrasaccharide 1-phosphate) and alpha-Kdo-(2-8)-alpha-Kdo-(2-4)-alpha-Kdo-(2-6)-beta-D-GlcpN-(1-6) -alpha-D- GlcpN 1-phosphate (pentasaccharide 1-phosphate). alpha-Kdo-(2-4)-alpha-Kdo-(2-6)-beta-D-GlcpN-(1-6)-alpha/beta- D-GlcpN 4'-phosphate (tetrasaccharide 4'-phosphate) and alpha-Kdo-(2-8)-alpha-Kdo-(2-4)-alpha-Kdo-(2-6)-beta-D-GlcpN-(1-6) -alpha/beta-D-GlcpN 4'-phosphate (pentasaccharide 4'-phosphate) were prepared from the 1,4'-bisphosphates isolated from the recombinant strain Escherichia coli F515-207 by treatment with alkaline phosphatase and purification by high-performance anion-exchange chromatography and gel-permeation chromatography. Their structures were characterised by chemical analysis, NMR spectroscopy, and fast-bombardment mass spectrometry.

Identification of subspecies- and serotype 1-specific epitopes on the 80- to 90-kilodalton protein region of Chlamydia psittaci that may be useful for diagnosis of Chlamydial induced abortion

Summary Genus-, subspecies-, and serotype 1-specific antigens of Chlamydia psittaci were characterized by immunoblot analysis, using monoclonal antibodies that recognize 2 C psittaci strains: AB7 isolated from an ewe that had aborted, and iBl isolated from feces of a healthy ewe. Genus-specific epitopes were detected on lipopolysaccharide, on a 47-kd protein, and on a 27- to 30-kd doublet. Subspecies-specific epitopes were located on a 30-kd protein, and a 80- to 90-kd protein region was identified, which bore subspecies- and serotype l-specific epitopes. These 80- to 90-kd proteins were highly reactive with serum from ewes that had aborted and could be a useful antigen for diagnosis of Chlamydial induced abortion of ruminants.