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The structure of the O-specific polysaccharide moiety of the lipopolysaccharide from Citrobacter 396 was elucidated by composition, methylation, and periodate oxidation studies. The repeating unit consists of four 2-linked mannoses and one 3-linked N-acetylglucosamine. One of the mannose units is substituted at C3 with alpha-glucose, and one is substituted at C3 with alpha-(2-O-acetyl)-abequose. All the mannosyl linkages appear to have the beta-configuration; the N-acetylglucosaminyl linkage has the alpha-configuration. In bacterial agglutination and passive hemagglutination in some Salmonella antisera, Citrobacter 396 as well as its O-antigenic lipopolysaccharide expressed the serological factors 5 and 6. In corroboration of our structural studies, this showed the presence of alpha-(2-O-acetyl)-abequosyl-1,3-mannose (factor 5) and alpha-glucosyl-1,3-mannose (factor 6).
The O-antigenic polysaccharide of phenol-water extracted Salmonella typhimurium (O antigens 4, 12) lipopolysaccharide was enzymatically cleaved by phage P22 endorhamnosidase. An octasaccharide with the (formula: see text) structure Gal-Man-Rha-Gal-Man-Rha was isolated and shown to retain the O-antigen 4 specificity of the native polysaccharide. After oxidation of the terminal reducing rhamnose residue to the corresponding aldonic acid, the octasaccharide was covalently linked to bovine serum albumin (OLS-BSA) by use of a water-soluble carbodimide. The resulting conjugate showed O-antigen 4 specificity in enzyme-linked immunosorbent assay (ELISA) ans passive hemagglutination inhibition tests. Immunization of rabbits with the OLS-BSA conjugate gave rise to antibodies directed toward both the octasaccharide and the carrier protein. ELISA titration with synthetic disaccharide-protein conjugates as antigens revealed that the antibody titer against the mannose-rhamnose structure was higher than against the abequose-mannose structure. In rabbits immunized with heat-killed whole bacteria the titers against the two disaccharides were equal. The reason for this difference is not obvious. It is evident, however, that the OLS-BSA conjugate elicited in rabbits O-antibodies with the same specificity as whole bacteria.
The somatic (O-) antigens of the type strains of the providencia antigenic scheme were examined for their biochemical reactions and their O-specificities. The scheme of 62 O-antigens was reconstituted from 52 original type strains and 10 strains substituted for originals that either were biochemically atypical of the genus or showed inappropriate serological reactions. Thirty-six type strains showed no significant relations with other type strains, and antisera could be used for typing without absorption. Among 26 type strains, significant reciprocal relations were demonstrated, and each cross-reacting antigen was examined for specificity and for its distribution among the type strains. Antisera to these strains required absorption with cell suspensions of other type strains for production of specificity in O-typing. Each typing antiserum, at low dilution, was shown to agglutinate homologous, but not heterologous, cell suspensions of type strains, and this result demonstrated the required specificity for typing on the basis of the O-antigens.
Studied was the effect of gamma-rays at rates of 1 to 20 MR on the toxic, antigenic, and immunogenic properties of the O-antigen extracted from Salmonella gallinarum-pullorum by the method of Boivin. It was found that the irradiation of aqueous solutions of the O-antigen lowered the toxic, antigenic, and immunogenic capacity with the increase in the dose of treatment. Strongest detoxication of the endotoxin was produced through irradiation at the rate of 10 MR, and especially at 20 MR. The antigenic activity, established through the hyperimmunization of rabbits was expressed best at irradiation rates of 1 and 5 MR. At such treatments the immunogenic capacity remained intact as followed up by the murine protection test.
The somatic O-antigens of 583 French isolates of Serratia marcescens were studied with the aid of the tube O-agglutination technique sensitized by centrifugation. Flagella antigens were identified by the H-immobilization test. Five new O-antigens (O16 to O20) were found. An antigenic factor (Co) common to isolates of serotypes O12:H9, O14:H12, O14:H20, and O13:H17 is described. Agar cultures of serotypes O12:H9 and O14:H12 dissociated into two colonial types: iridescent colonies, and non-iridescent colonies. Non-iridescent colonies, heated at 100 degrees C, are agglutinated by anti-Co serum, whereas heat-treated iridescent colonies are not. Close relationship between serogroup antigens O6 and O14 observed by traub and Kleber is confirmed. Serogroup O9 was subdivided into two subgroups. In France, 49 different serotypes were identified, with a predominance of serotypes O14:H12, O13:H17 and O3:H12. Recently, several new H-antigens were independently described by Traub and Kleber, and by us. To avoid confusion, these new H-antigens have been herein redesignated in accord with Dr Traub.
The cross-reactivity of O antigens prepared from Bacteroides fragilis, other Bacteroides species and from Fusobacterium has been examined by indirect haemagglutination and inhibition of haemagglutination. Fifteen of 20 B. fragilis ss. fragilis strains showed O-antigenic cross-reactivity with one or more of the test strains of B. fragilis ss. fragilis: NCTC 9343, Lille E 323 and SBL B55. The same applies also to 3 strains classified as B. coagulans, B. hypermegas and B. putredinis. The multispecificity of B. fragilis O antigens is pronounced. Test systems for demonstration of 9 specificities, all harboured by one or more of the 3 test strains, have been worked out.
Previous studies of different Shigella flexneri O-antigens indicate that their O-specific region is composed of oligosaccharide repeating units containing a basic tetrasaccharide structure, to which alpha-D-glucopyranosyl groups and O-acetyl groups may be attached to different positions. Structural studies of O-antigens from variant X, type 5a and type 5b lend further support to this assumption. These antigens contain terminal alpha-D-glucopyranosyl groups, one each per repeating unit in X and 5a, two in 5b. The location of these groups in the repeating unit has been determined.
We have developed a solid-phase radioimmunoassay technique for specific gram-negative bacterial lipopolysaccharide (LPS) O antigens. The method exploits the high-titer, specific immunoglobulin M response of the rabbit to LPS immunization to measure as little as 5 ng of homologous LPS per ml with less than 0.5% cross-reactivity toward heterologous LPS or culture supernatants. We found that O antigen in complete LPS was less available for antibody binding than O antigen in the soluble polysaccharide derived by mild acid hydrolysis of LPS and that triethylamine-induced disaggregation of complete LPS increased its activity in the assay. Quantitation of O antigen with the assay was thus influenced by the physical state of LPS or "free" O antigen.
Non-nodulating mutant strains of Rhizobium japonicum lacked a surface antigen that was present on the wild type. This surface antigen is associated with the O antigen portion of the lipopolysaccharide. Paper chromatography of hydrolyzed lipopolysaccharide and O antigen revealed three major component differences between the non-nodulating strains and the wild type.
A modified method of obtaining the purified K- and O-antigens of Sh. newcastle with the use of ultracentrifugation is described. Serologically individual K- and O-antigens were obtained and their chemical composition was studied. Polysaccharide of the K-antigen consisted of the galacturonic acid, galactose, glucose, rhamnose xylose and hexosamine; it contained no heptoses. The O-antigen contained galactose, glucose, mannose, rhamnose, heptose and hexosamine; in difference to K-antigen was equal to 150000--160000. Thus, the K- and the O-antigen differed not only in the electrophoretic and diffuse mobility and immunological specificity, but also in the chemical composition.
During a systemic investigation into O-antigenic relations within the family Enterobacteriaceae, with special emphasis on serological relationships between O-antigens (or O-groups) of E. coli and various O-groupps of Salmonella, Arizona, and Citrobacter, our tests confirmed some findings previously descried by other authors, some were not confirmed, and other antigenic relationships were discovered which have not yet been published. Individually tested were O-Antigens of 142 serologically defined E. coli types by using O-factorsera of Salmonella, Arizona, and Citrobacter for the slide agglutination. The slide agglutination was applied in preference to the tube-agglutination, not only because the former has been generally adopted but it also shows broader reactions...
In Salmonella, ilv-linked rfe genes participate in the biosynthesis of the enterobacterial common antigen (CA) as well as of certain types of O antigen (serogroups C1 and L). rff genes, probably in the same cluster with rfe, are required for CA synthesis (P.H. Mäkelä et al., in preparation). Several Escherichia coli strains were studied to determine whether they also have rfe-rff genes that are involved in the synthesis of O antigen and CA, or of CA only. In a first approach, E, coli K-12 F-prime factors carrying the genes ilv and argH or argE and presumably rfe-rff genes were introduced into CA-negative Salmonella mutants that are blocked in CA synthesis because of mutated rfe or rff genes. All resulting ilv+ hybrids were CA positive. In recipients with group C1-derived rfb genes, the synthesis of O6,7-specific antigen was also restored. This result shows that E. coli K-12 has rfe and rff genes providing the functions required in the synthesis of CA and Salmonella 6,7-specific polysaccharide. By introduction of defective rfe regions from suitable Salmonella donors into E. coli O8, 09, and O100 strains, the synthesis of CA as well as of the O-specific polysaccharides was blocked. This indicates that in the E. coli strains tested the rfe genes are involved in the synthesis of both O antigen and CA. This suggestion was confirmed by the finding of E. coli rough mutants that had simultaneously become CA negative. In transduction experiments it could be shown that the appearance of the rough and CA- phenotype was due to a defect in the ilv-linked rfe region.
The structures of the O-antigens of all known serotypes and subserotypes of Shigella flexneri have been reinvestigated. The results support the assumption that these antigens are composed of a basic tetrasaccharide repeating unit (1), to which alpha-D-glucopyranosyl and/or O-acetyl groups are attached at different positions. Leads to 3)-beta-D-GlcNAcp-(1 leads to 2)-alpha-L-Rhap-(1 leads to 2)-alpha-L-Rhap-(1 leads to 3)-alpha-L-Rhap-(1 leads to The immunological determinants responsible for O-factors I, II, IV, V and 7, 8 contain alpha-D-gluco-pyranosyl groups, the locations of which have been determined. O-Factor 6 is due to O-acetyl groups, linked to O-2 of the 3-substituted alpha-L-rhamnopyranosyl residue in unit 1 and O-factor III seems to be due to the same groups. The chemical natures of the determinants responsible for O-factors 4 and 3, 4 are still obscure. The structural studies indicate that the immunological classification of Sh. flexneri serotypes and subserotypes, as regards these O-factors, may need revision.
BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) keratitis can progress rapidly to vision-threatening disease, even with intensive therapy. Virulence-associated genes are key determinants of ocular-surface pathogenesis. We therefore sought to develop a composite wbp-exo genotyping framework for risk stratification and to guide wbp-dependent, LPS-directed, levofloxacin-polymyxin B (LVX-POL) combination therapy for high-risk corneal infections. METHODS: A well-characterised clinical P. aeruginosa keratitis cohort was integrated with whole-genome sequencing. Based on comprehensive virulence-gene identification and annotation, the relationship between strain-level genetic features and clinical prognosis was analysed. The differences between WBP1 strains and WBP2 strains in adhesion, invasion, and biofilm formation in corneal epithelial cells were further evaluated. To establish biological plausibility, wbp genotypes were correlated with LPS O-antigen electrophoretic profiles and in vivo corneal inflammatory phenotypes in murine infection, including the observation of leucocyte recruitment and cytokine responses. To further confirm the key role of wbp gene status and LPS O-antigen in pathogenicity, wbpL knockout and reconstitution strains were constructed. Their appearances in vitro and in vivo were evaluated. Finally, a mechanistic rationale for an LPS-directed LVX-POL regimen was tested in a high-risk WBP1 P. aeruginosa murine keratitis. FINDINGS: Whole-genome sequencing was performed on 46 clinical P. aeruginosa isolates and identified an average of 332 virulence- and fitness-associated genes per strain. The exo and wbp gene families were significantly associated with patient prognosis. A fusion model (AUC = 0.86) outperformed single-gene-family models (EXO: 0.66; WBP: 0.72) for predicting clinical outcomes. Intact wbp cassettes were enriched in poor-outcome isolates, and electrophoretic LPS profiles indicated that WBP1 strains produce highly polymerised O-antigen associated with sustained neutrophil recruitment and cytokine production. Murine experiments further implicated wbp genes in clinical pathogenesis, showing stronger immune responses and higher expression of TLR4, MyD88, TRAF6, p65, p-p65, IL-6, TNF-α, and IL-1β throughout the inflammatory course. After knocking out wbpL gene, the WBP1 strain got stronger in biofilm formation and adhesion but weaker in inflammation and ocular surface survival. In the high-risk WBP1 P. aeruginosa keratitis model, LVX-POL combinations achieved complete ulcer resolution and markedly improved stromal infiltration and hypopyon, outperforming LVX monotherapy. INTERPRETATION: The wbp gene family was identified as a key genetic factor that contributes to the LPS O-antigen structure, inflammatory intensity, bacterial ocular surface survival and poor prognosis in P. aeruginosa keratitis. A WBP1-targeted, LPS-directed LVX-POL regimen was proposed as a mechanistically informed option for high-risk strains. FUNDING: This research was supported by Beijing Public Health High-level Talent Training Program (Phase III-03-14), Prevention and Control of Emerging and Major Infectious Diseases-National Science and Technology Major Project (2026ZD01909300) and Beijing Natural Science Foundation "QiYan" Undergraduate Research Fund (QY26496).
O antigen extracted from whole cells of Bacteroides fragilis ss. fragilis NCTC 9343 with 45 per cent aqueous phenol has been purified by gel filtration and chromatography. First, the water phase was treated with RNase and DNase and passed through a column of agarose. The chromatographic procedures included ion exchange on a column of DEAE-cellulose and adsorption to hydroxylapatite. The O antigen was eluted from the DEAE-cellulose with a gradient of NaCl, and from the column of hydroxylapatite with 1 M phosphate buffer, pH 6.8. Inhibition of indirect haemagglutination was used to detect the O antigen in the eluates.
The authors discuss the results of studies on the choice of highly-reproducible and sufficiently informative methods of 9-antigen standardization in a new chemical vaccine against cholera (cholerogen-toxoid); materials were collected under conditions of controlled epidemilogical trial. Titration of O-antigen in industrial batches of the preparation with the aid of precipitation in gel against standard O-cholera serum could be used for its standardization. Quantitative limits of O-antigen responsible for the formation of vibration (antimicrobial) antibodies in the persons vaccinated were found by studying the immunogenicity and reactogenicity of a number of cholerogen-toxoid batches contrast by O-antigen content described by means of the precipitation test.
The quality of lipopolysaccharide has previously been shown to influence the mouse virulence of Salmonella so that strains with O antigen 4,12 were more virulent than their O-9,12 sister strains. Immunosuppression did not alter this O-antigen-dependent difference in virulence. I have now constructed smooth O-4,12 and O-6,7 sister hybrid strains of Salmonella typhimurium. No other phenotypic differences were found between these strains; they were all "common antigen" positive. In intraperitoneal infection, the O-4,12 strains were more mouse virulent than their O-6,7 sisters. The difference in virulence correlated with a difference in clearance rates; the O-6,7 hybrids were removed from the blood more rapidly than their O-4,12 sisters. No natural bactericidal antibodies were found in the sera of the mice.