Determination of Salmonella typhimurium antigens in the excreta of children with salmonellosis using the passive hemagglutination reaction

1,390 samples of different excreta obtained from salmonellosis patients have been tested for the presence of S. typhimurium O- and H-antigens. S. typhimurium antigens, detected with the use of antibody diagnostica, have been found to occur more frequently than S. typhimurium cells. Particulate O- and H-antigens capable of agglutinating antibody diagnostica are excreted differently with saliva and urine. Salmonella antigens are best detected in feces in the passive hemagglutination test with the use of antibody diagnostica, but not in the antibody neutralization test. The combination of the passive hemagglutination test, carried out with the use of antibody diagnostica, and bacteriological study considerably enhances the efficiency of diagnosing salmonellosis in children in comparison with bacteriological study alone.     

Methods of using the antibody neutralization test in intestinal coli-infections]

Specificity and sensitivity of the antibody neutralization test intended for detection of the O-antigen of enteropathogenic escherichia were checked under experimental conditions. Only 3 strains of the Klebsiella genus proved to neutralize the antibodies to the enteropathogenic escherichia of the serological group O20:K84. In the rest of the cases a positive result was obtained only in homologous combinations. In comparative study of the microbial cultures of the infected feces on hard nutrient media by means of bacteriological and serological methods the latter was found to be more sensitive, capable of detecting the homologous O-antigen with the bacterial concentration of not less than 5-10(5) microbial cells per 1 ml.     

The rapid diagnosis of salmonellosis by a determination of the antigen in the biosubstrates of patients using the coagglutination reaction]

The diagnostic potential of the coagglutination test was checked with the aim of improving the laboratory diagnosis of Salmonella infections by the detection of Salmonella specific antigen in different biological materials (feces, urine, saliva and immune complexes in blood sera). The study of all specimens resulted in the confirmation of the diagnosis in 78% of patients, often during the first days of the disease. The proportion of nonspecific reactions, as shown in the control groups of healthy donors and patients with dysentery and other acute enteric infections, did not exceed 5%.     

Systemic immunological memory in enteral immunization with the O antigen from S. sonnei

Mice received S. sonnei O-antigen at various concentrations (0.01-20,000 micrograms/ml) in drinking water. Systemic immunological memory, induced by feeding with O-antigen, was manifested by secondary immune response to parenteral boosting with homologous O-antigen or ribosomal vaccine. A pronounced priming effect was also produced by O-antigen at concentrations as low as 0.01 micrograms/ml after courses of feeding as short as 1-3 days. Even high doses of the antigen had no tolerogenic activity. The state of immunological memory was formed at least 12 days after the first feeding and lasted for a long period (at least 4 months after the last feeding). The specificity of immunological memory was proved in experiments with heterologous O-antigen (Salmonella typhimurium): the insignificant stimulating action of this antigen was revealed only when high concentrations of the antigen (1000 micrograms/ml) were used for feeding.     

Quantitative determination of the soluble antigens of intestinal bacteria using a method of immunoenzyme analysis. I. Processing of the parameters of the method

The parameters of the assay based on the quantitative evaluation of the neutralization of specific antibodies by the antigen under study and the subsequent detection of free antibodies on the fixed reference antigen with the aim of the quantitative determination of the specific O-antigens of Salmonella, groups D, B, C1, as well as those of Shigella sonnei and Shigella flexneri, have been developed. The proposed method makes it possible to detect the O-antigen of the causative agent at concentrations of 0.001 micrograms/ml to 100 micrograms/ml.     

Immunoenzyme analysis of the serological activity of the structural modifications of a synthetic antigen simulating the O-determinant 4 of Salmonellae

The comparison of the copolymer of 2-O-(alpha-abequosy)-alpha-alyl-alpha-mannopyranoside and acrylamide (AMA), used as a synthetic antigen, with its natural prototype, Salmonella typhimurium lipopolysaccharide, in the enzyme immunoassay (EIA) has revealed that the serological activity of AMA is related to the specific content of antigenic determinants in its molecule. The analysis of the serological specificity of AMA indicates that this specificity corresponds to factor 4 of Salmonella O-antigen. The high activity and monofactor specificity of the synthetic antigen AMA suggest that the use of this antigen in EIA as a diagnostic preparation holds good promise.     

The biological properties of Salmonella strains isolated from adult patients at different seasons of the year]

Biological properties of Salmonella strains, isolated in different seasons from patients with the corresponding disease of moderate severity, were compared. Their morphological, biochemical, serologic properties, sensitivity to antibiotics, capacity for synthesizing O-antigen, as well as their virulence for experimental animals, have been studied. Seasonal changes in the virulence of Salmonella strains have been established: the strains isolated in autumn have proved to be more virulent than those isolated in winter. In winter the isolation rate of Salmonella strains resistant to the therapeutic doses of antibiotics is significantly higher than in other seasons. In spring and summer Salmonella O-antigen is synthesized more intensively.     

肠道沙门氏菌O-抗原多糖的研究进展

O-抗原是由多糖重复单元组成的多聚糖,表达于细菌的外膜,具有多样性,是划分沙门菌血清型的重要依据。O-抗原多糖由多基因协同作用而合成,这些基因在沙门菌基因组上成簇存在,形成O-抗原基因簇。O-抗原多糖也是重要的毒力因子,在沙门菌入侵宿主、体内存活、定殖等致病过程中均发挥着重要的作用。此外,O-抗原还是沙门菌主要的保护性抗原,能激发宿主产生高水平抗体并发挥免疫保护作用,成为疫苗研究的靶点。本文综述O-抗原多糖的基因结构和合成、生物学功能及其在疫苗研制中的应用与前景。     

Altering the length of the lipopolysaccharide O antigen has an impact on the interaction of Salmonella enterica serovar Typhimurium with macrophages and complement

A panel of isogenic Salmonella enterica serovar Typhimurium strains that vary only in the length of the O antigen was constructed through complementation of a wzz double mutant (displaying unregulated O-antigen length) with one of two homologous (wzzST and wzzfepE) or three heterologous (wzzO139 of Vibrio cholerae and wzzSF and wzzpHS-2 of Shigella flexneri) wzz genes. Each gene was functional in the S. enterica serovar Typhimurium host and specified production of O-antigen polymers with lengths typical of those synthesized by the donor bacteria (ranging from 2 to >100 O-antigen repeat units). By use of this panel of strains, it was found that O-antigen length influences invasion/uptake by macrophage cells; this is the first time this has been shown with Salmonella. O-antigen length was confirmed to be related to complement resistance, with a minimum protective length of >4 and <15 repeat units. O antigen of 16 to 35 repeat units was found to activate complement more efficiently than other lengths, but this was unrelated to complement resistance. No evidence was found to suggest that modifying the length of the O-antigen polymer affected expression of the O1, O4, or O5 antigenic factors.     

Structural and genetic characterization of enterohemorrhagic Escherichia coli O145 O antigen and development of an O145 serogroup-specific PCR assay

Enterohemorrhagic Escherichia coli O145 strains are emerging as causes of hemorrhagic colitis and hemolytic uremic syndrome. In this study, we present the structure of the E. coli O145 O antigen and the sequence of its gene cluster. The O145 antigen has repeat units containing three monosaccharide residues: 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamidoylamino-2,6-dideoxy-L-galactose, and N-acetylneuraminic acid. It is very closely related to Salmonella enterica serovar Touera and S. enterica subsp. arizonae O21 antigen. The E. coli O145 gene cluster is located between the JUMPStart sequence and the gnd gene and consists of 15 open reading frames. Putative genes for the synthesis of the O-antigen constituents, for sugar transferase, and for O-antigen processing were annotated based on sequence similarities and the presence of conserved regions. The putative genes located in the E. coli O145 O-antigen gene cluster accounted for all functions expected for synthesis of the structure. An E. coli O145 serogroup-specific PCR assay based on the genes wzx and wzy was also developed by screening E. coli and Shigella isolates of different serotypes.     

The determination of the antibodies and the somatic O-antigen of Salmonella group B by using the complement-bound lysis of liposomes]

A simple and sensitive method for the determination of group B Salmonella O-antigen and specific antibodies to group B Salmonella by means of the complement-dependent lysis of liposomes sensitized with S. typhimurium lipopolysaccharide (LPS) is proposed. The factors affecting the sensitivity of the method during the determination of antibodies and free antigen have been studied. The method permits the determination of soluble LPS antigen in concentrations of 0.5-200 micrograms/ml.     

Evidence for energy-dependent transposition of core lipopolysaccharide across the inner membrane of Salmonella typhimurium.

The uncoupler 2,4-dinitrophenol blocks the final step of lipopolysaccharide assembly--transfer of O antigen from undecaprenyl pyrophosphate to core lipopolysaccharide--in intact Salmonella typhimurium but not in isolated membrane fractions. The O-antigen ligase enzyme is not inhibited by dinitrophenol in vitro, and core lipopolysaccharide synthesized in the presence of uncoupler in vivo is functional as acceptor of O antigen in vitro. The evidence strongly suggests that maintenance of proton motive force is required for transmembrane transposition of core lipopolysaccharide to the active site of O-antigen ligase at the periplasmic face of the inner membrane.     

Isolation of monospecific Salmonella anti-O-4 antibodies by affinity chromatography and their use in immunoenzyme reactions

An immunosorbent with Salmonella typhimurium 1021 O-antigen has been obtained on the basis of polymer-modified porous glass. Binding has been effected due to the presence of NH2-groups on the carrier and aldehyde groupings on O-antigen previously oxidized with sodium periodate. Specific anti-O-4 antibodies have been isolated with the use of this immunosorbent, and on the basis of these antibodies peroxidase conjugates have been obtained. This preparation has made it possible to establish the parameters of EIA for the detection of Salmonella O-4 antigens in the assay involving the use of the sandwich technique. The method has proved to be sufficiently simple, sensitive and specific. It is recommended for the detection of Salmonella O-4 antigen in biological fluids.     

Somatic Antigen 2 Inheritance in Salmonella Groups B and D

Somatic (O) antigen 2 of Salmonella paratyphi A replaced somatic antigen 4 of an S. typhimurium recipient as the consequence of mating with an S. paratyphi A var. durazzo Hfr strain. The genetic determinants of these O antigens behaved in this cross as alleles of a common O locus, which is linked to the determinant of histidine biosynthesis, his. By employing phage lysates obtained by growth of P22 on an S. typhimurium hybrid which had received his and O-factor 2 determinants from the S. paratyphi A Hfr, it was possible to cotransduce the his and O-antigen 2 genes to both S. typhimurium and S. typhosa. S. typhimurium transductants which received somatic antigen 2 concurrently lost O-antigen 4, and S. typhosa transductants receiving O-antigen 2, lost their native O-antigen 9. These results indicate that the genetic determinants of O-antigens 2, 4, and 9 occupy the same O locus in S. paratyphi A, S. typhimurium, and S. typhosa, respectively, and are probably allelic.     

Molecular cloning and expression in Escherichia coli K-12 of the rfb gene cluster determining the O antigen of an E. coli O111 strain

The O antigen of Escherichia coli O111 is identical in structure to that of Salmonella enterica serovar adelaide. Another O-antigen structure, similar to that of E. coli O111 and S. enterica serovar adelaide is found in both E. coli O55 and S. enterica serovar greenside. Both O-antigen structures contain colitose, a 3,6 dideoxyhexose found only rarely in the Enterobacteriaceae. The O-antigen structure is determined by genes generally located in the rfb gene cluster. We cloned the rfb gene cluster from an E. coli O111 strain (M92), and the clone expressed O antigen in both E. coli K-12 and a K-12 strain deleted for rfb. Lipopolysaccharide analysis showed that the O antigen produced by strains containing the cloned DNA is polymerized. The chain length of O antigen was affected by a region outside of rfb but linked to it and present on some of the plasmids containing rfb. The rfb region of M92 was analysed and compared, by DNA hybridization, with that of strains with related O antigens. The possible evolution of the rfb genes in these O antigen groups is discussed.     

Immunoelectrophoretic analysis of the antigenic composition of Yersinia

The antigenic composition of 24. Y. pseudotuberculosis newly isolated and reference strains, 7 Y. enterocolitica strains, as well as Y. pestis vaccine strain EV, has been studied by the method of immunoelectrophoresis in agar. The antigenic composition of these bacteria has been found to be complicated and to comprise not less than 8-11 antigens, and among them nonspecific protein antigens common for enterobacteria, the common generic antigen, the antigen common with Y. pestis, as well as O-antigens specific for each serovar are identified. Immunoelectrophoretic study has shown the possibility of Y. pseudotuberculosis O-antigen, serovar I, with Salmonella sera, serogroup A, and Y. enterocolitica 09 with brucellar and cholera sera.     

The activity of a putative polyisoprenol-linked sugar translocase (Wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat

During O antigen lipopolysaccharide (LPS) synthesis in bacteria, transmembrane migration of undecaprenylpyrophosphate (Und-P-P)-bound O antigen subunits occurs before their polymerization and ligation to the rest of the LPS molecule. Despite the general nature of the translocation process, putative O-antigen translocases display a low level of amino acid sequence similarity. In this work, we investigated whether complete O antigen subunits are required for translocation. We demonstrate that a single sugar, GlcNAc, can be incorporated to LPS of Escherichia coli K-12. This incorporation required the functions of two O antigen synthesis genes, wecA (UDP-GlcNAc:Und-P GlcNAc-1-P transferase) and wzx (O-antigen translocase). Complementation experiments with putative O-antigen translocases from E. coli O7 and Salmonella enterica indicated that translocation of O antigen subunits is independent of the chemical structure of the saccharide moiety. Furthermore, complementation with putative translocases involved in synthesis of exopolysaccharides demonstrated that these proteins could not participate in O antigen assembly. Our data indicate that recognition of a complete Und-P-P-bound O antigen subunit is not required for translocation and suggest a model for O antigen synthesis involving recognition of Und-P-P-linked sugars by a putative complex made of Wzx translocase and other proteins involved in the processing of O antigen.     

Determination of the O antigen of Shigella sonnei by a competitive immunoenzyme method

A technique for immunoenzymatic diagnosis of dysentery by Shigella sonnei O-antigen was developed. For induction of antibodies to O-antigen rabbits were immunized by intravenous administration of a commercial antidysentery vaccine. Specific antibodies to O-antigen belonging to class G immunoglobulins and not binding to O-antigens of Sh. flexneri and Salmonella typhimurium were obtained. beta-Lactamase of Bacillus licheniformis 749/c was used as a marker enzyme in the immunoenzymatic assay. To increase the sensitivity, beta-lactamase molecules were preliminarily linked with glutaric aldehyde into oligomers. Conjugates of Sh. sonnei O-antigen with the oligomers of B. licheniformis 749/c beta-lactamase were prepared with the periodate method by oxidizing O-antigen. The conjugate was used in competing solid phase immunoenzymatic assay for determination of Sh. sonnei O-antigen in blood serum of patients with dysentery. The sensitivity of the assay is 0.5-1 ng per 1 ml of O-antigen.     

Functional characterization of WaaL, a ligase associated with linking O-antigen polysaccharide to the core of Pseudomonas aeruginosa lipopolysaccharide

The O antigen of Pseudomonas aeruginosa B-band lipopolysaccharide is synthesized by assembling O-antigen-repeat units at the cytoplasmic face of the inner membrane by nonprocessive glycosyltransferases, followed by polymerization on the periplasmic face. The completed chains are covalently attached to lipid A core by the O-antigen ligase, WaaL. In P. aeruginosa the process of ligating these O-antigen molecules to lipid A core is not clearly defined, and an O-antigen ligase has not been identified until this study. Using the sequence of waaL from Salmonella enterica as a template in a BLAST search, a putative waaL gene was identified in the P. aeruginosa genome. The candidate gene was amplified and cloned, and a chromosomal knockout of PAO1 waaL was generated. Lipopolysaccharide (LPS) from this mutant is devoid of B-band O-polysaccharides and semirough (SR-LPS, or core-plus-one O-antigen). The mutant PAO1waaL is also deficient in the production of A-band polysaccharide, a homopolymer of D-rhamnose. Complementation of the mutant with pPAJL4 containing waaL restored the production of both A-band and B-band O antigens as well as SR-LPS, indicating that the knockout was nonpolar and waaL is required for the attachment of O-antigen repeat units to the core. Mutation of waaL in PAO1 and PA14, respectively, could be complemented with waaL from either strain to restore wild-type LPS production. The waaL mutation also drastically affected the swimming and twitching motilities of the bacteria. These results demonstrate that waaL in P. aeruginosa encodes a functional O-antigen ligase that is important for cell wall integrity and motility of the bacteria.