Serotype-specific Polysaccharide Antigen Research Articles

Characterization of a new serotype g isolate of Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is usually isolated from the oral cavity where it is associated with active periodontitis. The species can be divided into six serotypes (a-f) according to their surface carbohydrate antigens. However, some clinical isolates cannot be grouped within these six serotypes. Gram-negative, facultative anaerobic, catalase-positive coccobacilli were isolated from a patient with periodontitis and identified by employing genetic, biochemical and serological analyses. Phenotypic data identified the isolate as A. actinomycetemcomitans. Serotype-specific polysaccharide antigen from the isolate was untypeable by immunodiffusion testing in comparison with reference A. actinomycetemcomitans serotype a to f strains. Biofilm formation by the isolate was strong but cytotoxic activity was low. Gas chromatography/mass spectroscopy analysis of partially methylated alditol acetates from surface polysaccharide showed the presence of 2,4-di-O-methyl-rhamnose and 2,3,6-tri-O-methyl-glucose, with a 1 : 1 m ratio. The (1)H- and (13)C-nuclear magnetic resonance spectra of the antigen showed that both constituent glycoses had alpha-anomeric configuration. It is proposed that the untyped strain is a new A. actinomycetemcomitans serotype, designated serotype g.

Demonstration of Streptococcus mutans with a cell wall polysaccharide specific to a new serotype, k, in the human oral cavity.

Streptococcus mutans organisms are occasionally isolated from the blood of patients with infective endocarditis, though the mechanisms of invasion and survival remain to be elucidated. Two of four blood isolates from patients with bacteremia or infective endocarditis (strains TW295 and TW871) were serologically untypeable by immunodiffusion testing, which was due to a lack of the glucose side chain of the serotype-specific polysaccharide antigen of S. mutans. Immunodiffusion analyses using antisera against these strains demonstrated that 2 of 100 isolates from 100 subjects showed a positive reaction, while further analysis of 2500 isolates from 50 subjects revealed that all 50 isolates from a single subject were not reactive with anti-c, -e, and -f antisera, though they were reactive with anti-TW295 and -TW871 antisera. The oral isolates showed biological properties similar to those of the reference S. mutans strain MT8148, including high levels of sucrose-dependent adhesion and cellular hydrophobicity, along with expression of glucosyltransferases and a protein antigen, PA. We designated these organisms serotype k. A glucose side chain-defective mutant strain was then constructed by insertional inactivation of the gluA gene of strain MT8148, which showed biological properties similar to those of serotype k of S. mutans. Serotype k oral isolates were less susceptible to phagocytosis, as were the gluA-inactivated mutant of strain MT8148 and blood isolates. These results indicate that S. mutans serotype k strains are present in the oral cavity in humans and may be able to survive longer in blood owing to their low susceptibility to phagocytosis.

Attenuation of glucan-binding protein C reduces the cariogenicity of Streptococcus mutans: analysis of strains isolated from human blood.

A blood isolate of Streptococcus mutans strain TW871 shows relatively low homology with MT8148, a reference oral isolate strain, and lacks the serotype-specific polysaccharide antigen, suggesting that other cell-surface structures correlate with cariogenicity. We compared cariogenicity of TW871 with MT8148 (serotype c) and blood isolate TW964 (serotype f) in rats. Strain TW871 showed significantly lower cariogenicity than MT8148 or TW964 and expressed significantly lower sucrose-independent cellular adhesion to saliva-coated hydroxyapatite and dextran-binding activity than strain MT8148. Strains TW871 and TW964 showed a defect in the gbpA gene by Southern hybridization analysis, while sequencing analysis revealed gbpC variation in TW871. These results suggest that variation in GbpC may alter cellular adherence properties and can be correlated with the cariogenicity of S. mutans in this strain.

Biochemical and genetic characterization of serologically untypable Streptococcus mutans strains isolated from patients with bacteremia.

Four out of 522 streptococcal isolates from the peripheral blood of patients with bacteremia exhibited typical properties of Streptococcus mutans in terms of sucrose-dependent adhesion, expression of glucosyltransferases, fermentation profiles of sugars, the presence of surface protein antigen, and DNA-DNA hybridization. Two strains were determined as serotype f and e by immunodiffusion, whereas the other two isolates did not react with the specific antiserum to S. mutans serotype c. e. or f of the eight different serotypes of mutans streptococci. The latter two untypable isolates, however, expressed a new antigenic determinant that was different from serotype c/e/f specificity as revealed by immunodiffusion. Analysis of the cell wall polysaccharides revealed very low contents of glucose in the untypable isolates. Furthermore, Southern blot analysis demonstrated that the untypable strains lacked at least one gene corresponding to a glucose-adding enzyme. These results indicate that the serologically untypable nature is due to the loss of glucosidic residue from the serotype-specific polysaccharide antigens of S. mutans.

Identification of Actinobacillus actinomycetemcomitans serotypes by multiplex PCR.

Oligonucleotide primers specific for gene clusters involved in the biosynthesis of serotype-specific polysaccharide antigens were designed to identify Actinobacillus actinomycetemcomitans serotypes a to e using the multiplex PCR. This method may be useful for serotype-specific genotyping rapidly and directly from clinical samples containing various organisms.

A gene cluster for the synthesis of serotype d-specific polysaccharide antigen in Actinobacillus actinomycetemcomitans

The serotype d antigen of Actinobacillus actinomycetemcomitans consists of d-glucose, d-mannose, and l-rhamnose in a molar ratio of 1:2:1. A gene cluster involved in the synthesis of serotype-specific polysaccharide antigen was cloned from the chromosomal DNA of A. actinomycetemcomitans IDH 781 (serotype d). This cluster consisted of 12 open reading frames. Insertional inactivation of six genes in this cluster resulted in loss of ability of A. actinomycetemcomitans IDH 781 cells to produce the polysaccharide. Comparing the structure of the gene cluster with similar clusters from other serotypes of A. actinomycetemcomitans, showed that eight genes are unique to serotype d; the other four genes are involved in the biosynthesis of dTDP- l-rhamnose. These results suggest that the synthesis and structure of serotype d-specific polysaccharide of A. actinomycetemcomitans is quite different from those of other serotype strains.

A Novel NDP-6-deoxyhexosyl-4-ulose Reductase in the Pathway for the Synthesis of Thymidine Diphosphate-d-fucose

The serotype-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans Y4 (serotype b) consists of D-fucose and L-rhamnose. Thymidine diphosphate (dTDP)-D-fucose is the activated nucleotide sugar form of D-fucose, which has been identified as a constituent of structural polysaccharides in only a few bacteria. In this paper, we show that three dTDP-D-fucose synthetic enzymes are encoded by genes in the gene cluster responsible for the synthesis of serotype b-specific polysaccharide in A. actinomycetemcomitans. The first and second steps of the dTDP-D-fucose synthetic pathway are catalyzed by D-glucose-1-phosphate thymidylyltransferase and dTDP-D-glucose 4,6-dehydratase, which are encoded by rmlA and rmlB in the gene cluster, respectively. These two reactions are common to the well studied dTDP-L-rhamnose synthetic pathway. However, the enzyme catalyzing the last step of the dTDP-D-fucose synthetic pathway has never been reported. We identified the fcd gene encoding a dTDP-4-keto-6-deoxy-D-glucose reductase. After purifying the three enzymes, their enzymatic activities were analyzed by reversed-phase high performance liquid chromatography. In addition, nuclear magnetic resonance analysis and gas-liquid chromatography analysis proved that the fcd gene product converts dTDP-4-keto-6-deoxy-D-glucose to dTDP-D-fucose. Moreover, kinetic analysis of the enzyme indicated that the Km values for dTDP-4-keto-6-deoxy-D-glucose and NADPH are 97.3 and 28.7 microM, respectively, and that the enzyme follows the sequential mechanism. This paper is the first report on the dTDP-D-fucose synthetic pathway and dTDP-4-keto-6-deoxy-D-glucose reductase.

Binding of the capsule-like serotype-specific polysaccharide antigen and the lipopolysaccharide from Actinobacillus actinomycetemcomitans to human complement-derived opsonins.

We investigated the molecular mechanism of resistance of Actinobacillus actinomycetemcomitans to complement-dependent chemiluminescence response by human polymorphonuclear leukocytes. Whole cells of serotype b-specific polysaccharide antigen-defective mutants ST2 and ST5 were constructed by inserting transposon Tn916 into A. actinomycetemcomitans strain Y4. These strains induced strong chemiluminescence response by human polymorphonuclear leukocytes and markedly bound to human complement-derived opsonins. In contrast, strain Y4 induced weak chemiluminescence response and weakly bound to complement-derived opsonins. The biosensor analysis revealed that lipopolysaccharide from strain Y4 strongly bound to human C3b, but serotype b-specific polysaccharide antigen did not. The serotype b-specific polysaccharide antigen molecule might sterically hinder the interaction between complement-derived opsonins and lipopolysaccharide to reduce complement-dependent chemiluminescence response by human polymorphonuclear leukocytes.

A gene cluster for 6-deoxy- l-talan synthesis in Actinobacillus actinomycetemcomitans

The serotype c antigen of Actinobacillus actinomycetemcomitans consists of 6-deoxy- l-talose. A gene cluster involved in the synthesis of serotype-specific polysaccharide antigen was cloned from the chromosomal DNA of A. actinomycetemcomitans NCTC 9710 (serotype c). This cluster consisted of 17 open reading frames. Escherichia coli produced the polysaccharide that reacts with the serotype c-specific antibody when transformed with a plasmid containing the cluster. Comparing the structure of the gene cluster with a similar cluster from A. actinomycetemcomitans Y4 (serotype b), which produces a polysaccharide consisting of l-rhamnose and d-fucose, revealed that a 5.7 kb region containing seven genes in the cluster from strain Y4 was replaced by a 3.8 kb region containing three genes in strain NCTC 9710. The results suggest that these region, as well as dTDP-6-deoxy- l-talose-forming dTDP-4-keto- l-rhamnose reductase, is essential to the production of extracellular polysaccharide specific to serotype c.

Identification of a genetic locus essential for serotype b-specific antigen synthesis in Actinobacillus actinomycetemcomitans.

A large gene cluster associated with the biosynthesis of the serotype-specific polysaccharide antigen (SPA) of Actinobacillus actinomycetemcomitans Y4 (serotype b) was cloned and characterized. Western blot analysis showed that Escherichia coli DH5alpha, containing a plasmid carrying this cluster, produced a polysaccharide which reacted with a monoclonal antibody directed against the SPA of A. actinomycetemcomitans Y4. High-performance liquid chromatography analysis indicated that the polysaccharide produced by an E. coli transformant, as well as A. actinomycetemcomitans Y4 SPA, was composed of rhamnose and fucose. Furthermore, using various derivatives of the plasmid, we demonstrated that the cloned 13-kb BssHII-BspHI fragment was indispensable for SPA synthesis in E. coli DH5alpha. The 24,909-bp nucleotide sequence, which included this fragment and its flanking regions, was determined. In the sequenced area, 24 open reading frames (ORFs) with the same orientation were found. Most of these were located sequentially within a short distance of each other. Many of the deduced amino acid sequences were similar to the gene products of the polysaccharide synthetic genes of other bacteria. The average G+C content (37.7%) of all 24 ORFs in the sequenced area was lower than that (45.6%) of the whole chromosome of A. actinomycetemcomitans Y4. It is noteworthy the average G+C content of the nine ORFs in the 8.5-kb central region of the 13-kb BssHII-BspHI fragment indispensable for SPA synthesis in E. coli was found to be especially low (27.0%).

Biological function of the dTDP-rhamnose synthesis pathway in Streptococcus mutans.

We have cloned a new gene locus that comprises three genes concerned with the biosynthesis of the serotype c-specific polysaccharide antigen in Streptococcus mutans. The genes encode proteins exhibiting significant homology to the rfbA, rfbB, and rfbD gene products that are involved in the anabolism of dTDP-L-rhamnose from D-glucose-1-phosphate. This anabolism pathway pertains to biosynthesis of the O antigen of lipopolysaccharide in gram-negative bacteria. The cell extract of Escherichia coli expressing each of the cloned genes of S. mutans exhibited enzymatic activity corresponding to the homologous counterpart of the rfb gene products. Rhamnose was not detected in the cell wall preparation purified from the mutant in which each of the three cloned genes was insertionally inactivated. Rabbit antiserum against S. mutans serotype c-specific antigen did not react with the autoclaved extracts from these mutants. These results indicate that the gene products identified in the present study are involved in the dTDP-L-rhamnose synthesis pathway and that the pathway relates to the biosynthesis of the serotype-specific polysaccharide antigen of S. mutans. Southern hybridization analysis revealed that genes homologous to the cloned genes involved in the dTDP-L-rhamnose synthesis pathway were widely distributed in a variety of streptococci. This is the first report of the biological function of the dTDP-rhamnose pathway in streptococci.

Purification of serotype d-- and e--specific antigens from Actinobacillus actinomycetemcomitans and seroclassification of clinical isolates from periodontal patients

Oral Actinobacillus actinomycetemcomitans strains have been classified into five serotypes. The aim of this study was to determine the compositions of A. actinomycetemcomitans serotype d- and e- specific antigens. The serodistribution of clinical isolates from the patients with periodontitis were also investigated. Serotype-specific polysaccharide antigens of A. actinomycetemcomitans IDH 781 (serotype d) and OMZ 534 (serotype e) were extracted from whole cells by autoclaving. The extracts were purified by chromatography on DEAE-Sepharose CL-6B and Sephacryl S-300HR columns. The serotype d antigen was composed of rhamnose (17.1%), mannose (45.5%), galactose (2.0%) and glucose (35.5%). On the other hand, the serotype e antigen was composed of rhamnose (23.9%), mannose (29.1%), galactose (11.0%), glucose (13.5%) and unidentified sugar (22.5%). Immunodiffusion tests revealed that the purified polysaccharide antigen form a single precipitin line with the corresponding rabbit antiserum. A total of 157 A. actinomycetemcomitans clinical isolates from diseased sites of 39 patients with periodontitis were serotyped by using serotype-specific rabbit antisera against A. actinomycetemcomitans serotype a, b, c, d and e strains. In the immunodiffusion assay, the autoclaved extracts of 42, 12, 34, 8 and 41 A. actinomycetemcomitans clinical isolates reacted with serotype a, b, c, d and e antisera, respectively. These findings indicate that the extraction of serotype antigens by autoclaving is useful and definite for the serotyping of A. actinomycetemcomitans clinical isolates.

An enzyme-linked immunosorbent assay for measuring antibodies to serotype-specific polysaccharide antigens of Actinobacillus actinomycetemcomitans

An enzyme-linked immunosorbent assay utilizing avidin-biotin binding has been developed for measuring antibodies to serotype-specific polysaccharide antigens of Actinobacillus actinomycetemcomitans. The serotype a, b and c polysaccharide antigens were coupled with biotin via an adipic acid hydrazide functional group. The biotinylated antigens immobilized on the avidin-precoated wells reacted specifically with rabbit antisera to whole cells of the corresponding serotype of A. actinomycetemcomitans. Antibodies to the antigens in 1:6,400 to 1:12,800 dilutions of human sera from subjects with periodontitis could be detected by using the assay.

Development and Evaluation of a Mixed-Antigen ELISA for Serodiagnosis of Actinobacillus Pleuropneumoniae Serotypes 1, 5, and 7 Infections in Commercial Swine Herds

A mixed-antigen enzyme-linked immunosorbent assay (ELISA) containing serotype-specific polysaccharide antigens from serotypes 1, 5, and 7 of Actinobacillus pleuropneumoniae was developed. This ELISA was evaluated using sera from experimentally infected pigs. With a negative cutoff value of 0.250 (optical density at 405 nm), sensitivity and specificity were determined to be 96% and 99.5%, respectively. The ELISA was further evaluated using sera from commercial swine. Sows and suckling piglets were found to be the best age groups for detection of positive reactors to A. pleuropneumoniae. The mixed-antigen ELISA could be used in a herd health monitoring program for detection of A. pleuropneumoniae infections.

6-Deoxy-D-talan and 6-deoxy-L-talan. Novel serotype-specific polysaccharide antigens from Actinobacillus actinomycetemcomitans.

Serotype-specific polysaccharide antigens from Actinobacillus actinomycetemcomitans ATCC 29523 (serotype a) and NCTC 9710 (serotype c) were extracted from whole cells by autoclaving and purified by ion-exchange chromatography and gel filtration. Analysis of component sugars by gas-liquid chromatography-mass spectrometry, high performance liquid chromatography, and NMR together with optical rotation data showed that the serotype a antigen was composed solely of 6-deoxy-D-talose, whereas the serotype c antigen consisted of 6-deoxy-L-talose. Structural analysis indicated that both of these antigens were composed of closely related repeating units, -3)-6-deoxy-alpha-D-Talp-(1-2)-6-deoxy-alpha-D-Talp-(1-(sero type a) and -3)-6-deoxy-alpha-L-Talp-(1-2)-6-deoxy-alpha-L-Talp-(1-(sero type c). 1H and 13C NMR analysis showed that both of these serotype antigens contained one acetyl group/2 sugar residues. These acetyl groups localized at the O-2 position of 3-linked 6-deoxy-D-talose (serotype a) or O-4 position of 3-linked 6-deoxy-L-talose residues (serotype c), respectively. These results coupled with our previous findings on the serotype b antigen (Amano, K., Nishihara, T., Shibuya, N., Noguchi, T., and Koga, T. (1989) Infect. Immun. 57, 2942-2946) showed that the serotype antigens from A. actinomycetemcomitans are a group of novel polysaccharides with structural features closely related biosynthetically.

Murine macrophage interleukin-1 release by capsularlike serotype-specific polysaccharide antigens of Actinobacillus actinomycetemcomitans

Serotype-specific polysaccharide antigens (SPAs) were extracted from whole cells of Actinobacillus actinomycetemcomitans ATCC 29523 (serotype a), Y4 (serotype b), and NCTC 9710 (serotype c) by autoclaving and purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300 columns. Y4 SPA induced interleukin-1 (IL-1) release by P388D1 murine macrophages. Polymyxin B had virtually no effect on the release of IL-1. Rabbit anti-murine IL-1 serum strongly suppressed the proliferation of C3H/HeJ mouse thymocytes induced with the culture supernatants of Y4 SPA-stimulated P388D1 cells and a submitogenic dose of concanavalin A. Gel filtration of the culture supernatants of Y4 SPA-stimulated macrophages on Sephacryl S-200 showed that an IL-1 peak at a point corresponding to approximately 16.5 kDa was eluted. The ability of SPAs from strains ATCC 29523 and NCTC 9710 to induce the release of IL-1 was lower than that of Y4 SPA. The IL-1-releasing ability of serotype a and c antigens was enhanced by deacetylation of both polysaccharides, suggesting that acetyl groups of these antigens might hinder the interaction between the antigens and macrophages.

Immunochemical and structural characterization of a serotype-specific polysaccharide antigen from Actinobacillus actinomycetemcomitans Y4 (serotype b)

A serotype-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans Y4 (serotype b) was extracted from whole cells by autoclaving. The extract was purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300 columns. The purified polysaccharide antigen formed a single precipitin line with anti-type b serum but not with anti-type a serum and anti-type c serum. The antigen was composed of 43.9% L-rhamnose, 49.1% D-fucose, and a trace amount of fatty acid. Methylation analysis, Smith degradation, and optical rotation data showed that the antigen was a polymer consisting of a disaccharide repeating unit, ----3)-alpha-D-fucopyranosyl-(1----2)-beta-L-rhamnopyranosyl-(1----. In quantitative precipitin inhibition tests, D-fucose and L-rhamnose showed very low inhibition, but the partial hydrolysate of the purified antigen was an effective inhibitor, suggesting that the serotype b specific antiserum recognizes the larger oligosaccharide units.