The type antigen III of group F streptococci separation of group and type antigens and partial characterization of type III antigen

Polysaccharides extracted from Z₃III streptococci either with formamide or with dilute hydrochloric acid or isolated from the growth medium could be fractionated in type III- and group Z₃ antigens by alcohol precipitation. No good separation could be obtained from TCA extracts. When the same extractions and fractionations were applied to streptococci carrying type III antigen, but different group antigens, good separations were again obtained of all formamide extracts, but not of all hydrochloric acid extracts. The group antigens showed a rhamnose content of at least 50% and contained hexosamines. Type III antigens contained mainly rhamnose, glucose and galactose in relative amounts of approximately 1:2:3. Analysis of the methylated type III antigen suggests it to be a polysaccharide with a linear structure. Type III antigen isolated from the medium was characterized not only by a different sugar ratio, but also by its fucose content of 20%. In some cases the purified polysaccharides contained considerable amounts of glycogen-like material. Partial acid hydrolysis of the type III antigen extracted with formamide yielded a great number of oligosaccharides. Analyses, inhibition reactions and methylation studies gave indications that the most probable structure of a determinant group of type III antigen is β-glucosyl-(1-6)-galactosyl-(1-6)-galactosyl-(1-3)-rhamnose. The possibility of the existence of a second determinant group is discussed.     

Mechanism of type 3 capsular polysaccharide synthesis in Streptococcus pneumoniae

The glycosidic linkages of the type 3 capsular polysaccharide of Streptococcus pneumoniae ([3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->](n)) are formed by the membrane-associated type 3 synthase (Cps3S), which is capable of synthesizing polymer from UDP sugar precursors. Using membrane preparations of S. pneumoniae in an in vitro assay, we observed type 3 synthase activity in the presence of either Mn(2+) or Mg(2+) with maximal levels seen with 10-20 mM Mn(2+). High molecular weight polymer synthesized in the assay was composed of Glc and glucuronic acid and could be degraded to a low molecular weight product by a type 3-specific depolymerase from Bacillus circulans. Additionally, the polymer bound specifically to an affinity column made with a type 3 polysaccharide-specific monoclonal antibody. The polysaccharide was rapidly synthesized from smaller chains and remained associated with the enzyme-containing membrane fraction throughout its synthesis, indicating a processive mechanism of synthesis. Release of the polysaccharide was observed, however, when the level of one of the substrates became limiting. Finally, addition of sugars to the growing type 3 polysaccharide was shown to occur at the nonreducing end of the polysaccharide chain.     

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 gene cluster for the synthesis of serotype g-specific polysaccharide antigen in Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is an important pathogen related to aggressively progressive periodontal breakdown in adolescents and adults. The species can be divided into six serotypes (a–f) according to their surface carbohydrate antigens. Recently, a new serotype g of A. actinomycetemcomitans was proposed. The aim of the present study was to sequence the gene cluster associated with the biosynthesis of the serotype g-specific polysaccharide antigen and develop serotype-specific primers for PCR assay to identify serotype g strains of A. actinomycetemcomitans. The serotype-specific polysaccharide (SSPS) gene cluster of the NUM-Aa 4039 strain contained 21 genes in 21,842-bp nucleotides. The similarity of the SSPS gene cluster sequence was 96.7 % compared with that of the serotype e strain. Seventeen serotype g genes showed more than 90 % homology both in nucleotide and amino acids to the serotype e strain. Three additional genes with 1,579 bp in NUM-Aa 4039 were inserted into the corresponding ORF13 of the serotype e strain. The serotype g-specific primers were designed from the insertion region of NUM-Aa 4039. Serotypes of the a–f strains were not amplified by serotype-specific g primers; only NUM-Aa 4039 showed an amplicon band. The NUM-Aa 4039 strain was three genes in the SSPS gene cluster different from those of serotype e strain. The specific primers derived from these different regions are useful for identification and distribution of serotype g strain among A. actinomycetemcomitans from clinical samples.     

Quantitation of M antigen in Lancefield extracts of group A streptococci type 12 using electro-immuno assay

Anti-type 12 serum incorporated in agarose-polyethylene glycol gel in a concentration of 1.5% (vol/vol) was found to enable a distinct "rocket" precipitate in electro-immuno assay using hot hydrochloric acid extract of type 12 group A streptococci. This precipitate was removed by trypsin treatment of the extract and on addition of anti-M12 typing serum but not of five other typing sera to the extract. The streptococcal component responsible for this precipitate was eluted from a CM-cellulose ion exchange column at pH 6.5. These findings demonstrated that the precipitate was caused by the M12 antigen. Crossed immuno-electrophoresis of hot hydrochloric acid extracts of three different type 12 group A streptococci showed that the electrophoretic mobility of the M12 antigens was similar in the three extracts. A linear correlation was obtained between the concentration of the M12-antigen and the height of the precipitate obtained in the electro-immuno assay using different dilutions of a standard type 12 extract. M12 antigen could thus be quantitated by the electro-immuno assay. In quantitation experiments, uniformly prepared extracts of five randomly selected, freshly-isolated type 12 strains were found to contain from 130 to 1850% of M12 antigen, respectively (expressed in % of the content of the standard type 12 extract).     

Co-operative inhibition by fluoride and zinc of glucosyl transferase production and polysaccharide synthesis by mutans streptococci in suspension cultures and biofilms

Fluoride and zinc, alone or in combination at concentrations of 0.2 mM, inhibited production-secretion of glucosyltranferases by Streptococcus mutans UA159 growing in suspension cultures. Inhibition did not involve growth inhibition or starvation. Fluoride and zinc also inhibited glucan production, especially insoluble glucan, in fed-batch biofilms. Inhibition of biofilms appeared to be associated with starvation as indicated by markedly decreased ATP pools and iodophilic polysaccharide levels in biofilm cells. As insoluble glucans are important for virulence of mutans streptococci, the inhibitory actions of fluoride and zinc could significantly affect cariogenicity.     

Genetic bases and medical relevance of capsular polysaccharide biosynthesis in pathogenic streptococci

Many streptococci are human and/or animal pathogens and the frequent cause of life-threatening diseases. Among various streptococcal virulence factors, capsular polysaccharides (CPs) are recognized as essential to prevent phagocytosis by macrophages and neutrophils. In the last decade, an impressive advance on the knowledge of the genetic bases underlying capsule formation has been achieved. The capsular gene cluster driving the formation of the CP of Streptococcus pyogenes and other hyaluronate-producing streptococci, represents one of the simplest cases of gene organization to synthesize a capsule. A more complex situation has been found in Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus suis, and other streptococci. On the whole, there exists a direct relationship between the structural and chemical complexity of the repeating unit of the polysaccharide and the number of genes found in the corresponding capsular locus. Streptococcal vaccines, either polysaccharide or conjugate, are currently being tested in clinical trials to overcome the rise of worldwide antibiotic resistance, although, for different reasons, none of these vaccines are expected to provide the required full coverage in a near future. This concern has prompted to explore alternative possibilities with an improved therapeutic potential against streptococcal diseases.