Pneumococcal type III

A simple procedure for determining the specificity of jS-o-glucan hydrolases involves the use of pneumococcal type III polysaccharide as a substrate. Whereas cellulase, laminarinase, and lichenase are inactive towards this polysaccharide, the two last enzymes hydrolyse its reduced and esterified form. Comparisons of the activities of the enzymes towards these and other substrates e.g. laminarin, lichenin, carboxymethylcellulose) permit them to be distinguished, provided the oligosaccharides released are identified. 

A derivatized form of pneumococcal type III polysaccharide has been used to distinguish between three jS-D-glucan hydrolases (including cellulase) from a Streptomyces species. Oligosaccharides also released from carboxymethyl-cellulose, lichenin, and oat glucan by the enzymes were identified, allowing the substrate specificities of the enzymes to be deduced. 

Bacillus subtilis lichenase hydrolysed lichenin, oat glucan, and reduced pneumococcal type III polysaccharide. Unlike laminarinase, the enzyme did not degrade laminarin and carboxymethyl-laminarin. 

By 1945, Stacey speculated about the possibility of a structural relationship between Pneumococcus capsular polysaccharides and those produced by other organisms. With Miss Schliichterer, he had examined the capsular polysaccharide of Rhizobium radicicolum. This polysaccharide gave a precipitin reaction in high dilution, not only with Type III Pneumococcus antiserum, but also mixed with antisera from other Pneumococcus types. The chemical evidence indicated that the polysaccharide resembled the specific polysaccharides of Types I and II Pneumococcus. A decade later, the acidic capsular polysaccharide from Azoto-bacter chroococcum, a soil organism, was studied. It, too, produced serological cross-reactions with certain pneumococcal specific antisera. Although the molecular structure of the polysaccharide was not established, adequate evidence was accumulated to show a structural relationship to Type III Pneumococcus-specific polysaccharide. This was sufficiently close to account for the Type III serological cross-relationship. 

J5. Jaton, J. C., Huser, H., Brown, D. C., Schlessinger, J., Pecht, I., and Givol, D., Conformational changes induced in a homogeneous anti-Type III pneumococcal antibody by oligosaccharides of increasing size. Biochemistry 14, 5312-5315 (1975). 

For most antigens, the production of antibody (immunoglobulin) is based on the cooperative interaction of two types of lymphocyte, called T-cells (thymus-derived) and B-cells (bone marrow-derived). The T-cells, preprimed with macrophage-presented antigen, stimulate the B-cells to secrete copious quantities of antibody. However, on the basis of animal studies, such polysaccharide antigens as the type III pneumococcal polysaccharide have been considered to be T-cell-independent, as they are capable of triggering B-cells to produce antibody (IgM) in T-cell-deficient mice.167 These studies also indicated... 

Anti-type III pneumococcal polysaccharide O Anti-type VIII pneumococcal polysaccharide Anti-streptococcal group A variant carbohydrate A Anti-streptococcal group C carbohydrate + Anti-p-azophenylarsonate x Anti-Micrococcus lysodeikticus " Mouse kappa light chain. c Human kappa light chain subgroup IV. d K9-335 has an identical sequence as K9-338. 

SCHEME 44. Inverse regioselectivity of the ditritylated acceptors in the synthesis119 of Group B type III pneumococcal oligosaccharide 228. 

Avery, O. T., C. M. MacLeod, and M. McCarthy. 1944. Studies on the chemical nature of the substance inducing transformation of pneumococcal types I induction of transformation by DNA from Pneumococcus type III. J. Exp. Med. 79, 137... 

The discovery " of a so-called atypical Type III strain of Pneumococcus which was immunologically related to, but not identical with, Type III provided the first instance of a cross-reaction of a type-specific, pneumococcal polysaccharide with a heterologous, pneumococcal antiserum. The atypical Type III was later reclassified as Tjrpe VIII. 

The cross-reactions of Types III and VIII Pneumococcus in horse and rabbit antisera were studied quantitatively by Heidelberger and his colleagues. - At least three distinct kinds of anticarbohydrate (after removal of antibodies to the somatic C-substance) were evoked in horses in response to the stimulus of the type-specific pneumococcal antigen. Two of these, usually from one-quarter to one-third of the total, were completely precipitable by SIII or SVIII. However, the principal antibody fraction... 

A partially fractionated, cell-free, enzyme preparation from Type III pneumococcal cells (which had been grown in the presence of Sill depolymerase to lower the proportion of pre-existing Sill) incorporated d-glucose and D-glucuronic acid in the ratio of 1 1 from carbou-14-labeled... 

Capsules of pneumococci consist largely of a polysaccharide slime, but they contain in addition some protein and small amounts of other substances. The type-specificity and virulence of the pneumococci are due to their capsular polysaccharides (see Chapter VIII). The specificity of reaction for different pneumococcal types is due to variation in the capsular polysaccharide molecules. The antigenic polysaccharides are isolable from the bacteria but are more often prepared from the culture broth into which they are liberated by bacterial autolysis. Though there are more than 70 known types of pneumococci, little is known of the structures of their capsular polysaccharides save that from type III pneumococcus. This is shown to be a linear molecule of D-glucose and D-glucuronic acid units in equal amounts. They are linked alternately so that the molecule may be regarded as a chain of aldobiouronic acid units 131) ... 

The numerous examples of regular complex copolysaccharides often involve familiar-looking material for cellulose chemists. Figure 10 shows two pneumococcal polysaccharides. Types III and VIII, also known as "specific soluble substance", which in the 1920 s and early 1930 s were shown to be antigenic although they were free of nitrogen and did not possess any of the properties of peptides. The knowledge achieved by the extensive studies on cellulose and carbohydrates in the first decades of this century was responsible for the early establishment of the chemical structures of Types III and VIII. The revolutionary work on bacterial transformation, in which Avery, MacLeod and McCarty in 1944 identified DNA as the... 

Fig. 10 Type III and Type VIII pneumococcal polysaccharides which are regular structures with a dimeric and tetrameric repeating unit, respectively. The structures are drawn from left to right corresponding to "nonreducing" end at the left. Both structures contain one glucuronic acid unit per repeat and two repeating units are shown for the Type III.

In further studies (Prusansky and Axelrod, 1954) on the same deficiency states discussed above the serum complement was markedly depressed by the intrapeiitoneal injection of pneumococcal polysaccharide type III (Sill) followed 1 hour later by the intravenous injection of a neutralizing amount of specific rabbit antiserum. This depletion process was repeated daily for 3 days. The regeneration of serum complement was followed by determining the serum titer 22 hours after the final injection of rabbit antiserum. The rate of serum complement regeneration by the deficient animals was not significantly different from that of corresponding controls. 

The Pneumococcal Polysaccharides. Specific polysaccharides from Types I, II and III Pneumococcus, which had been obtained in relatively undegraded forms, have been examined by Stacey and Record.109 In contrast to the results of Tennent and Watson, both the sedimentation and the diffusion constants were found to be very dependent on the concentration. The polysaccharides were polydisperse, with molecular weights in the range (10)B—(10) and they possessed an elongated form in solution (Table XV). 

S. pneumoniae has more than 80 sero-types. The current polysaccharide vaccine consists of 23 serotypes and covers about 87% of all pneumococcal diseases in the United States. Current vaccine development is based on conjugate technology and concentrates on the most prevalent 7—9 serotypes. Three multivalent vaccine candidates are in clinical trials. All are based on conjugating the polysaccharide to a T-dependent protein carrier. The results of phase I and II trials in infants have demonstrated the safety and immunogenicity (56—58) of these vaccines. Phase III trials to demonstrate efficacy against systematic diseases and otitis media are in progress and final approval of this vaccine for infant immunization will be by the year 2000. 

Stacey has written an excellent review on the subject of mucopolysaccharides, which he classified on the basis of their containing both hexosamine and hexuronic acid residues, one or the other of these sugar derivatives, or neither. Hyaluronic acid, chondroitinsulfuric acid. Type I pneumococcal polysaccharides, and heparin are members of the first class. Types II, III, and VIII pneumococcal polysaccharides are examples containing hexuronic acid but no hexosamine. Chi tin and Types IV and XIV pneiuno-coccal polysaccharides contain hexosamine but no hexuronic acid and bacterial dextrans, mold polysaccharides, and levans contain neither hexosamine nor hexuronic acid. 

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