Localization of group A streptococcal nontype-specific antigens and their occurrence among streptococci of different groups]

It was shown by immunodiffusion methods that nontypespecific antigens revealed in the HCl extracts of streptococcus, group A, were localized in the cell wall. In B, E, H, K, L, M, P, S, T streptococci groups there was revealed only one, and in C and G streptococci groups--two antigens identical to the HTC antigens of streptococci, group A. Besides, an antigen, which was apparently specific specific for group A streptococcus only, was detected. The data obtained should be taken into consideration in the elaboration of improved method of grouping and typing group A streptococcus.     

Cross-reacting antigens of human thymus myoid cells and stable L-forms of group A streptococci

Indirect immunofluorescence has shown a similarity between the antigen components of group A streptococcus L-forms and human thymus myoid cells. An analogous antigen (or antigens) is present in the cytoplasmic membrane of human myocardial cell fibers. The depletion of antiserum to the streptococcal L-forms both by the culture of L-forms grown in meat or casein media and by the homogenate of the cardiac muscle leads to the inhibition of immunofluorescence. The depletion of serum by the homogenate of other tissues (liver) or by L-form culture does not virtually affect the immunofluorescence intensity. According to the authors' opinion, the similarity of antigens of group A streptococcus L-forms to the antigenic components of organ tissues is likely to be responsible for long-term persistence of the microorganisms under consideration and to favour, in some cases, the occurrence of autoantibodies. The latter circumstance might lead to pathological changes in organs containing cross-reacting antigens.     

Osmotic fragility of the group A streptococcal L form

The osmotic fragility, expressed in terms of survival, of two group A streptococcal L-form strains was examined by suspending the L form in sodium chloride and sucrose solutions of graded concentrations. An immediate and marked reduction in viability followed suspension in sodium chloride solutions of less than 0.7m. A wide distribution of osmotic fragility within the L-form population was observed. The two L-form strains (GL-8 and AED) differed in that the AED L-form strain appeared to be consistently more resistant to osmotic lysis, and survived considerably better in sodium chloride solutions up to 90 minutes. Sucrose solutions of tonicities comparable to those of the sodium chloride solutions used, however, stabilized the labile GL-8 L form completely. Magnesium chloride (0.05m) and serum (10% v/v) substantially increased L-form survival in sodium chloride. The results are interpreted to indicate a difference in the cell envelope of the two L-form strains, the AED limiting membrane possessing a greater intrinsic stability. The significantly greater resistance to sonic oscillation of the AED L form as compared to the GL-8 L form, is in agreement with and supports this conclusion. The possibility that the difference in physical properties of the two L-form strains is related to a difference in chemical composition of their limiting envelopes is discussed.The author wishes to thank Dr. W. Hijmans for his interest and advice, and Miss H. L. Ensering and Miss M. J. W. Kastelein for technical assistance.     

Submicroscopic structure of the streptococcal L forms isolated from a living body]

The authors studied in the L-forms of streptococcus induced in the living organism. Submicroscopic structure of the L-forms under study was analogous to the L-forms of the museum streptococcus strain and to the L-forms of some other bacteria. As revealed on the ultra-thin sections of the protoplast-like cells the intracytoplasmic membrane structures were located close to the cytoplasmic membrane and also passed through the whole cell in the form of a band. The latter was in contact with the nucleotide. The elemental bodies were found in the vesicular and the protoplastic cells, and also in the space between the cells; sometimes they formed groups surrounded by a membrane.     

Resistance of the group A streptococcal L form to sonic oscillation

The mechanical fragility of the L form of two group A streptococcal strains was assessed by subjecting L-form suspensions to sonic oscillation (60 W, 20 kc/sec). To evaluate the effect of the sonic energy applied, the original streptococcal strains and aSerratia marcescens strain were subjected to the same treatment. The killing of the organisms by sonic oscillation followed the expression log N_o/N_t=–K·t^1/2. The mortality rate constants K of the streptococcus,S. marcescens and the L form of the AED and GL-8 streptococcal strains were –0.19,–0.98,–1.14 and–1.52 min^–1/2. respectively. The mortality rates of the L form of the two streptococcal strains differed significantly (P=0.01). From a comparison of these data, and taking into account the difference in cell envelope between the bacterial and the L form, it is concluded that the limiting envelopes of the group A streptococcal L-form elements probably possess a relatively marked stability. The factors which might be responsible for the difference in mortality rates between the L form of the streptococcal strains are discussed.The author wishes to thank Dr. W. Hijmans (Institute for Rheumatism Research, Leiden) for his contribution to and constant interest in the work and Mr. J. C. Houwelingen (Department of Mathematical Statistics, Utrecht) for the statistical analysis. This study could not have been accomplished without the able and conscientious technical assistance of Miss H. L. Ensering (Institute for Rheumatism Research, Leiden), which is gratefully acknowledged.     

Epitopes of group A streptococcal M protein shared with antigens of articular cartilage and synovium

Rabbit antisera evoked by purified pepsin-extracted group A streptococcal M proteins were screened for the presence of joint cross-reactive antibodies by indirect immunofluorescence using thin sections of mouse knee joints. Pep M1, M5, and M18 antisera contained antibodies that cross-reacted with chondrocytes, cartilage, and synovium. Immunofluorescence inhibition assays showed that some of the joint cross-reactive epitopes were shared among the three heterologous serotypes of M protein. The pep M5 joint cross-reactive epitopes were localized to three different synthetic peptides of the C-terminal region of pep M5. Immunoblot analyses showed that the M5 joint cross-reactive antibodies recognized two proteins of human synovium and cartilage of molecular mass 56 and 58 kDa. The cross-reactive antibodies binding to the 56-kDa protein were inhibited by purified vimentin in immunoblot inhibition experiments. M protein-specific antibodies from patients with acute rheumatic fever were also shown to cross-react with joint tissue in a pattern similar to the rabbit antisera. Rabbit and human M protein-specific antibodies that were bound to articular cartilage activated significant levels of complement when compared to control serum, suggesting that M protein joint cross-reactive antibodies could potentially be involved in the pathogenesis of ARF and arthritis.     

Serum antibodies to group A streptococcal extracellular and cell-associated antigens in Egyptians with post-streptococcal diseases

We investigated serum antibodies to a comprehensive array of group A streptococcal antigens and superantigens in Egyptian subjects. Antibodies to Streptococcus pyogenes cell-associated proteins and to proteins released by rapidly dividing S. pyogenes were compared in four patient groups with different post-streptococcal diseases and in healthy controls. Enzyme-linked immunosorbent assays showed that total Ig and IgG to extracellular antigens were significantly higher in patients with acute rheumatic fever (ARF) compared to healthy controls, but no differences were found in either total Ig or IgG titres to cell-associated proteins between any of the groups. Western blotting showed that multiple extracellular and cell-associated antigens, covering a wide range of molecular masses, were recognised by all sera, including healthy controls. No evidence was obtained for putative dominant antigens associated with any disease group, although a low molecular mass cell-associated protein (approximately 4 kDa) was clearly recognised by two-thirds of subjects irrespective of disease status. These findings demonstrate that raised serum Ig and IgG titres to extracellular, but not cell-associated, S. pyogenes antigens are a feature of ARF in this population, and suggest that multiple S. pyogenes antigens contribute to this response.     

Use of transformation for studying the nature of formation of stable bacterial L forms]

The authors obtained a stable variant of the L-forms of Bacillus subtilis capable of exponential growth of the minimal and synthetic medium. An electron-microscopic study of different stages of the L-form formation was carried out by the method of ultra-thin sections. A possibility was shown of the transfer of the L-form formation sign by the method of transformation. DNA isolation from the L-forms by soft lysis considerably facilitated and simplified the genetic analysis of the L-form formation by the transformation method.     

Characteristics of group A streptococcal bacteriophages

Friend, Patric L. (Northwestern University Medical School, Chicago, Ill.), and Hutton D. Slade. Characteristics of group A streptococcal bacteriophages. J. Bacteriol. 92:148-154. 1966.-A medium for the growth of group A streptococcal phages is described, consisting of Brain Heart Infusion broth supplemented with 0.2% yeast extract, 10(-4)m CaCl(2), and 10 mug/ml of dl-tryptophan. Cell and phage growth in this medium was excellent, and did not require the addition of serum or other proteins as indicated by other workers. Growth of one phage has also been achieved in a completely synthetic medium. The adsorption characteristics of two group A phages in protein broth and synthetic broth were studied, and the initial adsorption of phage was found to be more extensive in synthetic broth. However, the final amounts of adsorption in both were similar. The addition of purified group A carbohydrate antigen to the adsorption mixture in synthetic broth had no effect on the adsorption, and cells containing type-specific M protein adsorbed phage at the same rate as those lacking M protein. It was concluded that neither the group antigen nor the type antigen was the primary site of phage adsorption. One-step growth curves of the two phages showed a second step or burst occurring. Sonic oscillation of the bacterial cultures, which broke up the chains to single cells, abolished the second step of the growth curve. It appears that the second step is a function of the chain formation of streptococcal cells.     

ABH blood group antigens in O-glycans of human glycophorin A

The major O-linked oligosaccharide structures attached to human glycophorin A (GPA) have been extensively characterized previously. Our own recent findings, obtained by immunochemical methods, suggested the presence of blood group A and B determinants in O-glycans of human glycophorin originating from blood group A or B erythrocytes, respectively. Here, we elucidate the structure of O-glycans, isolated from GPA of blood group A, B, and O individuals by reductive beta-elimination, carrying A, B or H blood group epitopes, respectively. Structural studies based on nanoflow electrospray-ionization tandem mass spectrometry and earlier reported data on the carbohydrate moiety of GPA and ABH antigens allowed us to conclude that these blood group epitopes are elongations of the beta-GlcNAc branch attached to C-6 of the reducing GalNAc. The galactose linked to C-3 of the reducing GalNAc carries NeuAcalpha2-3 linked residue. Identified here O-glycans were found in low amounts, their content estimated at about one percent of all GPA O-glycans. These O-glycans with type-2 core, carrying the blood group A, B or H determinants, have not been identified in GPA so far. Our results demonstrate the efficacy of nanoESI MS/MS in detecting minor oligosaccharide components present in a mixture with much more abundant structures.     

Reactions of antibodies to streptococcus group A antigens with fibroblasts of human heart interstitial connective tissue]

The indirect immunofluorescence method was applied to the study of the serum of rabbits immunized with fractions containing nontype-specific antigens of streptococcus, group A, belonging to the cell wall proteins. Antibodies reacting with fibroblasts of the interstitial connective tissue of the human heart were revealed in the sera. On the basis of the experimental results of absorption a supposition was put forward on the presence of a cross-reacting antigen common with the fibroblast antigen in some hydrochloric extracts obtained from streptococci, groups A and C.     

Ultrastructure of L forms. IV. L forms of nonagglutinating vibrios]

A study was made of the ultrastructure of stable L-forms of Nag vibrios aged 24 hours. Cells of all types of the L-forms had cytoplasmic membranes, and a three-layered structure, which was found not everywhere. Externally of the cytoplasmic membrane, in some areas of the individual cells there were revealed a plastic layer of cell wall and a basal membrane. However, in difference to bacterial forms of the vibryos, rigidity of the cell wall was disturbed, and the links between the cell wall and the cytoplasmic membrane were indetectable. There were regularly revealed lamellar of myelin-like membranous structures in the cytoplasm, which did not occur in bacterial forms, and also lamellar mesosomes. The latter were found in the sites of cell division. Viability of small bodies as the minimal reproductive forms of the L-cultures is confirmed by the presence in them of a nucleoid and of the binary division.