ABSTRACT
An extracellular product of group A streptococci which induces lymphocyte blastogenesis has been purified to homogeneity by DEAE-cellulose and CM-cellulose chromatography. The protein, termed streptococcal blastogen A, has a mol wt of approximately or equal to 17,500 and is inactivated by protease treatment and by heating at 100 degrees C. The purified blastogen gave rise to multiple protein bands on nondenaturing polyacrylamide gel electrophoresis, only two of which possessed blastogenic activity. Treatment of the protein with dithiothreitol before electrophoresis resulted in the apparent conversion of the multiple forms to a single active species. Blastogen A differs in electrophoretic mobility from the streptococcal pyrogenic exotoxins and its lymphocyte stimulating activity is not inhibited by rabbit antisera to the exotoxins. An enzyme immunoassay has been developed to measure human antibodies against blastogen A. A selection of sera with varying levels of anti-DNase B contained antiblastogen A-IgG.
Subject(s)
Bacterial Proteins/analysis , Lymphocyte Activation , Mitogens/analysis , Streptococcus pyogenes , Antibodies, Bacterial/analysis , Bacterial Proteins/isolation & purification , Electrophoresis, Polyacrylamide Gel , Exotoxins/analysis , Hot Temperature , Humans , Mitogens/isolation & purification , Molecular WeightABSTRACT
Nearly all group B streptococcal strains representing the five major serotypes were found to produce extracellular nucleases by screening with an agar-well-diffusion technique in DNA-methyl green agar plates. Three different nucleases have been isolated and partially purified by DEAE-and carboxymethyl-cellulose chromatography. They possessed different mobilities on polyacrylamide gel electrophoresis and different molecular weights. These nucleases, designated I, II, and III, are optimally activated by cations of calcium and manganese and exhibited RNase as well as DNase activity. Despite differences in their physical and biochemical properties, nucleases II and III appear antigenically similar, but distinct from nuclease I. These group B streptococcal nucleases are immunologically different from the nucleases of group A streptococci. Neutralizing activity, probably antibody, to nucleases II and III was found in human sera, and was most prevalent in sera of pregnant women colonized with group B streptococci and in their newborn infants.
Subject(s)
Deoxyribonucleases/metabolism , Extracellular Space/enzymology , Streptococcus agalactiae/enzymology , Bacterial Proteins/analysis , Calcium , Female , Humans , Hydrogen-Ion Concentration , Infant, Newborn , Manganese , Molecular Weight , Pregnancy , Ribonucleases/metabolism , Streptococcus agalactiae/immunology , Streptococcus pyogenes/immunologyABSTRACT
The antigenic relationships of hyaluronidases, bound and free, associated with temperate bacteriophages of group A streptococci were examined with antibody against purified whole phage and with antibody against phage-bound enzyme released by urea and purified to homogeneity. Studies performed by double diffusion in agar (ouchterlony) with antibody against the homologous purified enzyme from a temperate phage of a type 49 streptococcus indicated that the bound and free enzyme gave a single line of identity and that the free hyaluronidase activities in induced lysates of four strains of M type 49 streptococci were immunologically indistinguishable but different from the enzyme in induced lysates of a heterologous type. The four M type 49 strains were from widely different geographical or temporal sources and of different phage subtypes as determined by lyxic patterns. These findings were confirmed in studies that employed a functional assay of enzyme neutralization. An immunoglobulin preparation of antiserum against the purified enzyme as well as one against homologous purified whole phage neutralized the hyaluronidase activity produced by induction of the M type 49 strains and present either phage-bound or soluble in phage-free lysates. These immunoglobulin preparations had little effect on the hyaluronidase activities present in phage-lysates of other M types of group A streptococci. Inhibition of propagation of temperate phages by antibody against the purified phage hyaluronidase paralleled the neutralization of phage-associated enzyme activity by this antibody, indicating that antibody to the purified enzyme can inhibit phage infection. Antibody preparations against the purified phage-bound enzyme or against purified whole phage did not neutralize the extracellular hyaluronidase in the supernate of an uninduced culture of M type 4 streptococci. A human serum strongly inhibitory for the extracellular enzyme of this strain or on the purified phage enzyme from an M type 49 strain. The results support the view that the hyaluronidases associated with the temperate bacteriophages from various M types of group A streptococci do not share common antigenic determinants but that an immunological specificity exists that parallels the serologic specificity of the M protein of the host strains.
Subject(s)
Bacteriophages/enzymology , Hyaluronoglucosaminidase/immunology , Antibody Specificity , Antigen-Antibody Reactions , Antigens, Viral/analysis , Bacteriophages/growth & development , Solubility , Streptococcus pyogenes/enzymologyABSTRACT
Lymphocytes, from randomly selected individuals having normal immune function, when incubated in vitro with varying concentrations of streptococcal antigens, responded in three ways: (a) response over the entire antigen concentration range, i.e., responders; (b) low response to only the highest antigen concentrations; and (c) no response at any antigen concentration. Frequency distribution analysis of these groups indicated that a significant association occurred between the ability to respond and HL-A 5.
Subject(s)
Antigens, Bacterial , HLA Antigens , Histocompatibility Antigens , Lymphocytes/immunology , Streptodornase and Streptokinase/immunology , Adult , Aged , Humans , Immunogenetics , Lymphocyte Activation , Lymphocytes/metabolism , Middle Aged , Streptococcus/immunology , Thymidine/metabolism , TritiumABSTRACT
A bactericidal substance previously isolated from phage type 71 Slaphylococcus aureus has been further identified and characterized. Staphylococci belonging to phage type 71 produce the substance in higher titers than staphylococci lysed by other phages in group II in addition to phage 71. Other staphylococci do not produce the bactericidal substance. The bactericidal substance shares several of the properties of bacteriocins but differs from this group of antibiotic substances in some respects. A combination of ammonium sulfate fractionation and gel filtration on a Sephadex G-100 column resulted in considerable degree of purification of the bactericidal substance. The substance is a previously unrecognized product of S. aureus and is distinct from other extracellular products of this organism.
Subject(s)
Bacteria/drug effects , Bacteriocins/isolation & purification , Staphylococcus/analysis , Bacteriocins/pharmacology , Bacteriophage Typing , Chemical Precipitation , Chromatography, Gel , Mitomycins/pharmacology , Ultraviolet RaysABSTRACT
A bacteriocin, streptocin A, was isolated from the supernatant fluid of tryptic soy broth cultures of Group A streptococcus strain FF-22. Evidence was obtained which supports the view that the failure to recover active streptocin A after growth of the producer strain in certain fluid media is due to the inactivation of the bacteriocin by concomitantly synthesized streptococcal proteinase. The bacteriocin was purified 139-fold and the active product appeared to be of uniform size, having a molecular weight of approximately 8,000. Streptocin A was bactericidal, but not lytic, for a susceptible Group A streptococcus and the lethal effect was markedly temperature dependent. The bacteriocin inhibited the synthesis of DNA, RNA, and protein, and also prevented the uptake and incorporation of glucose by the sensitive cells. Degradation of RNA occurred, but appeared to be less than that produced by a staphylococcal bacteriocin. This effect may be due to differences in the killing potency of the two bacteriocins in preparations having similar inhibitory activity when measured by lawn culture assays.
Subject(s)
Bacteriocins/biosynthesis , Streptococcus/metabolism , Alanine/metabolism , Bacterial Proteins/biosynthesis , Bacteriocins/antagonists & inhibitors , Bacteriocins/isolation & purification , Bacteriocins/pharmacology , Carbon Radioisotopes , Culture Media , DNA, Bacterial/biosynthesis , Glucose/metabolism , Peptide Hydrolases/metabolism , RNA, Bacterial/biosynthesis , Streptococcus/drug effects , Streptococcus/enzymology , Streptococcus/growth & development , Thymidine/metabolism , Tritium , Uridine/metabolismABSTRACT
The lymphocyte transformation responses to purified preparations of two extracellular products of group A streptococci (blastogen A and nuclease B), to phytohemagglutinin, and to Candida albicans antigen were measured in tonsillar and peripheral blood lymphocytes from patients with rheumatic heart disease (RHD) and suitably matched nonrheumatic (control) subjects. The mean phytohemagglutinin dose responses of tonsillar and peripheral lymphocytes from RHD patients were essentially indistinguishable from those of controls. In contrast, the responses of tonsillar and peripheral blood lymphocytes to the two extracellular products of group A streptococci were significantly lower in RHD patients than in nonrheumatic control subjects. Candida antigen produced very little stimulation of lymphocytes in any of the subjects. The geometric means of antibody levels against streptolysin O, nuclease B, and nicotinamide adenine dinucleotidase showed no consistent differences between the control group and the group of RHD subjects. Group A streptococci were isolated from the tonsils of approximately 25% of both groups of subjects. The RHD patients clearly had a depressed cellular immune response to the two purified streptococcal extracellular antigens. The equal frequency in recovery of group A streptococci from tonsils and the absence of consistent difference in titers of humoral antibodies to streptococcal extracellular antigens, particularly nuclease B, suggest that this differential response is not due to a lower level of stimulation by repeated exposure to group A streptococcal products.
Subject(s)
Immunity, Cellular , Rheumatic Heart Disease/immunology , Streptococcus pyogenes/immunology , Antigens, Bacterial , Extracellular Space/physiology , Humans , Lymphocyte Activation , Lymphocytes/immunology , Palatine Tonsil/immunology , Streptococcus pyogenes/enzymologyABSTRACT
The ability of monoclonal antibodies (MAb) against a human B lymphocyte alloantigen has been suggested to discriminate between rheumatic fever "susceptible" individuals and persons with a lower risk of developing RF. However, while such MAb have been reported to identify a majority of RF/RHD patients in some populations, a reduced discriminatory ability has been observed in others. Antigenic variation in the RF marker(s) may exist among ethnic groups which reduce the discriminatory ability of these monoclonal antibodies. We developed MAb using B lymphocytes from RF patients of North Indian ethnic origin. In this same population we compared the new MAb (PGI/MN II) with a previously described MAb of Caucasian ethnic origin (D8/17). In three groups: acute rheumatic fever patients (no evidence of previous attacks of rheumatic fever), patients with chronic rheumatic heart disease and normal controls from the same population, we found a greater discriminating ability of PGI/MNII MAb to identify Indian RF/RHD patients than with the D8/17 MAb. Further, sixty percent of 142 siblings of the RF/RHD patients were "positive" when tested with PGI/MN II. The data from these studies suggest that before such MAb can be used for identification of RF "susceptibles" in public health programs, variation among ethnic populations must be assessed.
Subject(s)
Antibodies, Monoclonal/analysis , Rheumatic Fever/genetics , Adolescent , Adult , Antibodies, Monoclonal/genetics , Child , Child, Preschool , Clone Cells , Disease Susceptibility , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Genetic Markers , Genetic Testing , Humans , India/epidemiology , Male , Rheumatic Fever/ethnology , Rheumatic Fever/immunology , Rheumatic Heart Disease/ethnology , Rheumatic Heart Disease/genetics , Rheumatic Heart Disease/immunology , Sensitivity and SpecificitySubject(s)
Bacterial Proteins/metabolism , Chlorophyll/biosynthesis , DNA, Bacterial/metabolism , RNA, Bacterial/metabolism , Rhodopseudomonas/metabolism , Acridines/pharmacology , Carbon Isotopes , Cell Membrane/metabolism , Macromolecular Substances , Rhodopseudomonas/growth & development , Thymidine/metabolism , Uracil/metabolism , Valine/metabolismSubject(s)
Bacterial Proteins/biosynthesis , RNA, Bacterial/biosynthesis , RNA, Ribosomal/biosynthesis , Rhodobacter sphaeroides/metabolism , Chloramphenicol/pharmacology , Electrophoresis, Polyacrylamide Gel , Kinetics , Methionine/metabolism , Methionine/pharmacology , Mutation , Rhodobacter sphaeroides/drug effects , Time Factors , Tritium , Uridine/metabolismSubject(s)
RNA, Transfer/metabolism , Rhodopseudomonas/growth & development , Aerobiosis , Amino Acids/isolation & purification , Amino Acids/metabolism , Amino Acyl-tRNA Synthetases , Anaerobiosis , Benzoates , Carbon Isotopes , Chromatography, DEAE-Cellulose , Phenylalanine/isolation & purification , Phenylalanine/metabolism , Photosynthesis , RNA, Transfer/isolation & purification , Rhodobacter sphaeroides/enzymology , Rhodobacter sphaeroides/growth & development , Rhodobacter sphaeroides/metabolism , Tritium , Tryptophan/isolation & purification , Tryptophan/metabolismABSTRACT
Group A streptococcal pyrogenic exotoxins A, B, and C (also known as scarlet fever toxins and erythrogenic toxins) were evaluated for relatedness to another streptococcus-derived lymphocyte mitogen, blastogen A. Streptococcal pyrogenic exotoxin A and blastogen A were immunologically cross-reactive and shared the same molecular weight, N-terminal amino acid sequence, and capacity to stimulate rabbit splenocyte proliferation nonspecifically.
Subject(s)
Bacterial Proteins/analysis , Exotoxins/analysis , Membrane Proteins , Mitogens/analysis , Pyrogens/analysis , Streptococcus pyogenes/immunology , Electrophoresis, Polyacrylamide Gel , Immunoblotting , Immunodiffusion , Streptococcal Infections/immunologyABSTRACT
A group of at least four distinct nucleases designated DcI through DcIV were isolated from cellular extracts of group A streptococcal strain S43 and shown to be antigenically similar to streptococcal extracellular deoxyribonuclease (DNase) D. These cellular endonucleases degraded single- and double-stranded deoxyribonucleic acid (DNA) as well as ribonucleic acid (RNA) to acid-soluble oligonucleotides. The products of digestion of DNA bore 5'-terminal phosphates, and in partial digests pdX-pdG linkages were most susceptible and pdA-pdX linkages were most resistant to nuclease action. The enzymes had pH optima of 8.0 to 8.5, were inhibited by NaCl, were unaffected by sulfhydryl modifying reagents, and absolutely required a divalent cation. Nucleases DcIII and DcIV were apparently hydrophobic in nature since they required the presence of detergents for migration on nondenaturing polyacrylamide gels. All four nucleases were electrophoretically distinct on such gels, from each other, and from DNase D. Molecular weights of DcI and DcII were similar to that of DNase D, suggesting that the mobility differences of these enzymes at least are reflections of differing net charges. It is suggested that the cellular nucleases represent a group of processing intermediates in the maturation and excretion of DNase D.
Subject(s)
Deoxyribonucleases/isolation & purification , Endonucleases/isolation & purification , Streptococcus pyogenes/enzymology , Deoxyribonucleases/immunology , Deoxyribonucleases/metabolism , Endonucleases/immunology , Endonucleases/metabolism , Epitopes , Exonucleases/immunology , Molecular Weight , Streptococcus pyogenes/immunology , Subcellular FractionsABSTRACT
The synthesis of various cell components was examined during the anaerobic photosynthetic growth of synchronous populations of Rhodopseudomonas spheroides. Net deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein increased continuously as did the rate of incorporation of radioactive precursors into protein. The rates of incorporation of radioactive precursors into RNA and DNA were marked by abrupt discontinuities. It is not clear whether these discontinuities represent changes in rates of synthesis or fluctuations in precursor pools. Although the synthesis of bacteriochlorophyll occurred in a continuous manner, those enzymes examined which are involved in the synthesis of tetrapyrroles, i.e., succinyl CoA thiokinase, delta-aminolevulinic acid synthetase, and delta-aminolevulinic acid dehydrase, increased discontinuously. Two other enzymes not involved in tetrapyrrole biosynthesis were examined. Alkaline phosphatase increased in a stepwise manner during the division cycle, whereas the synthesis of ornithine transcarbamylase increased rapidly before leveling off for a period of time until synthesis began again. In each instance of discontinuous enzyme synthesis, increases occurred at regular and characteristic times during the division cycle. Ammonium sulfate precipitation was employed to remove low molecular weight end product inhibitors from enzyme preparations. These studies suggested that the stepwise increases in enzyme activity observed in the present investigation were not affected by periodic end product inhibition. A temporal map of enzyme synthesis during the division cycle was constructed. Both delta-aminolevulinic acid synthetase and delta-aminolevulinic acid dehydrase appeared early in the division cycle, whereas alkaline phosphatase and succinyl CoA thiokinase appeared later on.
Subject(s)
Bacterial Proteins/biosynthesis , Cell Division , DNA, Bacterial/biosynthesis , RNA, Bacterial/biosynthesis , Rhodopseudomonas/metabolism , Alkaline Phosphatase/metabolism , Carbon Isotopes , Chlorophyll/analysis , Hydro-Lyases/metabolism , Ligases/metabolism , Ornithine Carbamoyltransferase/metabolism , Pyrroles/biosynthesis , Rhodopseudomonas/enzymology , Thymidine/metabolism , Tritium , Uracil/metabolismABSTRACT
A method has been developed for the preparation of streptococcal nuclease B by batch adsorpton to diethylaminoethyl-cellulose. The enzyme is homogeneous with respect to nuclease activity and is suitable for use as an antigen in measurement of anti-deoxyribonuclease B levels in sera.
Subject(s)
Deoxyribonucleases/isolation & purification , Streptococcus pyogenes/enzymology , Adsorption , Antibodies, Bacterial/analysis , Antigens, Bacterial , DEAE-Cellulose , Deoxyribonucleases/immunology , Diagnosis, Differential , Humans , Streptococcal Infections/diagnosis , Streptococcus pyogenes/immunologyABSTRACT
A simple method for preparation and fractionation of the streptococcal nucleases by polyacrylamide gel electrophoresis is presented. The procedure is carried out with ammonium sulfate-precipitated supernatant fluids from cultures of beta-hemolytic streptococci grown to stationary phase. Electrophoresis on polyacrylamide gel and subsequent elution of the fractionated enzymes allows the preparation of sufficient homogeneous nuclease B for a large number of anti-nuclease B titrations.
Subject(s)
Deoxyribonucleases/isolation & purification , Electrophoresis, Disc , Streptococcus/enzymology , Ammonium Sulfate , Chemical Precipitation , DNA/metabolism , Deoxyribonucleases/metabolism , Indicators and Reagents , Methods , Micropore Filters , Streptococcus pyogenes/enzymology , Streptococcus pyogenes/growth & developmentABSTRACT
The phenylalanine tRNA of Rhodopseudomonas sphaeroides was fractionated on benzoylated diethylaminoethyl-cellulose into four isoaccepting species (tRNAPheI to IV). tRNAPheIII represented 80% of the total tRNAPhe in anaerobic, photosynthetically grown organisms, whereas in cultures grown aerobically for prolonged periods, tRNAPheII represented 80% of the total. In cultures adapting to aerobic growth, the addition of rifampin resulted in a tRNAPhe profile characteristic of anaerobic-photosynthetic conditions due to the conversion of tRNAPheII to tRNAPheIII. In fully adapted aerobic cultures, this conversion was inhibited in the presence of chloramphenicol or rifampin. The conversion of tRNAPheIII to tRNAPheII was not observable in vivo. It is proposed that an enzymic activity synthesized during anaerobic-photosynthetic growth was responsible for the conversion.