Dynamics of the immune response against extracellular products of group A streptococci during infection

The immune response against the infecting group A streptococcus (GAS) extracellular products (EP) was determined in acute- and convalescent-phase sera from 75 patients with different clinical manifestations of GAS infection. All EP elicited a high proliferative response in human peripheral blood mononuclear cells. In patients with bacteremia, low neutralization in acute-phase sera was associated with development of streptococcal toxic shock syndrome. Lack of neutralization in acute-phase sera was more common in patients infected with the T1emm1 serotype. The majority of patients did not develop the ability to neutralize the mitogenic activity of their infecting isolate despite a significant increase in enzyme-linked immunosorbent assay titer in early convalescent-phase sera. In patients with the ability to neutralize GAS EP, the immune response remained high over at least 3 years. In contrast, the neutralization capacity conferred by intravenous immunoglobulin and/or plasma treatment disappeared within 3 months.     

Cytokine induction by extracellular products of oral viridans group streptococci.

During an etiological study of Kawasaki disease (mucocutaneous lymph node syndrome [MCLS]), we found that dominant viridans streptococcal strains on tooth surfaces and in the throat of both MCLS patients and non-MCLS control children formed erythrogenic and biologically active, extracellular products. In this study, we demonstrated that erythrogenic culture supernatant concentrates of representative strains (two Streptococcus mitis and two Streptococcus oralis), when injected intravenously, induced serum tumor necrosis factor alpha, interleukin-6 (IL-6), and gamma interferon in muramyldipeptide- or Propionibacterium acnes-primed C3H/HeN mice. The concentrates also induced tumor necrosis factor alpha, IL-6, and thymocyte-activating factor (essentially IL-1) in murine peritoneal macrophage, human monocyte, and human whole-blood cultures. An erythrogenic, heat-labile extracellular protein fraction (F-1) that was concentrated from the culture supernatants of a representative S. mitis strain exhibited the above-mentioned cytokine-inducing activity. This partially purified F-1 fraction also induced thymocyte-activating factor and IL-6 in human umbilical vascular endothelial cell and gingival fibroblast cultures.     

Modulation of human lymphocyte functions by group A streptococci

Intact (heat-inactivated) bacteria and isolated cellular components of pathogenic group A (M type 5 or 12). B. C. and G streptococci were used to evaluate the in vitro reactivity of mononuclear cells (MNC) from peripheral blood of healthy donors and from human tonsils. High doses of A-streptococcal cells, cell walls, and cell membranes stimulated DNA synthesis, production of leukocyte migration inhibitory factor (LIF), and immunoglobulin (Ig) secretion in MNC from all donors. A streptrococci stimulated higher proliferation rates and larger numbers of plaque-forming cells (PFC) in tonsil cell cultures than in blood MNC cultures. Polyclonal activation of both tonsil and blood B lymphocytes by A-streptococcal cell components was T cell and monocyte dependent, thus showing a similarity between these structures and pokeweed mitogen (PWM), which is a polyclonal T-cell activator (PTA). Cocultivation studies demonstrated that, in the presence of A streptococci, precultured MNC and T cells can suppress the blastogenic and PFC responses of autologous fresh MNC stimulated by phytomitogen or antigen, which is very similar to the concanavalin A (Con A)-induced activation of suppressor cells. In contrast, similar group B-, C-, and G-streptococcal cell envelope biostructures failed to activate blood or tonsil lymphocytes to proliferate, differentiate, or produce LIF.     

Properties of extracellular neuraminidase produced by group A streptococcus.

Extracellular neuraminidase production by group A streptococci was examined in 92 strains. Fourteen of these strains produced appreciable amounts of enzyme; 12 of the neuraminidase-producing strains belonged to T types 1, 4, and 12. Production of the enzyme paralleled bacterial growth in culture and was maximal in medium containing 0.2% glucose. The enzyme produced by one of these strains was partially purified by ammonium sulfate fractionation and filtration on G-200 Sephadex. Its molecular weight was estimated at 90,000. Activity was optimal at pH 5.7 and in the presence of 0.01 to 0.03 M calcium and magnesium cations. The enzyme was stable at temperatures of 4 and 37 degrees C for at least 24 h but was inactivated within 10 min at temperatures of 50 and 65 degrees C. The enzyme hydrolyzed 40% of the sialic acid in bovine submaxillary mucin, but was inactive on sialyl-lactose, porcine submaxillary mucin, oligosaccharides derived from porcine mucin, or human orosomucoid. The Km value for this enzyme with bovine submaxillary mucin as substrate was in the order of 3.6 x 10(-4) M.     

In vitro lymphocyte proliferation in response to type III group B streptococci.

The in vitro cell-mediated responses to group B streptococci (GBS) and the relationship of cell-mediated immunity to specific humoral immunity to type III GBS were investigated. Blood was obtained from 20 adult volunteers, and lymphocytes were isolated and cultured in microtiter plates. Each well contained 2 x 10(5) lymphocytes, 15% autologous serum, and either GBS (cell-to-organism ratio of 1:10, 1:1, or 1:0.1), phytohemagglutinin, streptokinase-streptodornase, or RPMI 1640. Cells were harvested at 5, 6, or 7 days, and DNA synthesis was quantitated. Serum antibody titers were determined with an enzyme-linked immunosorbent assay. Maximal lymphocyte responses occurred at 6 days of culture and at a cell-to-organism ratio of 1:1. Individuals with significant antibody titers to type III GBS, as well as those with undetectable antibody, responded to GBS (stimulation index greater than 10). There was a significant difference (P less than 0.001) between mean antibody concentrations in responders (stimulation index greater than 10) and nonresponders (stimulation index less than 10). Thus, the in vitro responses to GBS may be both to a specific antigen and to a nonspecific mitogen and may be important in host immunity to GBS.     

Cell-associated collagenolytic activity by group B streptococci.

Group B streptococci (GBS) are important pathogens in neonatal sepsis, pneumonia, and meningitis. The ability of GBS to invade the collagen-rich amniotic membrane of the placenta has been shown in vitro. In the presence of GBS, the collagen fibrils of the amnion appear disordered, suggesting a role for GBS in premature rupture of membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Sephadex G-200 column chromatography, and gelatin zymograms were used in this study to characterize cell-associated collagenolytic activities of GBS. The synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which mimics the primary structure of collagen, was degraded by GBS USF704, a clinical isolate from the placenta of a septic newborn. Cells of GBS USF704 (9 x 10(7) CFU/ml) hydrolyzed 902 nmol of FALGPA over a 24-h period. As reported for zinc metalloenzymes such as collagenase, the hydrolysis of FALGPA by GBS was inhibited by addition of EDTA or 1,10-phenanthroline. Boiling of the cells resulted in loss of activity, while higher activity was observed with crude GBS cell lysates (hydrolysis of 970 nmol of FALGPA in 1.5 h). Antiserum raised against collagenase from Clostridium histolyticum was found to cross-react with cell-associated proteins produced by GBS and to inhibit GBS FALGPA hydrolysis. Twenty-five additional GBS clinical isolates were screened and found to have various levels of FALGPA hydrolytic activity. These observations suggest a cell-associated collagenolytic activity by GBS which may be involved in premature rupture of membranes and neonatal disease.     

Hyaluronidase activity of bacteriophages of group A streptococci.

A sensitive dye-binding assay was employed to study the hyaluronidase associated with temperate and virulent phages infected group A streptococci. Some enzyme was detectable in each purified phage preparation examined, but differences of several orders of magnitude separated the lower enzyme levels in virulent phages that required the addition of hyaluronidase for plaque formation and the higher levels in temperate phages that did not. Infection by virulent phage A25 was accompanied by the production of levels of hyaluronidase proportionate to the average burst size. Hyaluronidase was produced during infection by temperate phages at a much higher level than could be accounted for by the number of phage particles formed. The major portion of this hyaluronidase was free and apparently unassociated with phage or phage fragments. The phage-associated enzyme was tightly bound but could be released and solubilized by treatment with urea.     

Phage influence on the synthesis of extracellular toxins in group A streptococci.

Phage conversion of group A streptococci to produce streptococcal exotoxins was shown to occur more widely than has been previously reported. Toxigenic conversion was found in 19 newly constructed lysogenic and pseudolysogenic strains resulting in synthesis of exotoxin types A and B. Conversion was accomplished by a positive conversion effector, which was a phage characteristic expressed by the prophage and vegetatively reproducing phage. Exotoxin production was determined by the rabbit skin test and by countercurrent immunoelectrophoresis with type-specific antisera. New lysogens and pseudolysogens were constructed with strains which failed to produce at least one exotoxin type. Phages were obtained from toxigenic strains isolated from cases of scarlet fever. Conversions were consistent and repeatable; loss of the recently introduced phage was accompanied by loss of the newly acquired toxin productivity. Conversion resulted in production of additional exotoxin type or types and never affected existing toxin synthesis. Converting phages were characterized by electron microscopy and negatively stained preparations and were all found to be of morphological class B1. All phage nucleic acid was double-stranded DNA. Though similar in structure, each converting phage had a different host range, and the nine new converting phages identified here did not react with antiserum prepared against the originally reported converting phage.     

Characterization of neuraminidases produced by various serotypes of group B streptococci.

Neuraminidase produced by 11 strains of group B streptococci (GBS), from serotypes Ia, Ib, Ic, II, and III, were characterized according to molecular weight, antigenic identity, and substrate specificity. Following growth in a chemically defined medium, ammonium sulfate-concentrated culture supernatants were assayed for activity with bovine submaxillary mucin as substrate. Neuraminidase produced by GBS strain 122 (serotype III) was purified by a combination of salt fractionation, affinity chromatography with Affi-Gel Blue, ion-exchange chromatography with DEAE-cellulose, and gel filtration on Sephadex G-200. Purified neuraminidase was used to immunize rabbits, and the resultant antiserum reduced the activity of purified neuraminidase from strain 122 by 87.7%. The antiserum also reduced the activity of neuraminidases produced by the other four serotypes by between 78.3 and 90%. Molecular weight estimates of the neuraminidases produced by the various serotypes were obtained by gel filtration chromatography on Sephadex G-200. The molecular weights obtained for the neuraminidases from the representative strains of each serotype ranged from 110,000 to 180,000. In addition, all of the GBS neuraminidases examined (regardless of the producing serotype) were active only on bovine submaxillary mucin. On the basis of these results, it appears that the neuraminidases produced by different GBS serotypes are quite similar.     

Streptococcal cysteine protease augments lung injury induced by products of group A streptococci.

Streptococcus pyogenes infections in humans may be associated with severe clinical manifestations, including adult respiratory distress syndrome and a toxic shock-like syndrome. These observations have led to the investigation of products of group A streptococci that may contribute to increased virulence. Streptococcal pyrogenic exotoxin B is a highly conserved precursor of an extracellular cysteine protease that is secreted by S. pyogenes. We investigated the ability of this streptococcal cysteine protease (SCP) to act synergistically with either streptococcal cell wall antigen (SCW) or streptolysin-O (SLO) to augment lung injury in rats. Intratracheal administration of either SCW or SLO alone caused lung injury, as measured by pulmonary vascular leak. Bronchoalveolar lavage (BAL) fluid analysis showed that SCW induced neutrophil accumulation and appearance of interleukin-1beta and tumor necrosis factor alpha. In contrast, SLO induced neither neutrophil influx nor significant cytokine elevations in BAL fluids. Intratracheal administration of SCP with either SCW or SLO resulted in synergistic augmentation of lung vascular permeability and accumulation of BAL neutrophils. The synergy was reduced when SCP was either heat inactivated or coinstilled with a peptide inhibitor of the protease. SCP in the presence of SCW resulted in a significant increase in BAL fluid tumor necrosis factor alpha content but not in immunoreactive interleukin-1beta. Moreover, the copresence of SAP with SAW resulted in increased BAL fluid nitrite-nitrate levels, indicative of nitric oxide production. These data demonstrate that SCP acts synergistically with other S. pyogenes products (SCW or SLO) to increase tissue injury and provide additional evidence that SCP may function as an important virulence factor in group A streptococcal infections.     

Virulent human strains of group G streptococci express a C5a peptidase enzyme similar to that produced by group A streptococci.

Specific proteolytic destruction of the human chemotaxin, C5a, is a property of group A and B streptococcal pathogens. Here we show that virulent group G streptococci from human sources also express C5a peptidase activity. The enzyme responsible for this activity is approximately the same size as and is antigenically similar to that produced by group A streptococci. On the basis of Southern hybridization analysis with an internal fragment of the group A C5a peptidase gene (scpA) as a probe, a copy of this gene was found in the genome of all group G human isolates tested. Comparison of partial restriction maps of scpA and scpG revealed significant similarity between the two genes. Group G strains isolated from dogs and cows were found to lack C5a peptidase activity and did not hybridize to the scpA-specific probe. The association of this activity with three streptococcal species suggests that elimination of phagocyte chemotactic attractants is a more universal virulence mechanism than originally anticipated.     

Typing of group A streptococci by random amplified polymorphic DNA analysis.

Random amplified polymorphic DNA (RAPD) analysis was evaluated in comparison with restriction endonuclease analysis (REA) of genomic DNA and serotyping in the typing of 160 epidemiologically unrelated group A streptococci (GAS). Amplification of genomic DNA of GAS was performed with a single primer with an arbitrarily selected nucleotide sequence of 12 nucleotides. In total, 31 RAPD patterns and 15 REA patterns were observed among the isolates studied. The results of RAPD analysis were in accordance with the results of REA for 86% of the isolates, as both methods identified 15 different strains among 138 isolates. However, RAPD analysis differentiated 16 additional strains among 22 isolates. RAPD analysis was somewhat better than REA for differentiation of isolates of the same and different serotypes. However, not all of the serotypes were differentiated by RAPD analysis either. In conclusion, RAPD analysis provides a practical alternative for genomic typing of GAS. It can be recommended for the typing of GAS, especially if used in parallel with serotyping.     

Quantitative assay of glycocalyx produced by viridans group streptococci that cause endocarditis.

A quantitative method to determine glycocalyx production by strains of viridans group streptococci from patients with endocarditis is presented. There is good correlation between this new tryptophan quantitative assay and qualitative assays employing polysaccharide stains (ruthenium red, periodic acid-Schiff, and Cellufluor) or the Molisch test. The quantification of the glycocalyx production in glucose substrate in vitro by viridans group streptococci correlates with the size of cardiac vegetation and ease of antimicrobial sterilization in experimental endocarditis. The relationship of in vitro quantification of glycocalyx to maintenance of infection, morbidity of infection, and antimicrobial treatment is discussed.     

Intracellular and extracellular cytokine production by human mixed mononuclear cells in response to group B streptococci

Group B streptococci (GBS) are a major cause of severe infection in newborns, pregnant females, and other immunocompromised hosts. Infection often includes septicemia, shock, pneumonia, and respiratory failure. In previous studies, we have reported that GBS induce marked production of tumor necrosis factor alpha (TNF-alpha) by human mononuclear cells. The present study was designed to measure the production of TNF-alpha as well as additional cytokines, including interleukin 1beta (IL-1beta), IL-6, IL-8, IL-12, and gamma interferon (IFN-gamma) but also to determine from what cells and at what time point during incubation with GBS that these cytokines are produced. Mixed mononuclear cells were incubated with heat-killed GBS, media alone, or 1 microg of Escherichia coli lipopolysaccharide (LPS). Brefeldin A was added to each sample prior to staining, which prevented the export of cytokines by the Golgi apparatus. The cells were then stained with the appropriate conjugated antibodies and analyzed by using a flow cytometer. Results indicate that intracellular cytokines appear, in almost all cases, simultaneous to or before secreted proteins are detected. In contrast to the response to LPS, where TNF-alpha, IL-1beta, IL-6, and IL-8 appear almost simultaneously, the human monocyte response to GBS results in the production of TNF-alpha but delayed appearance of IL-1beta, IL-6, and IL-8. The lymphocyte response to GBS was also strikingly different from that to LPS in that both secreted IFN-gamma and IL-12 was detected, while LPS failed to induce production of these critical cytokines. This suggests an important role for TNF-alpha, IFN-gamma, and IL-12 in GBS pathogenesis and/or immunity.     

Molecular species of R-protein antigens produced by clinical isolates of group B streptococci.

Clinical isolates of group B streptococci from body fluids and mucosal surfaces were examined for production of a trypsin-resistant antigen known as R protein. R protein was extracted with 1% trypsin from cells grown in a semidefined medium. The extracts were tested by immunodiffusion in agarose with a panel of antisera for detection and precise identification of the four species of R protein described by Wilkinson. R antigen was present in 49 of 131 (37%) of the strains tested. Analysis by serotype revealed that 0 of 2 type Ia, 0 of 11 Ib, 1 of 16 (6%) Ia/c, 12 of 15 (80%) II, 0 of 20 II/c, 35 of 49 (71%) III, 0 of 6 IV, and 1 of 12 (8%) nontypeable strains produced R antigen. Production of the R protein and the trypsin-resistant or alpha component of the c protein appeared to be mutually exclusive. R antigen was more prevalent in isolates from blood (50%) than in those from mucosal sites (27%) for type II strains; no difference was seen for type III strains from these sites. Concordant results were obtained with five paired body fluid-mucosal surface isolates from individual patients and with isolates from 17 mother-baby pairs. The most frequent species of R antigen was R4 (45 of 49), followed by R1 (4 of 49). These two species of R protein were biochemically (trypsin resistant and pepsin sensitive) and immunologically identical to the R-protein antigens produced by prototype strains of groups A, B, and C streptococci.     

Relationship of cellular potential hemolysin in group A streptococci to extracellular streptolysin S.

The relationship of streptolysin S (SLS) and a cellular potential hemolysin (PH) activatable by sonication was examined in strain C203S (a known high producer of SLS), its SLS(-) mutant (C203U), and in 20 other group A streptococci of various M and T serotypes. All strains shown to form SLS (ribobycleic acid (RNA)-core SLS) contained PH. The two strains lacking PH were the only ones that did not produce SLS In strain C203S, formation of SLS bycells incubated with RNA-core for 60 min at 47 C in a nongrowth basal medium (Bernheimer's basal medium) was followed by a marked decrease (99.6% loss) of PH titer. Without stimulation of SLS production by addition of RNA-sore, the same incubation resulted in a progressive but less marked fall (38.8%loss in 60 min) of PH titer: these cells produced disproportionately low titers of SLS on subsequent addition of RNA-core. This effect of prior incubation in Bernheimer's basal medium on SLS titer was partially nullified by use of fresh medium after 30 min, but not after 60 min, and did not occur during 60 min of incubation at OC. These results provide additional evidence for a precursor-product relationship between PH and SLS. They also suggest that a medium factor (or factors) is utilized or destroyed at 37 C and that this factor is essential to both the stability of PH and its efficient conversion to SLS.     

Sequencing emm-specific PCR products for routine and accurate typing of group A streptococci.

Rapid sequence analysis of specific PCR products was used to accurately deduce emm types corresponding to the majority of the known group A streptococcal (GAS) M serotypes. The study involved 95 M type reference GAS strains and a survey of 74 recent clinical isolates. A high percentage of agreement between M type serology and the previously published 5' sequences of the emm genes of M type reference strains was noted. The 5' sequences for six established M protein genes--the emm-32, emm-34, emm-38, emm-40, emm-42, and emm-71 genes--were determined to supplement the existing emm sequence database. Rapid sequence analysis differentiated serologically M-nontypeable strains and was used to establish the probable.     

Evaluation of the lymphocyte blastogenic response by rapid flow analysis.

The data presented show that the blastogenic response of human peripheral lymphocytes to mitogens and specific antigens can be easily evaluated with rapid flow analysis of mithramycin stained cells. The precision, reproducibility and rapidity of the method make it highly suitable to study lymphocyte response to antigens in patient populations.     

Identification of group D streptococci by SeroSTAT.

Clinical isolates of group D streptococci presumptively identified by biochemical methods were grouped by latex agglutination using a commercially prepared reagent specifically sensitized with group D antiserum (SeroSTAT; Scott Laboratories, Inc., Fiskeville, R.I.). Streptococcus species tested included S. faecalis, S. faecium, S. durans, S. avium, S. bovis, and S. equinus. Colonies of the organism to be tested were picked from agar plates, emulsified in a drop of glycine-buffered saline on a slide, and mixed with a drop of the latex reagent. Macroscopic agglutination occurred within 60 s. A total of 115 isolates of group D streptococci were tested; 103 (89.6%) gave positive reactions with SeroSTAT. Twelve strains failed to react with the latex reagent; these 12 strains also gave negative results with group D antiserum when tested by the Lancefield method. Two of 14 group A streptococci also reacted with the SeroSTAT group D reagent; after trypsinization, the cross-reaction was eliminated. Group B streptococci, viridans group streptococci, anaerobic streptococci, and staphylococci all gave negative reactions with the SeroSTAT reagent. The SeroSTAT reagent is a useful diagnostic tool for the prompt identification of enterococcal and non-enterococcal group D streptococci.