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NAD(+)-glycohydrolase from Streptococcus pyogenes was purified by successive chromatography on CM Sepharose CL-6B, Sephacryl S-200 HR and hydroxyapatite. The purified enzyme possessed synthesis and hydrolysis activities of cyclic ADP-ribose (cADPR), a newly found second messenger for Ca2+ mobilisation, along with cleavage activity of the ribose-nicotinamide bond in NAD+.
Hemolytic mutants of Lancefield strain SS-95 and ATCC 19615 Streptococcus pyogenes were produced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. These mutants contained the same levels of streptolysin O, nicotinamide adenine dinucleotidase, deoxyribonuclease, and hyaluronidase. The mutants were deficient in streptolysin S, as was the naturally occurring nonhemolytic Lowry strain. The mutants retained their pathogenicity for mice and, when reisolated from the dead animals, produced the mutant hemolytic pattern.
It was recently reported that strains of Streptococcus pyogenes are capable of inducing entry of the bacterium into epithelial cells; however, nothing is known regarding the gene(s) and the underlying mechanism(s) involved. Using isogenic mutants of S. pyogenes JRS4 strain that are defective in the expression of each of the surface proteins F1 and M6, it was demonstrated that both are required for efficient internalization. Expression of F1 on the surface of a poorly invading S. pyogenes strain significantly enhances its internalization efficiency. Protein F1-mediated internalization is inhibited by UR, the nonrepetitive fibronectin-binding domain of this protein, and to a lesser extent, by the repetitive fibronectin-binding domain, RD2. Polyclonal anti-human fibronectin antibodies completely abolish F1-mediated internalization; increasing fibronectin concentrations result in a significant enhancement of bacterial uptake. The findings shown here suggest that protein F1 mediates streptococcal internalization and that the M6 protein is required for more efficient entry of the bacterium.
An extracellular protein of Streptococcus pyogenes, streptococcal inhibitor of complement (SIC), and its variant, called DRS (distantly related to SIC), are expressed by some S. pyogenes strains. SIC from type 1 (M1) isolates of S. pyogenes interferes with complement-mediated cell lysis, reportedly via its interaction with complement proteins. In this study we demonstrate that S. pyogenes strains carrying emm12 and emm55 (the genes for the M12 and M55 proteins, respectively) express and secrete DRS. This protein, like SIC, binds to the C6 and C7 complement proteins, and competition enzyme-linked immunosorbent assay experiments demonstrate that DRS competes with SIC for C6 and C7 binding. Similarly, SIC competes with DRS for binding to the complement proteins. Despite this, the recombinant DRS preparation showed no significant effect on complement function, as determined by lysis of sensitized sheep erythrocytes. Furthermore, the presence of DRS is not inhibitory to SIC activity.
OBJECTIVE: To determine the antibiotic susceptibility of recent isolates of Streptococcus pyogenes and to evaluate the prevalence of macrolide-resistant phenotypes. MATERIAL AND METHODS: In 1999, we conducted a cross-sectional study at Mexico Children's Hospital "Federico Gomez", to analyze one hundred strains of S. pyogenes isolated from 1992 to 1998, in children with uncomplicated pharyngotonsillitis. Strains were frozen at the bacteriology lab until they were analyzed. Strains were tested for susceptibility against some beta-lactams, macrolides and clindamycin. Double-disk testing was carried out to evaluate erythromycin-resistant phenotypes. Data are presented using central tendency measures. RESULTS: All tested strains were not resistant to beta-lactams and clindamycin; 16% of the strains were resistant to macrolides and all of them belonged to phenotype M. CONCLUSIONS: Susceptibility testing is recommended to identify possible changes in antibiotic resistance to streptococci.
The tRNA-specific adenosine deaminase from the pathogenic bacteria Streptococcus pyogenes (spTAD) has been overexpressed in Escherichia coli and crystallized in the presence of Zn2+ ion at 295 K using ammonium sulfate as a precipitant. Flash-cooled crystals of spTAD diffracted to 2.0 A using 30%(v/v) glycerol as a cryoprotectant. X-ray diffraction data have been collected to 2.0 A using synchrotron radiation. The crystal belongs to the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 81.042, c = 81.270 A. The asymmetric unit contains one subunit of spTAD, with a corresponding crystal volume per protein weight (VM) of 3.3 A3 Da(-1) and a solvent content of 62.7%.
The case history of necrotizing fasciitis in a previously healthy man aged 37 years is presented. The disease ran a rapidly fatal course. The only finding on bacterial culture was Streptococcus pyogenes, Lancefield group A, serotype M1, ("killer streptococci"). The therapeutic possibilities are briefly outlined and the importance of rapid surgical intervention is emphasized. It is concluded that this patient was actually beyond therapeutic aid less than 12 hours after admission. Necrotizing fasciitis caused by Streptococcus pyogenes, Lancefield group A, serotype M1, is comparable with meningococcal disease. It appears probable that the disease had developed in this patient as a sequel of streptococcal arthritis.
An acidic glycoprotein (SAGP), purified from crude extract (CE) of Streptococcus pyogenes, inhibits the growth of murine fibrosarcoma (Meth A) cells in vitro and prolongs the life span of mice inoculated with the same cells. This study revealed that the growth inhibitory activity of SAGP on Meth A cells in vitro was lowered in the presence of sulfhydryl-oxidizing agents such as cystamine and 5, 5'-dithiobis (2-nitrobenzoic acid) in a dose dependent manner. The activity of SAGP was also diminished by pretreatment of SAGP with cystamine. Inactivation of SAGP by cystamine was reversed by dithiothreitol. Furthermore, the effect of CE on prolonging the life span of Meth A-bearing mice was reduced by the administration of cystamine. These findings suggest that SH-groups are involved in the expression of anti-tumor activity of SAGP or CE in vitro and in vivo.
Mickelson, M. N. (National Animal Disease Laboratory, Ames, Iowa). Chemically defined medium for growth of Streptococcus pyogenes. J. Bacteriol. 88:158-164. 1964.-Three strains of group A hemolytic streptococci representing three serological types were serially subcultured for an extended period (100 or more transfers) in a peptide-free and protein-free culture medium. An amino acid assay medium, modified by addition of small amounts of glutamine, ammonium acetate, and 0.1 m phosphate (pH 7), was used. In this medium, high concentrations of glutamic acid or glutamine were required, and biotin was stimulatory to growth. Biotin could be partially replaced with NaHCO(3). Maximal growth was obtained with NaHCO(3) when biotin was present, and aspartic acid and asparagine were omitted from the medium. In 24 to 48 hr of incubation at 37 C, luxuriant growth was obtained with complete removal and quantitative fermentation of 1% glucose to lactic acid. Optical densities of cultures were equivalent to those obtained with the best infusion media.
Fibronectin-binding protein I (SfbI) from Streptococcus pyogenes plays a key role in bacterial adhesion to, and invasion of, eukaryotic cells. In addition, SfbI exhibits a considerable potential as mucosal adjuvant and can trigger polyclonal activation of B cells. Here, we report that SfbI is also capable of binding human IgG in a nonimmune fashion. SfbI was reactive with IgG1, IgG2, IgG3, and IgG4 isotypes (type IIo IgG-binding profile). The affinity constant (Kd) of the SfbI-IgG interaction was in the range of 1-2 x 10(-5) M. Further studies demonstrated that the SfbI binding was mediated by the Fc component of the IgG molecule. Experiments performed using purified recombinant proteins spanning different domains of SfbI showed that the IgG-binding activity was restricted to the fibronectin-binding domains, and in particular to the fibronectin-binding repeats. Finally, the presence of recombinant SfbI resulted in an impairment of both phagocytosis of IgG-coated RBCs and Ab-dependent cell cytotoxicity by macrophages. These results demonstrated for the first time that, in addition to its major role during the colonization process, SfbI may also favor bacterial immune evasion after the onset of the infection by interfering with host clearance mechanisms.
Streptococcus pyogenes (group A streptococci, GAS) is an important and exclusively human pathogen. Adherence to and internalization into host cells significantly contributes to the pathogenesis of GAS infections. The adherence mechanism is a two-step process in which host extracellular matrix (ECM) proteins act as prime targets. GAS may express more than a dozen different microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that attach to fibronectin or collagen. One of them, protein F1/SfbI binds fibronectin and mediates adherence of GAS to host cells. Bound fibronectin acts as a bridging molecule towards host cell integrins, which in turn initialize the uptake process that leads to GAS internalization. In their safe intracellular niche GAS can persist protected from antibiotics and host defense, a scenario currently discussed in the context of treatment failure, asymptomatic GAS carriers and recurrent GAS infections. Patients with such low grade infections represent the main GAS reservoir from which the bacteria are spread in the general population. Due to their important function, expression of GAS MSCRAMMs is under control of several "stand alone" transcriptional regulators and two-component signal transduction systems. Several regulator genes are organized together with MSCRAMM genes on one of two potential pathogenicity islands, act together in a growth phase-dependent regulatory network and are expressed in a strain-specific manner. A detailed understanding of these mechanisms is crucial, since interference with MSCRAMM function alone or in conjunction with specific manipulations of regulators is an attractive goal for novel anti-infective strategies.
Induction of anti-IgG during hyperimmunization of rabbit with Streptococcus pyogenes (group A streptococci; GAS) was previously shown to require the presence of IgG Fc receptors (FcR) in the vaccine strain. In the present work, we examined whether streptococcal FcR activity might also be of importance for heart and kidney deposition of IgG, known to occur in poststreptococcal sequelae as well as during experimental immunization of animals. Each of three IgG-binding (GAS types M1, M12 and M22) and two non-binding (GAS type T27 and S. agalactiae (GBS) type Ia) streptococcal strains were used for intravenous immunization of rabbits during two periods of eight and six weeks, respectively, separated by an interval of one month. Before use, vaccine strains were treated with KSCN and carefully washed in order to remove any surface-bound immunoglobulins. No deaths occurred among injected rabbits. No tissue deposition was elicited by the GAS type T27 or the GBS strain. In contrast, the strains of types M1, M12 and M22 all induced deposits of IgG in kidney and heart tissue, beginning during the first immunization period. In two tested animals, receiving GAS of types M1 or M22, circulating immune complexes containing anti-IgG antibodies were also detected. Finally, serum autoantibodies reacting with preparations of heart and kidney, but not lung or liver, were demonstrated in each of six animals receiving M1 or M22, reaching maximum levels during reimmunization; such antibodies were not evoked by the two strains not binding IgG. Our results suggest that, in GAS with capacity for non-immune binding of IgG, triggering of anti-IgG acted to enhance tissue deposition of IgG or immune complexes in immunized rabbits. Furthermore tissue-specific antibodies were elicited only by the IgG-binding strains and occurred comparatively late during immunization, suggesting that those antibodies might have been triggered due to the exposition of hidden kidney and heart determinants.
Thirty strains of Streptococcus salivarius were beta-haemolytic when initially isolated from throat swabs and, because they were also bacitracin sensitive, they could be confused with the pathogenic Streptococcus pyogenes on primary culture plates. These strains were isolated form approximately 3000 throat swab cultures made in 6 mth, during which time 220 Lancefield group A organisms were also detected. Other features of these organisms, including loss of beta-haemolysis on subculture, penicillin resistance in half of the strains and ability to grow on MacConkey bile salt agar, are not shared by Strep. pyogenes, and their demonstration enables strains of the 2 species to be simply distinguished.
To investigate the role of putative virulence factors of Streptococcus pyogenes (group A streptococcus; GAS) in causing disease, we introduced specific mutations in GAS strain B514, a natural mouse pathogen, and tested the mutant strains in two models of infection. To study late stages of disease, we used our previously described mouse model (C3HeB/FeJ mice) in which pneumonia and systemic spread of the streptococcus follow intratracheal inoculation. To study the early stages of disease, we report here a model of long-term (at least 21 days) throat colonization following intranasal inoculation of C57BL/10SnJ mice. When the three emm family genes of GAS strain B514-Sm were deleted, the mutant showed no significant difference from the wild type in induction of long-term throat colonization or pneumonia. We inactivated the scpA gene, which encodes a complement C5a peptidase, by insertion of a nonreplicative plasmid and found no significant difference from the wild type in the incidence of throat colonization. However, there was a small but statistically significant decrease in the incidence of pneumonia caused by the scpA mutant. Finally, we demonstrated a very important effect of the hyaluronic acid capsule in both models. Following intranasal inoculation of mice with a mutant in which a nonreplicative plasmid was inserted into the hasA gene, which encodes hyaluronate synthase, we found that all bacteria recovered from the throats of the mice were encapsulated revertants. Following intratracheal inoculation with the hasA mutant, the incidence of pneumonia within 72 h was significantly reduced from that of the control strain (P = 0.006). These results indicate that the hyaluronic acid capsule of S. pyogenes B514 confers an important selective advantage for survival of the bacteria in the upper respiratory tract and is also an important determinant in induction of pneumonia in our model system.
Serological tests are commonly employed to aid the diagnosis of Streptococcus pyogenes infections, particularly when non-suppurative sequelae are suspected. Conventional laboratory practice is to measure antibody levels to various combinations of the extracellular group A Streptococcus (GAS) antigens streptolysin O (SLO), DNase B, streptokinase and hyaluronidase. Antibody to the extracellular cysteine proteinase streptococcal pyrogenic exotoxin B (SPE B) and its precursor zymogen is also produced in response to GAS infections. An indirect hemagglutination test for antibody to zymogen/SPE B was established and evaluated in serum samples from 168 patients with proven (n = 27) or suspected GAS (n = 141) infections, which were also screened for antibodies using the 4 conventional tests. For comparison, sera from 56 patients infected with a variety of other pathogens, as well as sera from 16 patients infected with either S. agalactiae or S. pneumoniae and 34 sera from healthy subjects, were tested. Statistical analysis confirmed that antibody to zymogen/SPE B is a serological marker that can discriminate GAS infections. It can be ranked with the anti-SLO titer, currently the most widely used test, as a marker of an antecedent GAS infection.
BACKGROUND: Defining the temporal dynamics of invasive Streptococcus pyogenes (group A Streptococcus) and differences between hyperendemic and lower-incidence regions provides crucial insights into pathogen evolution and, in turn, informs preventive measures. We aimed to examine the clinical and temporal lineage dynamics of S pyogenes across different disease settings in Australia to improve understanding of drivers of pathogen diversity. METHODS: In this retrospective, multicentre, clinical and genomic epidemiology study, we identified cases of invasive S pyogenes infection from normally sterile sites between Jan 1, 2011, and Feb 28, 2023. Data were collected from five hospital networks across low-incidence regions in temperate southeast Australia and the hyperendemic, tropical, and largely remote Top End of the Northern Territory of Australia. The crude incidence rate ratio (IRR) of bloodstream S pyogenes infection comparing the Top End and southeast Australia and in First Nations people compared with non-First Nations people was estimated by quasi-Poisson regression. We estimated odds ratios (ORs) of intensive care unit (ICU) admission, in-hospital mortality, and 30-day mortality for the Top End versus southeast Australia using logistic regression. Retrieved and successfully sequenced isolates were assigned lineages at whole-genome resolution. Temporal trends in the composition of co-circulating lineages were compared between the two regions. We used an S pyogenes-specific multistrain simulated transmission model to examine the relationship between host population-specific parameters and observed pathogen lineage dynamics. The prevalence of accessory genes (those present in 5-95% of all genomes) was compared across geographies and temporal periods to investigate genomic drivers of diversity. FINDINGS: We identified 500 cases of invasive S pyogenes infection in patients in the Top End and 495 cases in patients in southeast Australia. The crude IRR of bloodstream infection for the Top End compared with southeast Australia was 5·97 (95% CI 4·61-7·73) across the entire study period; in the Top End, infection disproportionately affected First Nations people compared with non-First Nations people (5·41, 4·28-6·89). The odds of in-hospital mortality (OR 0·43, 95% CI 0·26-0·70), 30-day mortality (0·38, 0·23-0·63), and ICU admission (0·42, 0·30-0·59) were lower in the Top End than in southeast Australia. Longitudinal lineage analysis of 642 S pyogenes genomes identified waves of replacement with distinct lineages in the Top End, whereas southeast Australia had a small number of dominant lineages that persisted and cycled in frequency. The transmission model qualitatively reproduced a similar pattern of replacement with distinct lineages when using a high transmission rate, small population size, and high levels of human movement-characteristics similar to those of communities in the hyperendemic Top End. Using a lower transmission rate, larger population size, and lower levels of migration similar to those of communities in urbanised southeast Australia, the transmission model qualitatively reproduced a pattern of dominant lineages that cycled in frequency. Despite distinct circulating lineages, the prevalence of accessory genes in the bacterial population was maintained across geographies and temporal periods. INTERPRETATION: In a hyperendemic setting, the replacement of distinct S pyogenes lineages occurred in waves, which could be linked to the disproportionate burden of disease and sparse human population in this setting. The maintenance of bacterial gene frequency could be consistent with multilocus selection. These findings suggest that lineage-specific interventions-such as vaccines under development-should consider disease setting and, without broad cross-protection, might lead to lineage replacement. FUNDING: National Health and Medical Research Council, and Leducq Foundation.
An inhibitor of Streptococcus mutans strain GS5 bacteriocin was released from a bacteriocin-sensitive strain of S. pyogenes by sonic treatment and partly purified by gel filtration on a Sepharose 6B column. The inhibitory factor was sensitive to heat, trypsin, and periodate and possessed a molecular weight of approximately 93,000. This factor may be the receptor for the S. mutans bacteriocin.
Group A Streptococcus pyogenes has surface-located fibronectin (Fn)-binding proteins known to be a major virulence factor, which adheres to and invades host cells. We present a novel Fn-binding protein of group A streptococcus serotype M3 and M18 strains isolated from patients with toxic shock-like syndrome (TSLS). By searching the whole genome sequence of an M3 strain from a TSLS patient, an open reading frame was found among the putative surface proteins. It possessed an LPXTG motif and Fn-binding repeat domains in the C-terminal region and was designated as FbaB (Fn-binding protein of group A streptococci type B). The fbaB gene was found in all M3 and M18 strains examined, although not in other M serotypes. Furthermore, FbaB protein was expressed on the cell surface of TSLS strains but not on non-TSLS ones. Enzyme-linked immunosorbent assay and ligand blotting revealed that recombinant FbaB exhibits a strong Fn-binding ability. An FbaB-deficient mutant strain showed 6-fold lower adhesion and invasion efficiencies to HEp-2 cells than the wild type. Moreover, mortality was decreased in mice infected with the mutant strain in comparison to the wild type. These data suggest that FbaB is etiologically involved in the development of invasive streptococcal diseases.