The role of CopA in Streptococcus pyogenes copper homeostasis and virulence.

Maintenance of intracellular metal homeostasis during interaction with host niches is critical to the success of bacterial pathogens. To prevent infection, the mammalian innate immune response employs metal-withholding and metal-intoxication mechanisms to limit bacterial propagation. The first-row transition metal ion copper serves critical roles at the host-pathogen interface and has been associated with antimicrobial activity since antiquity. Despite lacking any known copper-utilizing proteins, streptococci have been reported to accumulate significant levels of copper. Here, we report that loss of CopA, a copper-specific exporter, confers increased sensitivity to copper in Streptococcus pyogenes strain HSC5, with prolonged exposure to physiological levels of copper resulting in reduced viability during stationary phase cultivation. This defect in stationary phase survival was rescued by supplementation with exogeneous amino acids, indicating the pathogen had altered nutritional requirements during exposure to copper stress. Furthermore, S. pyogenes HSC5 ΔcopA was substantially attenuated during murine soft-tissue infection, demonstrating the importance of copper efflux at the host-pathogen interface. Collectively, these data indicate that copper can severely reduce the viability of stationary phase S. pyogenes and that active efflux mechanisms are required to survive copper stress in vitro and during infection.

Effect of Phosphatase Activity of the Control of Virulence Sensor (CovS) on Clindamycin-Mediated Streptolysin O Production in Group A Streptococcus.

Severe manifestations of group A Streptococcus (GAS) infections are associated with massive tissue destruction and high mortality. Clindamycin (CLI), a bacterial protein synthesis inhibitor, is recommended for treating patients with severe invasive GAS infection. Nonetheless, the subinhibitory concentration of CLI induces the production of GAS virulent exoproteins, such as streptolysin O (SLO) and NADase, which would enhance bacterial virulence and invasiveness. A better understanding of the molecular mechanism of how CLI triggers GAS virulence factor expression will be critical to develop appropriate therapeutic approaches. The present study shows that CLI activates SLO and NADase expressions in the emm1-type CLI-susceptible wild-type strain but not in covS or control of virulence sensor (CovS) phosphatase-inactivated mutants. Supplementation with Mg2+, which is a CovS phosphatase inhibitor, inhibits the CLI-mediated SLO upregulation in a dose-dependent manner in CLI-susceptible and CLI-resistant strains. These results not only reveal that the phosphorylation of response regulator CovR is essential for responding to CLI stimuli, but also suggest that inhibiting the phosphatase activity of CovS could be a potential strategy for the treatment of invasive GAS infection with CLI.

Molecular epidemiology, antimicrobial susceptibility, and characterization of macrolide-resistant Streptococcus pyogenes in Japan.

Here we report the molecular epidemiology of macrolide-resistant Streptococcus pyogenes (group A streptococci, GAS) isolated from children with pharyngotonsillitis between 2011 and 2013 in Japan. In 299 isolates, 124 (41.5%) isolates were macrolide-resistant. We characterized the isolates by emm typing, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Of 299 isolates, 124 (41.5%) were macrolide-resistant isolates, 76 (61.3%) possessed mefA and 46 (37.1%) possessed ermB. All 76 isolates with mefA possessed msrD. There were no isolates possessed ermTR in this study. Eight emm/MLST types were observed. The predominant type was emm1/ST28 (57 isolates, 46.0%), which possessed the mefA/msrD complex, presenting as the M phenotype. The second most predominant type was emm12/ST467, which possessed ermB, presenting as the cMLSB phenotype. Of the cMLSB phenotype isolates, types emm28/ST52 and emm12/ST36 had multiple genetic backgrounds. We found high proportions of macrolide-resistant GAS in the southwestern areas of Japan.

Inhibition of streptococcal pyrogenic exotoxin B using allicin from garlic.

Streptococcal pyrogenic exotoxin B (SpeB) is an important virulence factor of group A streptococci (GAS) and inactivation of SpeB results in the significantly decreased virulence of the bacterium. The protein is secreted as an inactive zymogen of 40 KDa (SpeBz) and undergoes proteolytic truncation to result in a 28 KDa mature active protease (SpeBm). In this study the effect of allicin on the proteolytic activity of SpeBm was evaluated using azocasein assay. Allicin neutralized the SpeBm proteolytic activity in a concentration dependent manner (IC50 = 15.71 ± 0.45 µg/ml). The loss of activity was completely reversed by subsequent treatment with a reducing agent, dithiothreitol (DTT; 10 mM final concentration), suggesting that allicin likely inhibits the SpeBm by forming a disulfide linkage with an active thiol group in its active site. This mechanism of action was further confirmed with the fact that DTT did not reverse the SpeBm activity in the presence of E-64, a cysteine protease-specific inhibitor, which works specially by forming a thioether linkage with free sulfhydryl groups in enzymes active site. The MIC of allicin against GAS was found to be 32 µg/ml. Exposure of GAS culture to allicin (25 µg/ml) inhibited maturation of SpeBz to the SpeBm. In conclusion, the results of this study suggest that allicin inhibits the maturation of SpeBz and proteolytic activity of SpeBm and could be a potential therapeutic agent for the treatment of GAS infections.

An Approach for Identification of Novel Drug Targets in Streptococcus pyogenes SF370 Through Pathway Analysis.

Streptococcus pyogenes is one of the most important pathogens as it is involved in various infections affecting upper respiratory tract and skin. Due to the emergence of multidrug resistance and cross-resistance, S. Pyogenes is becoming more pathogenic and dangerous. In the present study, an in silico comparative analysis of total 65 metabolic pathways of the host (Homo sapiens) and the pathogen was performed. Initially, 486 paralogous enzymes were identified so that they can be removed from possible drug target list. The 105 enzymes of the biochemical pathways of S. pyogenes from the KEGG metabolic pathway database were compared with the proteins from the Homo sapiens by performing a BLASTP search against the non-redundant database restricted to the Homo sapiens subset. Out of these, 83 enzymes were identified as non-human homologous while 30 enzymes of inadequate amino acid length were removed for further processing. Essential enzymes were finally mined from remaining 53 enzymes. Finally, 28 essential enzymes were identified in S. pyogenes SF370 (serotype M1). In subcellular localization study, 18 enzymes were predicted with cytoplasmic localization and ten enzymes with the membrane localization. These ten enzymes with putative membrane localization should be of particular interest. Acyl-carrier-protein S-malonyltransferase, DNA polymerase III subunit beta and dihydropteroate synthase are novel drug targets and thus can be used to design potential inhibitors against S. pyogenes infection. 3D structure of dihydropteroate synthase was modeled and validated that can be used for virtual screening and interaction study of potential inhibitors with the target enzyme.

Essential oil of Artemisia vestita exhibits potent in vitro and in vivo antibacterial activity: Investigation of the effect of oil on biofilm formation, leakage of potassium ions and survival curve measurement.

The aim of the present study was to investigate the chemical composition of the essential oil of Artemisia vestita and to determine the antibacterial activity of the essential oil and its two major components, grandisol and 1,8­cineole, against certain respiratory infection­causing bacterial strains, in vitro and in vivo. The chemical composition of the essential oil was analyzed using gas chromatography­mass spectrometry. A micro­well dilution method was used to determine the minimum inhibition concentration (MIC) values of the essential oil and its major constituents. A model of Streptococcus pyogenes infection in mice was used to determine its in vivo activities. Lung and blood samples were obtained to assess bacterial cell counts. Toxicity evaluation of the essential oil and its components was completed by performing biochemical analysis of the serum, particularly monitoring aspartate transaminase, alanine transaminase, urea and creatinine. The essential oil exhibited potent antibacterial activity, whereas the two major constituents were less potent. The essential oil exhibited MIC values between 20 and 80 µg/ml, while the values of the two constituents were between 130 and 200 µg/ml. Scanning electron microscopy results demonstrated that the essential oil inhibited biofilm formation and altered its architecture. Survival curves indicated that the essential oil led to a reduction in the viability of different bacteria. The essential oil also induced significant leakage of potassium ions from S. pyogenes. The essential oil (100 µg/mouse) and grandisol (135 µg/mouse) significantly reduced the number of viable bacterial cells in the lungs (P<0.01). However, intake of 100 µg/mouse of essential oil or grandisol 135 µg/mouse once or twice each day for 9 days did not produce any toxic effects in the mice. In conclusion, the in vitro and in vivo results suggested that the essential oil of A. vestita and one of its major constituents, grandisol, can significantly inhibit the growth of different bacterial strains.

Streptococcus pyogenes arginine and citrulline catabolism promotes infection and modulates innate immunity.

A bacterium's ability to acquire nutrients from its host during infection is an essential component of pathogenesis. For the Gram-positive pathogen Streptococcus pyogenes, catabolism of the amino acid arginine via the arginine deiminase (ADI) pathway supplements energy production and provides protection against acid stress in vitro. Its expression is enhanced in murine models of infection, suggesting an important role in vivo. To gain insight into the function of the ADI pathway in pathogenesis, the virulence of mutants defective in each of its enzymes was examined. Mutants unable to use arginine (ΔArcA) or citrulline (ΔArcB) were attenuated for carriage in a murine model of asymptomatic mucosal colonization. However, in a murine model of inflammatory infection of cutaneous tissue, the ΔArcA mutant was attenuated but the ΔArcB mutant was hyperattenuated, revealing an unexpected tissue-specific role for citrulline metabolism in pathogenesis. When mice defective for the arginine-dependent production of nitric oxide (iNOS(-/-)) were infected with the ΔArcA mutant, cutaneous virulence was rescued, demonstrating that the ability of S. pyogenes to utilize arginine was dispensable in the absence of nitric oxide-mediated innate immunity. This work demonstrates the importance of arginine and citrulline catabolism and suggests a novel mechanism of virulence by which S. pyogenes uses its metabolism to modulate innate immunity through depletion of an essential host nutrient.

Study of streptococcal hemoprotein receptor (Shr) in iron acquisition and virulence of M1T1 group A streptococcus.

Streptococcus pyogenes (group A streptococcus, GAS) is a human bacterial pathogen of global significance, causing severe invasive diseases associated with serious morbidity and mortality. To survive within the host and establish an infection, GAS requires essential nutrients, including iron. The streptococcal hemoprotein receptor (Shr) is a surface-localized GAS protein that binds heme-containing proteins and extracellular matrix components. In this study, we employ targeted allelic exchange mutagenesis to investigate the role of Shr in the pathogenesis of the globally disseminated serotype M1T1 GAS. The shr mutant exhibited a growth defect in iron-restricted medium supplemented with ferric chloride, but no significant differences were observed in neutrophil survival, antimicrobial peptide resistance, cell surface charge, fibronectin-binding or adherence to human epithelial cells and keratinocytes, compared with wild-type. However, the shr mutant displayed a reduction in human blood proliferation, laminin-binding capacity and was attenuated for virulence in in vivo models of skin and systemic infection. We conclude that Shr augments GAS adherence to laminin, an important extracellular matrix attachment component. Furthermore, Shr-mediated iron uptake contributes to GAS growth in human blood, and is required for full virulence of serotype M1T1 GAS in mouse models of invasive disease.

Peroxide stimulon and role of PerR in group A Streptococcus.

We have characterized group A Streptococcus (GAS) genome-wide responses to hydrogen peroxide and assessed the role of the peroxide response regulator (PerR) in GAS under oxidative stress. Comparison of transcriptome changes elicited by peroxide in wild-type bacteria with those in a perR deletion mutant showed that 76 out of 237 peroxide-regulated genes are PerR dependent. Unlike the PerR-mediated upregulation of peroxidases and other peroxide stress defense mechanisms previously reported in gram-positive species, PerR-dependent genes in GAS were almost exclusively downregulated and encoded proteins involved in purine and deoxyribonucleotide biosynthesis, heme uptake, and amino acid/peptide transport, but they also included a strongly activated putative transcriptional regulator (SPy1198). Of the 161 PerR-independent loci, repressed genes (86 of 161) encoded proteins with functions similar to those coordinated by PerR, in contrast to upregulated loci that encoded proteins that function in DNA damage repair, cofactor metabolism, reactive oxygen species detoxification, pilus biosynthesis, and hypothetical proteins. Complementation of the perR deletion mutant with wild-type PerR restored PerR-dependent regulation, whereas complementation with either one of two PerR variants carrying single mutations in two predicted metal-binding sites did not rescue the mutant phenotype. Metal content analyses of the recombinant wild type and respective PerR mutants, in addition to regulation studies in metal-supplemented and iron-depleted media, showed binding of zinc and iron by PerR and an iron requirement for optimal responses to peroxide. Our findings reveal a novel physiological contribution of PerR in coordinating DNA and protein metabolic functions in peroxide and identify GAS adaptive responses that may serve to enhance oxidative stress resistance and virulence in the host.

Inhibition of streptolysin O by allicin - an active component of garlic.

Streptolysin O (SLO) is a potent cytolytic toxin produced by almost all strains of group A streptococci and is considered an important virulence factor for this organism. In this study we investigated the effect of allicin and aqueous garlic extracts on the haemolytic activity of SLO. All tested materials potentially inhibited the SLO haemolytic activity. Allicin neutralized SLO in a dose- and time-dependent manner. A 15 min incubation of SLO with 35 microg allicin totally inhibited the haemolytic activity of SLO [IC(50) (concentration necessary to reach half maximum inhibition)=5.97 microg]. The inhibitory activity of an old extract of garlic was equipotent to pure allicin (IC(50)=6.27 microg; P<0.05). In contrast, fresh extract of garlic inhibited the SLO haemolytic activity at lower concentrations (IC(50)=1.59 microl; 1.9 microg allicin). The inhibitory effect of the allicin was restored by addition of reducing agent DTT at 2 mM, suggesting that allicin likely inhibits the SLO by binding to the cysteine residue in the binding site. These results indicate a new activity for allicin and allicin may be a potential alternative drug against streptococcal diseases.

Abrogation of streptococcal pyrogenic exotoxin B-mediated suppression of phagocytosis in U937 cells by Cordyceps sinensis mycelium via production of cytokines.

Streptococcal pyrogenic exotoxin B (SPE B) is a virulent factor in group A streptococcal infection. We previously showed that SPE B reduced phagocytosis in human monocytic U937 cells. Here we show that the mycelium extract of Cordyceps sinensis (CS), a Chinese immunomodulatory herbal medicine, increased phagocytosis in U937 cells. Neither heat nor trypsin pretreatment prevented CS extract from causing this increase. Further studies indicated that SPE B-mediated suppression of U937 cell phagocytic activity was abrogated by CS extract. Factors in the conditioned medium from CS-extract-treated U937 cells were responsible for blocking the SPE B-mediated suppression of phagocytosis. Heating the conditioned medium eliminated the increase, which suggested that the U937-cell protein products augmented phagocytosis. Analyzing cytokine mRNA expression of U937 cells revealed increases in interferon-gamma (IFN-gamma), interleukin (IL)-12 p35 and p40, and tumor necrosis factor-alpha (TNF-alpha), but not in IL-1beta, IL-6, or IL-8. Treating U937 cells with anti-IFN-gamma, IL-12, and TNF-alpha antibodies also eliminated the conditioned medium-induced increase in phagocytosis. Taken together, SPE B inhibited phagocytosis, but CS mycelium extract abrogated this inhibition by causing cytokine production.

Chemical composition and antibacterial activity of Cochlospermum planchoni Hook.f. ex Planch essential oil from Burkina Faso.

The water distilled oil obtained from rhizomes of Cochlospermum planchonii Hook.f.ex Planch (Apocynaceae) from Burkina Faso was examined by GC and GC/MS. Cochlospermum planchonii oil presents a particular chemical composition with a high rate of oxygenated components with predominance of ketones and esters (86.4%). The essential oil was tested against twelve strains of bacteria using a broth microdilution method. The results suggest that Cochlospermum planchonii essential oil has significant bactericidal activity.

Identification and characterization of HtsA, a second heme-binding protein made by Streptococcus pyogenes.

Group A streptococci (GAS) can use heme and hemoproteins as sources of iron. However, the machinery for heme acquisition in GAS has not been firmly revealed. Recently, we identified a novel heme-associated cell surface protein (Shp) made by GAS. The shp gene is cotranscribed with eight downstream genes, including spy1795, spy1794, and spy1793 encoding a putative ABC transporter (designated HtsABC). In this study, spy1795 (designated htsA) was cloned from a serotype M1 strain, and recombinant HtsA was overexpressed in Escherichia coli and purified to homogeneity. HtsA binds 1 heme molecule per molecule of protein. HtsA was produced in vitro and localized to the bacterial cell surface. GAS up-regulated transcription of htsA in human blood compared with that in Todd-Hewitt broth supplemented with 0.2% yeast extract. The level of the htsA transcript dramatically increased under metal cation-restricted conditions compared with that under metal cation-replete conditions. The cation content, cell surface location, and gene transcription of HtsA were also compared with those of MtsA and Spy0385, the lipoprotein components of two other putative iron acquisition ABC transporters of GAS. Our results suggest that HtsABC is an ABC transporter that may participate in heme acquisition in GAS.

MtsABC is important for manganese and iron transport, oxidative stress resistance, and virulence of Streptococcus pyogenes.

MtsABC is a Streptococcus pyogenes ABC transporter which was previously shown to be involved in iron and zinc accumulation. In this study, we showed that an mtsABC mutant has impaired growth, particularly in a metal-depleted medium and an aerobic environment. In metal-depleted medium, growth was restored by the addition of 10 microM MnCl(2), whereas other metals had modest or no effect. A characterization of metal radioisotope accumulation showed that manganese competes with iron accumulation in a dose-dependent manner. Conversely, iron competes with manganese accumulation but to a lesser extent. The mutant showed a pronounced reduction (>90%) of (54)Mn accumulation, showing that MtsABC is also involved in Mn transport. Using paraquat and hydrogen peroxide to induce oxidative stress, we show that the mutant has an increased susceptibility to reactive oxygen species. Moreover, activity of the manganese-cofactored superoxide dismutase in the mutant is reduced, probably as a consequence of reduced intracellular availability of manganese. The enzyme functionality was restored by manganese supplementation during growth. The mutant was also attenuated in virulence, as shown in animal experiments. These results emphasize the role of MtsABC and trace metals, especially manganese, for S. pyogenes growth, susceptibility to oxidative stress, and virulence.

Identification and characterization of a Streptococcus pyogenes operon involved in binding of hemoproteins and acquisition of iron.

The hemolytic Streptococcus pyogenes can use a variety of heme compounds as an iron source. In this study, we investigate hemoprotein utilization by S. pyogenes. We demonstrate that surface proteins contribute to the binding of hemoproteins to S. pyogenes. We identify an ABC transporter from the iron complex family named sia for streptococcal iron acquisition, which consists of a lipoprotein (siaA), membrane permease (siaB), and ATPase (siaC). The sia transporter is part of a highly conserved, iron regulated, 10-gene operon. SiaA, which was localized to the cell membrane, could specifically bind hemoglobin. The operon's first gene encodes a novel bacterial protein that bound hemoglobin, myoglobin, heme-albumin, and hemoglobin-haptoglobin (but not apo-haptoglobin) and therefore was named Shr, for streptococcal hemoprotein receptor. PhoZ fusion and Western blot analysis showed that Shr has a leader peptide and is found in both membrane-bound and soluble forms. An M1 SF370 strain with a polar mutation in shr was more resistant to streptonigrin and hydrogen peroxide, suggesting decreased iron uptake. The addition of hemoglobin to the culture medium increased cell resistance to hydrogen peroxide in SF370 but not in the mutant, implying the sia operon may be involved in hemoglobin-dependent resistance to oxidative stress. The shr mutant demonstrated reduced hemoglobin binding, though cell growth in iron-depleted medium supplemented with hemoglobin, whole blood, or ferric citrate was not affected, suggesting additional systems are involved in hemoglobin utilization. SiaA and Shr are the first hemoprotein receptors identified in S. pyogenes; their possible role in iron capture is discussed.

Zinc binding and dimerization of Streptococcus pyogenes pyrogenic exotoxin C are not essential for T-cell stimulation.

Streptococcal pyrogenic enterotoxin C (Spe-C) is a superantigen virulence factor produced by Streptococcus pyogenes that activates T-cells polyclonally. The biologically active form of Spe-C is thought to be a homodimer containing an essential zinc coordination site on each subunit, consisting of the residues His(167), His(201), and Asp(203). Crystallographic data suggested that receptor specificity is dependent on contacts between the zinc coordination site of Spe-C and the beta-chain of the major histocompatibility complex type II (MHCII) molecule. Our results indicate that only a minor fraction of dimer is present at T-cell stimulatory concentrations of Spe-C following mutation of the unpaired side chain of cysteine at residue 27 to serine. Mutations of amino acid residues His(167), His(201), or Asp(203) had only minor effects on protein stability but resulted in greatly diminished MHCII binding, as measured by surface plasmon resonance with isolated receptor/ligand pairs and flow cytometry with MHCII-expressing cells. However, with the exception of the mutants D203A and D203N, mutation of the zinc-binding site of Spe-C did not significantly impact T-cell activation. The mutation Y76A, located in a polar pocket conserved among most superantigens, resulted in significant loss of T-cell stimulation, although no effect was observed on the overall binding to human MHCII molecules, perhaps because of the masking of this lower affinity interaction by the dominant zinc-dependent binding. To a lesser extent, mutations of side chains found in a second conserved MHCII alpha-chain-binding site consisting of a hydrophobic surface loop decreased T-cell stimulation. Our results demonstrate that dimerization and zinc coordination are not essential for biological activity of Spe-C and suggest the contribution of an alternative MHCII binding mode to T-cell activation.

Effect of date extract on growth and hemolytic activity of Streptococcus pyogenes.

The effect of date extract on growth and hemolytic activity of S. pyogenes was examined. It was found that 5% DE caused 78 % growth inhibition. However, 20% DE inhibited the growth by 86%. 5% DE inhibited hemolysin and streptolysin O activities by 43% and 24% respectively,while 20% caused 95 and 91 %inhibition.

Evolving resistance patterns in community-acquired respiratory tract pathogens: first results from the PROTEKT global surveillance study. Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin.

In recent years, antibacterial resistance among respiratory pathogens implicated in community-acquired respiratory tract infections (RTIs) has spread worldwide at an alarming rate. Thus, there is a pressing need for new antibacterials that retain activity against resistant organisms, have a low potential to select for resistance and do not induce cross-resistance. Telithromycin is the first of a new class of antibacterials - the ketolides - that have been designed specifically to overcome resistance among respiratory tract pathogens. This paper presents the first results of the PROTEKT study (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin), a worldwide surveillance study initiated to chart the prevalence of important resistance phenotypes and genotypes and the comparative activity of telithromycin against such strains. Analysis of over 7,000 bacterial isolates by April 2001 has confirmed the notable prevalence of strains resistant to commonly prescribed RTI antibacterials for all the pathogens studied. Telithromycin demonstrates high activity against isolates of Streptococcus pneumoniae, irrespective of penicillin G, macrolide or fluoroquinolone resistance. Telithromycin is also highly active against other respiratory tract pathogens, including Streptococcus pyogenes and beta-lactamase-producing strains of Haemophilus influenzae and Moraxella catarrhalis. These data justify the assertion that telithromycin is a promising new candidate for the empirical treatment of community-acquired RTIs, particularly in the face of increasing antibacterial resistance.

Coley toxins immunotherapy: a retrospective review.

OBJECTIVE: Coley toxins are administered to cancer patients worldwide, though clinical studies assessing efficacy either alone or in combination with conventional cancer therapy are limited. This article provides an overview of Coley toxins immunotherapy and compares the survival experience of cancer patients who received Coley toxins for renal, ovarian, breast cancer, or soft-tissue sarcomas with patients who received conventional treatment other than radiation. DATA SOURCES: Cases were compiled from 5 of 18 monographs by Helen Coley Nauts. STUDY SELECTION: Using a retrospective cohort design with external controls, 128 Coley cases treated in New York from 1890 to 1960 were compared with 1675 controls from the Surveillance Epidemiology End Result (SEER) population-based cancer registry who received a cancer diagnosis in 1983. DATA EXTRACTION: Groups were matched on age, sex, ethnicity, site, stage, and treatment status (i.e., no radiotherapy). DATA SYNTHESIS: The Cox proportional hazards model controlled for stage and menopausal status (when applicable) and the hazard ratio and 95% CI defined the odds of site-specific survival from date of diagnosis to last follow-up. Compared to the SEER population, risk of death within 10 years was not significantly different in Coley patients treated for renal, ovarian, breast cancer, or soft-tissue sarcomas. CONCLUSIONS: This study suggests that patients treated with surgery and Coley toxins between 1890 and 1960 experienced survival rates comparable to those of patients diagnosed in 1983 and treated with nonradiotherapeutic conventional approaches. The study is limited by small sample sizes, possibly inaccurate technology for staging during Coley time, and potential selection bias with Coley patients.

Insertional inactivation of streptolysin S expression is associated with altered riboflavin metabolism in Streptococcus pyogenes.

Transposon Tn916 mutagenesis was used to create a mutant of Streptococcus pyogenes M type 3, designated ISS417, in which the ability to produce streptolysin S (SLS) and several other exoproteins was impaired. Concomitantly, the mutant became dependent upon riboflavin for growth and was able to grow in Todd Hewitt broth (THB) when supplemented with riboflavin or riboflavinrich yeast extract. The parent strain was apparently able to utilize THB-derived components as a substitute for riboflavin, while the mutant was not. Although the parent strain grew well in synthetic medium, it was unable to produce SLS, except when it was supplemented with a small amount of THB. Thus, a component of THB was able to "trigger" SLS formation in the parent strain. The mutant grew well in this medium, but was unable to produce SLS even when it was supplemented with THB. Southern hybridization analysis revealed that the ISS417 mutant harbours a single transposon insertion in its chromosome. Phage transduction experiments showed that the riboflavin dependency and the inability to make SLS phenotypes are co-transducible. The pleotrophic properties of the ISS417 mutant differ from those reported for insertional inactivation of the mga locus which regulates production of a number of surface proteins in S. pyogenes and the sar locus which regulates production of a number of exoproteins in Staphylococcus aureus. In view of the possibility that there exist a genetic linkage between the riboflavin biosynthetic pathway and expression of the oxygen-stable SLS, we hypothesize that SLS has a role in the growth economy of S. pyogenes.