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PubMed2003

Role of M3 protein in the adherence and internalization of an invasive Streptococcus pyogenes strain by epithelial cells.

Streptococcus pyogenes utilizes multiple mechanisms for adherence to and internalization by epithelial cells. One of the molecules suggested of being involved in adherence and internalization is the M protein. Although strains of the M3 serotype form the second largest group isolated from patients with severe invasive diseases and fatal infections, not much information is known regarding the interactions of M3 protein with mammalian cells. In this study we have constructed an emm3 mutant of an invasive M3 serotype (SP268), and demonstrated that the M3 protein is involved in both adherence to and internalization by HEp-2 cells. Fibronectin promoted both adherence and internalization of SP268 in an M3-independent pathway. Utilizing speB and speB/emm3 double mutants, it was found that M3 protein is not essential for the maturation of SpeB, as was reported for the M1 protein. Increased internalization efficiency observed in both the speB and emm3/speB mutants suggested that inhibition of S. pyogenes internalization by SpeB is not related to the presence of an intact M3 protein. Thus, other proteins in SP268, which serve as targets for SpeB activity, have a prominent role in the internalization process.

Adhesins, Bacterial↗
PubMed2006

Prevalence and persistence of certain serologic types of Streptococcus pyogenes in metropolitan Tokyo.

M-typing was conducted for Streptococcus pyogenes strains isolated within 1 week of admission from patients admitted to the Tokyo Metropolitan Toshima Hospital for scarlet fever in the 23-year period between 1956 and 1978. M12 was detected in the largest number of patients, followed by M6, M4, M3, and M1. T-typing was also conducted for Streptococcus pyogenes strains isolated from healthy schoolchildren in three different parts of the 20-year period between 1977 and 1998. T12 was detected in the largest number of children, followed by T28, T1, T4, and T6. These results suggested that strains of similar types are prevalent in scarlet fever patients and healthy carriers, that these strains become prevalent at intervals of several years, and that they are detected every year, albeit in a small number of persons, indicating that they are endemic. Isolated strains were typed by M-typing and T-typing. The results of typing were identical in more than 90% of cases. Comparison of major Streptococcus pyogenes strains isolated in Tokyo and three other major cities abroad showed that M12 was detected in the largest number of persons in Tokyo and New York and that major types were similar in the four cities.

Adolescent↗
PubMed2004

CovS inactivates CovR and is required for growth under conditions of general stress in Streptococcus pyogenes.

The gram-positive human pathogen Streptococcus pyogenes (group A streptococcus [GAS]) causes diseases ranging from mild and often self-limiting infections of the skin or throat to invasive and life-threatening illnesses. To cause such diverse types of disease, the GAS must be able to sense adverse environments and regulate its gene expression accordingly. The CovR/S two-component signal transduction regulatory system in GAS represses about 15% of the GAS genome, including many genes involved in virulence, in response to the environment. We report that CovR is still able to repress transcription from several promoters in the absence of the putative histidine kinase sensor for this system, CovS. We also show that a phosphorylation site mutant (D53A) of CovR is unable to repress gene expression. In addition, we report that a strain with a nonpolar mutation in CovS does not grow at a low pH, elevated temperature, or high osmolarity. The stress-related phenotypes of the CovS mutant were complemented by expression of covS from a plasmid. Selection for growth of a CovS mutant under stress conditions resulted in isolation of second-site mutations that inactivated covR, indicating that CovR and CovS act in the same pathway. Also, at 40 degrees C in the wild-type strain, CovR appeared to be less active on the promoter tested, which is consistent with the hypothesis that it was partially inactivated by CovS. We suggest that under mild stress conditions, CovS inactivates CovR, either directly or indirectly, and that this inactivation relieves repression of many GAS genes, including the genes needed for growth of GAS under stress conditions and some genes that are necessary for virulence. Growth of many gram-positive bacteria under multiple-stress conditions requires alteration of promoter recognition produced by RNA polymerase association with the general stress response sigma factor, sigma(B). We provide evidence that for GAS, which lacks a sigB ortholog, growth under stress conditions requires the CovR/S two-component regulatory system instead. This two-component system in GAS thus appears to perform a function for which other gram-positive bacteria utilize an alternative sigma factor.

Amino Acid Sequence↗
PubMed2006

RscA, a member of the MDR1 family of transporters, is repressed by CovR and required for growth of Streptococcus pyogenes under heat stress.

The ability of Streptococcus pyogenes (group A streptococcus [GAS]) to respond to changes in environmental conditions is essential for this gram-positive organism to successfully cause disease in its human host. The two-component system CovRS controls expression of about 15% of the GAS genome either directly or indirectly. In most operons studied, CovR acts as a repressor. We previously linked CovRS to the GAS stress response by showing that the sensor kinase CovS is required to inactivate the response regulator CovR so that GAS can grow under conditions of heat, acid, and salt stress. Here, we sought to identify CovR-repressed genes that are required for growth under stress. To do this, global transcription profiles were analyzed by microarrays following exposure to increased temperature (40 degrees C) and decreased pH (pH 6.0). The CovR regulon in an M type 6 strain of GAS was also examined by global transcriptional analysis. We identified a gene, rscA (regulated by stress and Cov), whose transcription was confirmed to be repressed by CovR and activated by heat and acid. RscA is a member of the MDR1 family of ABC transporters, and we found that it is required for growth of GAS at 40 degrees C but not at pH 6.0. Thus, for GAS to grow at 40 degrees C, CovR repression must be alleviated so that rscA can be transcribed to allow the production of this potential exporter. Possible explanations for the thermoprotective role of RscA in this pathogen are discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Consideration of cysteine protease activity for serological M-typing of clinical Streptococcus pyogenes isolates.

Clinical isolates of Streptococcus pyogenes were classified by serological typing of their surface M protein. Non-M typeable strains with the emm1 gene were characterized as the degradation of M protein caused by overproduction of the extracellular cysteine protease, SpeB. These events are dependent on the growth phase. M protein produced prior to expression of SpeB is degraded in the stationary phase when the active form of SpeB is detected. The proteolytic degradation of M protein should be considered for precise M typing analysis.

Antigens, Bacterial↗
PubMed2005

Comparison of emm typing and ribotyping with three restriction enzymes to characterize clinical isolates of Streptococcus pyogenes.

A total of 336 Streptococcus pyogenes isolates recently recovered from patients with pharyngitis from 13 countries were characterized by emm typing and riboprinting using an automated Riboprinter (Dupont/Qualicon) based on the patterns produced by three restriction enzymes, EcoRI, PstI, and HindIII. Three enzymes were necessary to increase the discrimination of ribogroups formed by each enzyme. A total of 40 ribogroups and 38 emm sequences (not counting allelic variations) were identified. Multilocus sequence typing was performed on a sampling of the isolates, and those results were consistent with those of both emm typing and ribotyping. Correlations were observed among all three methods.

Antigens, Bacterial↗
PubMed2000

Streptococcal erythrogenic toxin B abrogates fibronectin-dependent internalization of Streptococcus pyogenes by cultured mammalian cells.

Streptococcus pyogenes secretes several proteins that influence host-pathogen interactions. A tissue-culture model was used to study the influence of the secreted cysteine protease streptococcal erythrogenic toxin B (SPE B) on the interaction between S. pyogenes strain NZ131 (serotype M49) and mammalian cells. Inactivation of the speB gene enhanced fibronectin-dependent uptake of the pathogen by Chinese hamster ovary (CHO-K1) cells compared to that in the isogenic wild-type strain. Preincubation of the NZ131 speB mutant with purified SPE B protease significantly inhibited fibronectin-dependent uptake by both CHO-K1 and CHO-pgs745 cells. The effect was attributed to an abrogation of fibronectin binding to the surface of the bacteria that did not involve either the M49 protein or the streptococcal fibronectin-binding protein SfbI. In contrast, pretreatment of the NZ131 speB mutant with SPE B did not influence sulfated polysaccharide-mediated uptake by CHO-pgs745 cells. The results indicate that the SPE B protease specifically alters bacterial cell surface proteins and thereby influences pathogen uptake.

Adhesins, Bacterial↗
PubMed1980

Immunogenicity of ribosomes from enzymatically lysed Streptococcus pyogenes.

Ribosomal fractions isolated from Streptococcus pyogenes by physical and enzymatic disruption of the cell wall were found to provide protection in mice against challenge with the homologous M type. Although ribosomal fractions isolated by physical disruption of the cells also provided protection against challenge with several heterologous M types, ribosomal fractions from enzymatically lysed cells did not provide protection against any of the heterologous M types. Ribosomes isolated by either method were found to be contaminated with cell surface proteins. Chemical analysis of the ribosomes showed a greater protein:ribonucleic acid ratio in ribosomes from physically disrupted cells than in ribosomes from enzymatically disrupted cells (2:1 versus 1:1). Antisera to ribosomes isolated from physically disrupted cells detected many more antigenic determinants on ribosomes isolated from enzymatically disrupted cells than did the corresponding homologous antisera. Immunodiffusion analysis suggested that ribosomes isolated from physically disrupted cells may contain cell wall antigenic determinants which are present on ribosomes isolated from enzymatically disrupted cells in a partially degraded form. Washing of ribosomes in high-molarity salt solutions suggested that some of the contaminating cell wall proteins are tightly bound to the ribosomes.

Animals↗
PubMed1988

Molecular analysis of a composite chromosomal conjugative element (Tn3701) of Streptococcus pyogenes.

The plasmid-free Streptococcus pyogenes A454 contains a conjugative element, Tn3701, encoding resistance to erythromycin (Emr), tetracycline (Tcr), and minocycline (Mnr). We have mapped a 50-kilobase (kb) chromosomal region of A454 corresponding to the internal part of Tn3701. Tn3701 includes a 19.7-kb structure, designated Tn3703, on which the Emr Tcr Mnr determinants were localized. Tn3703 was very similar in structure to Tn916. Translocation of the Emr Tcr Mnr markers from A454 onto pIP964, an Enterococcus faecalis hemolysin plasmid, yielded different pIP964 derivatives. When the inserts of four of these derivatives were aligned with the 50-kb region of Tn3701, three of them were found to result from the transposition of Tn3703 and one resulted from the insertion of a 44.0-kb portion of Tn3701, including Tn3703. Tn3701 inserted, apparently without changing its structure, in the chromosomes of various streptococcal transconjugants, as well as in one of the 12 E. faecalis transconjugants studied. Tn3703 inserted at different chromosomal sites in four E. faecalis transconjugants, and one copy of Tn3701 plus an additional copy of Tn3703 were detected in the chromosomes of seven transconjugants.

Cloning, Molecular↗
PubMed1980

Regulation of beta-galactoside phosphate accumulation in Streptococcus pyogenes by an expulsion mechanism.

Streptococcus pyogenes pregrown on lactose took up glucose, lactose, or methyl beta-D-thiogalactopyranoside (MeSGal or TMG) by a phosphoenolpyruvate-dependent phosphotransferase system. MeSGal accumulated in the cell as MeSGal-phosphate (MeSGalP). Three effects were noted when various sugars were added to MeSGal preloaded cells: (i) no decrease in intracellular MeSGalP concentration after addition of fructose, sucrose, o-nitrophenyl-beta-D-galactoside, glycerol, 6-deoxyglucose, alpha-methyl D-glucoside, 2-deoxygalactose, glucose 1-phosphate, or glucose 6-phosphate; (ii) slow loss of preaccumulated MeSGalP evoked by lactose, 2-deoxy-D-glucose, or unlabeled MeSGal; and (iii) a short lag followed by extremely rapid expulsion of intracellular MeSGalP elicited by glucose or mannose and a slower expulsion elicited by glucosamine. The expelled compound was free MeSGal, indicating the involvement of dephosphorylation in the expulsion mechanism. Deoxyglucose inhibited the expulsion evoked by mannose, and prepoisoning of cells with fluoride or arsenate prevented the glucose-dependent expulsion. The expulsion is due to activation of an expulsion mechanism rather than to turnover of MeSGalP and leak of internal MeSGal with concomitant inhibition of MeSGal influx. The results suggest the need for phosphotransferase-dependent translocation of a preferential sugar or accumulation of the sugar catabolite for expulsion activation. The significance of the expulsion mechanism in synthesis regulation of enzymes involved in carbohydrate utilization is proposed.

Arsenates↗
PubMed1988

Heparin-inhibitable basement membrane-binding protein of Streptococcus pyogenes.

Solubilized surface proteins of Streptococcus pyogenes serotype M6 were found by indirect immunofluorescence assays to bind selectively to proteoglycan-containing regions of basement membranes of kidney and cardiac muscle in vitro. Epithelial, endothelial, and interstitial cells were unstained. Binding of streptococcal protein to basement membranes was competitively inhibited by heparin and, to a lesser extent, by heparan sulfate. Weak inhibition was also observed with other glycosaminoglycans, including dermatan sulfate, chondroitin sulfate, and hyaluronic acid. Type IV collagen, gelatin, serum fibronectin, glucuronic acid, and a selection of monosaccharides had no significant effects on binding. The heparin-inhibitable basement membrane-binding protein was purified by affinity chromatography on heparin-Sepharose 6-B. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea dissociated the affinity-purified protein into two polypeptides of 9,000 and 15,000 mrs. Chemical analyses revealed that the purified protein was devoid of cysteine, amino and neutral sugars, and phosphate. Thus, the polypeptides are not glycosylated or complexed with trace amounts of lipoteichoic acid or polysaccharide. Binding of purified protein to tissue was determined by direct radioassay and indirect immunofluorescence and was inhibitable by heparin. Although the in vivo effects of this streptococcal component remain to be determined, its deposition on basement membranes in vitro supports the hypothesis that it contributes to the pathogenesis of poststreptococcal glomerulonephritis or acute rheumatic fever.

Animals↗
PubMed1983

The relationship between opacity factor and M protein in Streptococcus pyogenes.

Lancefield acid extracts of Streptococcus pyogenes, type 22 (T12, M22, OF positive) gave good yields of M protein and little opacity factor (OF), but sodium dodecyl sulphate (SDS) extracts contained high titres of OF (greater than 20000) and little M protein. Acid-extracted OF could be separated from M protein by Sepharose 4B chromatography, but some of the OF-positive fractions that did not precipitate with the absorbed homologous anti-M rabbit serum, were able to neutralise opsonic antibodies present in human serum. The isoelectric-focusing profiles of the two antigens showed partial similarity. Some strains of the OF-positive serotypes, e.g., M-types 22 and 49, lost both M antigen and OF activity on serial transfer in Todd-Hewitt broth, but this was not seen in a representative of M-type 60, and no M-negative OF-negative variants could be detected after six subcultures. Among the OF-negative serotypes some, e.g., M-types 5 and 6, were completely stable, whereas others, e.g., M-types 12, 55 and 57, lost their M antigens after serial subculture. One explanation is that the genes that code for M antigen are plasmid borne in some serotypes and, moreover, are carried on the same plasmid as the gene for OF in some OF-positive serotypes. However, analysis of cell lysates by agarose-gel electrophoresis failed to demonstrate the presence of plasmid DNA in any of the strains tested.

Animals↗
PubMed2006

Population genetics and linkage analysis of loci within the FCT region of Streptococcus pyogenes.

The FCT regions of Streptococcus pyogenes strains encode a variety of cell wall-anchored surface proteins that bind the extracellular matrix of the human host and/or give rise to pilus-like appendages. Strong linkage is evident between transcription-regulatory loci positioned within the FCT and emm regions and the emm pattern genotype marker for preferred infection of the throat or skin. These findings provide a basis for the hypothesis that FCT region gene products contribute to tissue-specific infection. In an initial series of steps to address this possibility, the FCT regions of 13 strains underwent comparative sequence analysis, the gene content of the FCT region was characterized for 113 strains via PCR, and genetic linkage was assessed. A history of extensive recombination within FCT regions was evident. The emm pattern D-defined skin specialist strains were highly homogenous in their FCT region gene contents, whereas the emm pattern A-C-defined throat specialist strains displayed a greater variety of forms. Most pattern A-C strains harbored prtF1 (75%) but lacked cpa (75%). In contrast, the majority of emm pattern D strains had cpa (92%) but lacked prtF1 (79%). Models based on FCT and emm region genotypes revealed the most parsimonious pathways of evolution. Using niche-determining candidate genes to infer phylogeny, emm pattern E strains--the so-called generalists, which lack a strong tissue site preference--occupied a transition zone separating most throat specialists from skin specialists. Overall, population genetic analysis supports the possibility that the FCT region gives rise to surface proteins that are largely necessary, but not always sufficient, to confer tissue site preference for infection.

Evolution, Molecular↗
PubMed2005

Balanitis caused by Streptococcus pyogenes: a report of two cases.

Streptococcus pyogenes (the Lancefield group A streptococcus) is a cause of pharyngitis and impetigo. However, it has rarely been implicated as a sexually transmitted pathogen. We herein report two cases of severe balanitis due to S. pyogenes in sexually active men. It is postulated that penile cellulitis developed following the invasion of S. pyogenes through a traumatic abrasion acquired during fellatio performed by commercial sex workers. Both patients were treated successfully with oral administration of penicillin.

Adult↗
PubMed2003

The novel conjugative transposon tn1207.3 carries the macrolide efflux gene mef(A) in Streptococcus pyogenes.

The macrolide efflux gene mef(A) of the Streptococcus pyogenes clinical strain 2812A was found to be carried by a 52-kb chromosomal genetic element that could be transferred by conjugation to the chromosome of other streptococcal species. The characteristics of this genetic element are typical of conjugative transposons and was named Tn1207.3. The size of Tn1207.3 was established by pulsed-field gel electrophoresis (PFGE), and DNA sequencing analysis showed that the 7,244 bp at the left end of Tn1207.3 were identical to those of the pneumococcal Tn1207.1 element. Tn1207.3-like genetic elements were found to be inserted at a single specific chromosomal site in 12 different clinical isolates S. pyogenes exhibiting the M phenotype of resistance to macrolides and carrying the mef(A) gene. Tn1207.3 was transferred from S. pyogenes 2812A to Streptococcus pneumoniae, and sequence analysis carried out on six independent transconjugants showed that insertion of Tn1207.3 in the pneumococcal genome always occurred at a single specific site as in Tn1207.1. Using MF2, a representative S. pneumoniae transconjugant, as a donor, Tn1207.3 was transferred again by conjugation to S. pyogenes and Streptococcus gordonii. The previously described nonconjugative element Tn1207.1 of S. pneumoniae appears to be a defective element, part of a longer conjugative transposon that carries mef(A) and is found in clinical isolates of S. pyogenes.

Anti-Bacterial Agents↗
PubMed2000

Pyopneumothorax: a complication of Streptococcus pyogenes pharyngitis.

A 20-y-old African-American female with Streptococcus pyogenes pharyngitis presented with tension pyopneumothorax. Her illness began with fever and sore throat that persisted for several days. She then developed a left neck swelling, followed by difficult swallowing and cough. Subsequently, she developed shortness of breath that became severe. On physical examination fever (39.2 degrees C), exudative pharyngitis, tenderness and swelling in the left anterior cervical area were noted. Chest X-ray revealed left side pneumothorax, air-fluid level and near-complete collapse of the left lung with displacement of the heart and trachea to the right. Computed tomography scan of the neck revealed swelling and enhancement of the sternocleidomastoid muscle with loculated fluid collection, inflammation in the left anterior medial neck displacing the trachea extending into the mediastinum and the left apex. Thoracentesis revealed purulent fluid; Gram stain showed Gram-positive cocci in chains; culture yielded pure growth of Streptococcus pyogenes. She was treated with high dose penicillin, several chest tubes and intra-pleural injections of streptokinase with gradual resolution. This complication has not been described previously in Streptococcus pyogenes pharyngitis.

Adult↗
PubMed1987

Purification and characterization of Streptococcus pyogenes erythrogenic toxin type A produced by a cloned gene in Streptococcus sanguis.

The gene of Streptococcus pyogenes erythrogenic toxin type A (speA) has been previously cloned in Streptococcus sanguis (Challis) and produces extracellular erythrogenic toxin type A (ET A). The ET A produced and secreted by this heterologous host was purified to homogeneity and shown to have properties identical to ET A produced by S. pyogenes strain NY-5; i.e., serological identity in immunodiffusion, migration in SDS-polyacrylamide gel electrophoresis, mitogenic activity, inhibition of mitogenic activity by specific antibody, and precipitation by an international scarlatina antitoxin preparation. The cloned speA gene specified an ET A which had a molecular weight identical to that of ET A from S. pyogenes previously reported from this laboratory. NH2-terminal sequence determination of the purified protein showed the first nine residues to be gln gln asp pro asp pro ser gln leu; this is consistent with predictions made from the nucleotide sequence of the speA gene according to Weeks and Ferretti and different from the sequence published by Johnson et al.

Amino Acids↗
PubMed2002

Clonal relationships among isolates of erythromycin-resistant Streptococcus pyogenes of different geographical origin.

The clonal relationships among 261 erythromycin-resistant Streptococcus pyogenes isolates collected in 1986-1997 from nine countries in Europe and North and South America were studied by using two molecular typing methods: Vir typing and random amplified polymorphic DNA (RAPD) analysis. A total of 49 different Vir genotypes (VTs) and 33 different RAPD patterns were noted among the 261 isolates. Isolates that shared the same VT and RAPD pattern were considered to belong to the same clone. Although as many as 60 different clones were found among the isolates studied, only seven clones, comprising 157 of the 261 (60%) isolates, were found in more than one country. Five of these seven clones expressed the M phenotype known to be associated with the drug efflux mechanism, and only two clones expressed the macrolide-lincosamide-streptogramin B-resistance phenotype. The results indicate a polyclonal spread of erythromycin-resistant Streptococcus pyogenes. Furthermore, predominance of the seven clones indicates that erythromycin-resistant Streptococcus pyogenes of the same clonal origin may be widely distributed and found in several different countries.

Drug Resistance, Bacterial↗