[23] Molecular analysis of Streptococcus pyogenes adhesion

Hanski, Emanuel; Fogg, George; Tovi, Aviva; Okada, Nobuhiko; Burstein, Israel; Caparon, Michael G.

doi:10.1016/s0076-6879(95)53025-8

Cited by 23 publications

(15 citation statements)

References 44 publications

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“…Standard procedures for cloning, transformation, and analysis of E. coli clones were used (49). Plasmid pLU11 was used to transform competent cells of the S. pyogenes AP1 strain as previously described (30). Transformants were selected on THY plates containing kanamycin and analyzed by PCR and Southern blotting by using standard techniques (49).…”

Section: Methodsmentioning

confidence: 99%

The Regulator PerR Is Involved in Oxidative Stress Response and Iron Homeostasis and Is Necessary for Full Virulence of Streptococcus pyogenes

2002

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Ferric uptake regulator (Fur) and Fur-like proteins form an important family of transcriptional regulators in many bacterial species. In this work we have characterized a Fur-like protein, the peroxide regulator PerR, in an M1 serotype of Streptococcus pyogenes. To determine the role of PerR in S. pyogenes, we inactivated the gene by allelic replacement. PerR-deficient bacteria showed 48% reduction of 55 Fe incorporation from the culture medium. Transcriptional analysis revealed that mtsA, encoding a metal-binding protein of an ABC transporter in S. pyogenes, was transcribed at lower levels than were wild-type cells. Although total iron accumulation was reduced, the growth of the mutant strain was not significantly hampered. The mutant showed hyperresistance to hydrogen peroxide, and this response was induced in wild-type cells by growth in aerobiosis, suggesting that PerR acts as an oxidative stress-responsive repressor. PerR may also participate in the response to superoxide stress, as the perR mutant was more sensitive to the superoxide anion and had a reduced transcription of sodA, which encodes the sole superoxide dismutase of S. pyogenes. Complementation of the mutation with a functional perR gene restored 55 Fe incorporation, response to peroxide stress, and transcription of both mtsA and sodA to levels comparable to those of wild-type bacteria. Finally, the perR mutant was attenuated in virulence in a murine air sac model of infection (P < 0.05). These results demonstrate that PerR is involved in the regulation of iron homeostasis and oxidative stress responses and that it contributes to the virulence of S. pyogenes.

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Section: Methodsmentioning

confidence: 99%

The Regulator PerR Is Involved in Oxidative Stress Response and Iron Homeostasis and Is Necessary for Full Virulence of Streptococcus pyogenes

2002

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“…The fragment was treated with appropriate restriction enzyme, ligated into the streptococcal suicide plasmid pFW13 (46), a kind gift from Andreas Podbielski, to generate pFW-sclA, and transformed into DH5␣. The plasmid was purified, and 2 g of the plasmid was electroporated into the AP1 strain (19) for homologous recombination to occur. Transformants were plated on THY plus 15 g of agar/liter with 150 g of kanamycin/ml.…”

Section: Methodsmentioning

confidence: 99%

SclA, a Novel Collagen-Like Surface Protein ofStreptococcus pyogenes

2000

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Surface proteins of Streptococcus pyogenes are important virulence factors. Here we describe a novel collagenlike surface protein, designated SclA (streptococcal collagen-like surface protein). The sclA gene was identified in silico using the Streptococcal Genome Sequencing Project with the recently identified protein GRAB as the probe. SclA has a signal sequence and a cell wall attachment region containing the prototypic LPXTGX motif. The surface-exposed part of SclA contains a unique NH 2 -terminal domain of 73 amino acids, followed by a collagen-like region. The sclA gene was found to be positively regulated by Mga, a transcriptional activator of several S. pyogenes virulence determinants. A mutant lacking cell wall-associated SclA was constructed and was found to be as effective as wild-type bacteria in platelet aggregation, survival in fresh human blood, and adherence to pharyngeal cells. The sclA gene was found in all 12 S. pyogenes strains that were investigated using PCR. Sequence analysis revealed that the signal sequence and the cell wall attachment region are highly conserved. The collagen-like domain is variable in its NH 2 -terminal region and has conserved repeated domains in its COOH-terminal part. SclA proteins from most strains have additional proline-rich repeats spacing the collagen-like domain and the cell wall attachment sequence. The unique NH 2 -terminal region is hypervariable, but computer predictions indicate a common secondary structure, with two alpha helices connected by a loop region. Immune selection may explain the hypervariability in the NH 2 -terminal region, whereas the preserved secondary structure implies that this region has a common function. These features and the Mga regulation are shared with the M protein of S. pyogenes. Moreover, as with the gene encoding the M protein, phylogenetic analysis indicates that horizontal gene transfer has contributed to the evolution of sclA.Streptococcus pyogenes is an important pathogen causing the common diseases pharyngitis, erysipelas, and impetigo. Sometimes this organism causes severe invasive diseases, such as a toxic shock-like syndrome and necrotizing fasciitis. Nonsuppurative sequelae following infection with S. pyogenes include acute rheumatic fever and poststreptococcal glomerulonephritis. The cell wall-anchored proteins of S. pyogenes are regarded as major virulence determinants and share common features, including a signal sequence and a cell wall attachment signal containing a so-called LPXTGX motif (39). Among the bestcharacterized and most important cell wall-attached proteins are the alpha-helical coiled-coil M and M-like proteins (see references 15 and 51). Each S. pyogenes strain carries one to three genes encoding M or M-like proteins, and these genes are colocated on the chromosome with the mga gene encoding their positive regulator, Mga (7,22,36,42,45). The scpA gene encoding the cell wall-attached C5a-peptidase is also located at this chromosomal site (44, 60). The M and M-like proteins bind several plasma proteins...

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“…Plasmid DNA was purified by standard techniques and transformed into E. coli by the method of Kushner (22). S. pyogenes was transformed by electroporation as described previously (5,16). Restriction endonucleases, ligases, and polymerases were used according to the recommendations of the manufacturers.…”

Section: Methodsmentioning

confidence: 99%

Constitutive expression of fibronectin binding in Streptococcus pyogenes as a result of anaerobic activation of rofA

Fogg

Caparon

1997

J Bacteriol

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Protein F is a fibronectin-binding surface protein of Streptococcus pyogenes (group A streptococcus) that mediates adherence to host cells. A gene product encoded by rofA activates transcription of the gene that encodes protein F (prtF) and was identified in a strain of S. pyogenes that expressed high levels of protein F under all conditions tested. Insertional inactivation of rofA in this strain results in a phenotype similar to that of other strains where high-level transcription of prtF occurs only in response to increased oxygen tension. In this study, we have compared the regulation of prtF and rofA in O 2 -regulated and constitutive strains in order to gain further insight into the function of rofA. Comparison of the prtF and rofA transcripts by S1 nuclease and primer extension assays indicated that the same promoters for each transcript are used in both O 2 -regulated and constitutive strains. However, analyses of rofA-lacZ reporter alleles revealed that a key difference between strains involves regulation of rofA itself. In O 2 -regulated strains, expression of rofA was elevated following culture under conditions of reduced O 2 tension. However, a much more robust activation of rofA expression was observed when constitutive strains were grown under similar conditions. Exchange of reporter and rofA alleles between strains demonstrated that host genetic background, and not the sequence of the respective rofA allele or regulatory region, dictates the expression phenotype. Activation of rofA required RofA, and RofA was shown to bind specifically to DNA containing the promoters for rofA and prtF. Finally, overexpression of either allele of rofA caused constitutive expression of prtF regardless of host background. These data suggest a model where anaerobic expression of prtF in constitutive hosts is controlled at the level of transcription of rofA and implicate additional factors in this regulatory pathway.Studies of many different bacterial pathogens have revealed that complex regulatory networks which sense environmental signals and control expression of particular sets of virulenceassociated genes are essential for adaptation to different environments in the host during infection (reviewed in references 7, 24, and 26). Consequently, an understanding of how virulence factors are regulated can provide important insights into pathogenesis and the contribution of specific genes to different stages of the infection process.

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[23] Molecular analysis of Streptococcus pyogenes adhesion

Cited by 23 publications

References 44 publications

The Regulator PerR Is Involved in Oxidative Stress Response and Iron Homeostasis and Is Necessary for Full Virulence of Streptococcus pyogenes

The Regulator PerR Is Involved in Oxidative Stress Response and Iron Homeostasis and Is Necessary for Full Virulence of Streptococcus pyogenes

SclA, a Novel Collagen-Like Surface Protein ofStreptococcus pyogenes

Constitutive expression of fibronectin binding in Streptococcus pyogenes as a result of anaerobic activation of rofA

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