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Mouse skin passage of a Streptococcus pyogenes Tn917 mutant of sagA/pel restores virulence, beta-hemolysis and sagA/pel expression without altering the position or sequence of the transposon.

Eberhard TH, Sledjeski DD, Boyle MD - BMC Microbiol. (2001)

Bottom Line: Streptolysin S (SLS), the oxygen-stable hemolysin of Streptococcus pyogenes, has recently been shown to be encoded by the sagA/pel gene.Insertion of a Tn917 transposon into the promoter region of the sagA/pel gene of S. pyogenes isolate CS101 eliminated expression of SLS, as well as decreased expression of the streptococcal pyrogenic exotoxin B, streptokinase and M protein.Northern blot analysis of the kidney-recovered isolates confirmed that transcription of sagA/pel was restored; however the expression of all sagA/pel regulated genes was not restored to wild type levels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Medical College of Ohio, Toledo, Ohio 43614, USA. teberhard@mco.edu

ABSTRACT

Background: Streptolysin S (SLS), the oxygen-stable hemolysin of Streptococcus pyogenes, has recently been shown to be encoded by the sagA/pel gene. Mutants lacking expression of this gene were less virulent in a dermonecrotic mouse infection model. Inactivation of the sagA/pel gene affect the expression of a variety of virulence factors in addition to the hemolysin. Insertion of a Tn917 transposon into the promoter region of the sagA/pel gene of S. pyogenes isolate CS101 eliminated expression of SLS, as well as decreased expression of the streptococcal pyrogenic exotoxin B, streptokinase and M protein.

Results: In this study a mouse skin air sac model was utilized to analyze the effect of biological pressures on expression of SLS and other sagA/pel regulated gene products. The insertion delayed the lethal effect of S. pyogenes in a mouse skin infection model. Despite this, bacteria could be cultured from the kidneys 72 hours post infection. These kidney-recovered isolates were beta-hemolytic despite the transposon being present in its original location and had equivalent virulence to the wild type isolate when re-injected into naive mice. Northern blot analysis of the kidney-recovered isolates confirmed that transcription of sagA/pel was restored; however the expression of all sagA/pel regulated genes was not restored to wild type levels.

Conclusions: These results show that biological pressure present in the mouse can select for variants with altered expression of key virulence factor genes in S. pyogenes.

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Phenotypes of CS101 wt, CS101 sagA/pel::Tn917 and CS101 sagA/pel::Tn917 kidney-recovered (KR) variants. Panel A. Bacterial binding of 125I labeled fibrinogen CS101 wt (square), CS101 sagA/pel:: Tn917 (circle) and CS101 sagA/pel Tn917 kidney-recovered (triangle). Panel B. Streptococcal pyogenic exotoxin B (SpeB) activity of culture supernatants treated with DTT. CS101 wt (square), CS101 sagA/pel::Tn917 (circle) and CS101 sagA/pel::Tn917 kidney-recovered (triangle). Panel C. Streptokinase (SK) activity of culture supernatants grown in the presence of cysteine protease inhibitor E 64 (10 μM) to prevent destruction of SK by any secreted cysteine protease; CS101 wt (square), CS101 sagA/pel::Tn917 (circle) and CS101 sagA/pel::Tn917 KR (triangle).
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Figure 4: Phenotypes of CS101 wt, CS101 sagA/pel::Tn917 and CS101 sagA/pel::Tn917 kidney-recovered (KR) variants. Panel A. Bacterial binding of 125I labeled fibrinogen CS101 wt (square), CS101 sagA/pel:: Tn917 (circle) and CS101 sagA/pel Tn917 kidney-recovered (triangle). Panel B. Streptococcal pyogenic exotoxin B (SpeB) activity of culture supernatants treated with DTT. CS101 wt (square), CS101 sagA/pel::Tn917 (circle) and CS101 sagA/pel::Tn917 kidney-recovered (triangle). Panel C. Streptokinase (SK) activity of culture supernatants grown in the presence of cysteine protease inhibitor E 64 (10 μM) to prevent destruction of SK by any secreted cysteine protease; CS101 wt (square), CS101 sagA/pel::Tn917 (circle) and CS101 sagA/pel::Tn917 KR (triangle).

Mentions: Expression of surface fibrinogen-binding M and M-related proteins was monitored by the ability of intact bacteria to bind radiolabeled fibrinogen. The kidney-recovered mutant not only recovered fibrinogen binding potential, that was lost when the sagA/pel gene was inactivated, but also the level of fibrinogen-binding exceeded that of the wild type isolate (Fig. 4A). Analysis of culture supernatants for the presence of SpeB (Fig. 4B) or SK (Fig. 4C) indicated that the sagA/pel mutant and the kidney-recovered variant displayed a similar low level of expression when compared to the wild type. There were no significant changes in fibronectin binding among any of the variants tested (see Table 2). Consequently, restoration of expression of the larger sagA/pel transcript (Fig. 3) was not sufficient to revert all of the sagA/pel-associated phenotypes to wild type levels (see Table 2).


Mouse skin passage of a Streptococcus pyogenes Tn917 mutant of sagA/pel restores virulence, beta-hemolysis and sagA/pel expression without altering the position or sequence of the transposon.

Eberhard TH, Sledjeski DD, Boyle MD - BMC Microbiol. (2001)

Phenotypes of CS101 wt, CS101 sagA/pel::Tn917 and CS101 sagA/pel::Tn917 kidney-recovered (KR) variants. Panel A. Bacterial binding of 125I labeled fibrinogen CS101 wt (square), CS101 sagA/pel:: Tn917 (circle) and CS101 sagA/pel Tn917 kidney-recovered (triangle). Panel B. Streptococcal pyogenic exotoxin B (SpeB) activity of culture supernatants treated with DTT. CS101 wt (square), CS101 sagA/pel::Tn917 (circle) and CS101 sagA/pel::Tn917 kidney-recovered (triangle). Panel C. Streptokinase (SK) activity of culture supernatants grown in the presence of cysteine protease inhibitor E 64 (10 μM) to prevent destruction of SK by any secreted cysteine protease; CS101 wt (square), CS101 sagA/pel::Tn917 (circle) and CS101 sagA/pel::Tn917 KR (triangle).
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Figure 4: Phenotypes of CS101 wt, CS101 sagA/pel::Tn917 and CS101 sagA/pel::Tn917 kidney-recovered (KR) variants. Panel A. Bacterial binding of 125I labeled fibrinogen CS101 wt (square), CS101 sagA/pel:: Tn917 (circle) and CS101 sagA/pel Tn917 kidney-recovered (triangle). Panel B. Streptococcal pyogenic exotoxin B (SpeB) activity of culture supernatants treated with DTT. CS101 wt (square), CS101 sagA/pel::Tn917 (circle) and CS101 sagA/pel::Tn917 kidney-recovered (triangle). Panel C. Streptokinase (SK) activity of culture supernatants grown in the presence of cysteine protease inhibitor E 64 (10 μM) to prevent destruction of SK by any secreted cysteine protease; CS101 wt (square), CS101 sagA/pel::Tn917 (circle) and CS101 sagA/pel::Tn917 KR (triangle).
Mentions: Expression of surface fibrinogen-binding M and M-related proteins was monitored by the ability of intact bacteria to bind radiolabeled fibrinogen. The kidney-recovered mutant not only recovered fibrinogen binding potential, that was lost when the sagA/pel gene was inactivated, but also the level of fibrinogen-binding exceeded that of the wild type isolate (Fig. 4A). Analysis of culture supernatants for the presence of SpeB (Fig. 4B) or SK (Fig. 4C) indicated that the sagA/pel mutant and the kidney-recovered variant displayed a similar low level of expression when compared to the wild type. There were no significant changes in fibronectin binding among any of the variants tested (see Table 2). Consequently, restoration of expression of the larger sagA/pel transcript (Fig. 3) was not sufficient to revert all of the sagA/pel-associated phenotypes to wild type levels (see Table 2).

Bottom Line: Streptolysin S (SLS), the oxygen-stable hemolysin of Streptococcus pyogenes, has recently been shown to be encoded by the sagA/pel gene.Insertion of a Tn917 transposon into the promoter region of the sagA/pel gene of S. pyogenes isolate CS101 eliminated expression of SLS, as well as decreased expression of the streptococcal pyrogenic exotoxin B, streptokinase and M protein.Northern blot analysis of the kidney-recovered isolates confirmed that transcription of sagA/pel was restored; however the expression of all sagA/pel regulated genes was not restored to wild type levels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Medical College of Ohio, Toledo, Ohio 43614, USA. teberhard@mco.edu

ABSTRACT

Background: Streptolysin S (SLS), the oxygen-stable hemolysin of Streptococcus pyogenes, has recently been shown to be encoded by the sagA/pel gene. Mutants lacking expression of this gene were less virulent in a dermonecrotic mouse infection model. Inactivation of the sagA/pel gene affect the expression of a variety of virulence factors in addition to the hemolysin. Insertion of a Tn917 transposon into the promoter region of the sagA/pel gene of S. pyogenes isolate CS101 eliminated expression of SLS, as well as decreased expression of the streptococcal pyrogenic exotoxin B, streptokinase and M protein.

Results: In this study a mouse skin air sac model was utilized to analyze the effect of biological pressures on expression of SLS and other sagA/pel regulated gene products. The insertion delayed the lethal effect of S. pyogenes in a mouse skin infection model. Despite this, bacteria could be cultured from the kidneys 72 hours post infection. These kidney-recovered isolates were beta-hemolytic despite the transposon being present in its original location and had equivalent virulence to the wild type isolate when re-injected into naive mice. Northern blot analysis of the kidney-recovered isolates confirmed that transcription of sagA/pel was restored; however the expression of all sagA/pel regulated genes was not restored to wild type levels.

Conclusions: These results show that biological pressure present in the mouse can select for variants with altered expression of key virulence factor genes in S. pyogenes.

Show MeSH
Related in: MedlinePlus