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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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Related in: MedlinePlus

Virulence of wild type isolate CS101 (square) and the isogenic β-hemolysis negative sagA/pel mutant, CS101 sagA/pel::Tn917 (circle) and a β-hemolytic kidney recovered CS101 pel::Tn917 KR sagA/pel mutant variant (triangle). Groups of 10 outbred CD1 mice were injected with 1 × 109 cfu into a skin air sack. Time to death was monitored and statistical significance was determined by use of Student's t test (wt vs. sagA/pel::Tn917 p = 0.54; sagA/pel::Tn917 vs. KR p = 0.018).
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Figure 5: Virulence of wild type isolate CS101 (square) and the isogenic β-hemolysis negative sagA/pel mutant, CS101 sagA/pel::Tn917 (circle) and a β-hemolytic kidney recovered CS101 pel::Tn917 KR sagA/pel mutant variant (triangle). Groups of 10 outbred CD1 mice were injected with 1 × 109 cfu into a skin air sack. Time to death was monitored and statistical significance was determined by use of Student's t test (wt vs. sagA/pel::Tn917 p = 0.54; sagA/pel::Tn917 vs. KR p = 0.018).

Mentions: Based on the M and M-related protein phenotypic characteristics of the β-hemolytic positive kidney-recovered variant of the sagA/pel mutant in vitro, we predicted this variant would be virulent in a mouse skin infection model. To test this prediction the kidney-recovered variant, the wild type and the original sagA/pel mutant were tested for virulence using the skin air sac model. The results present in figure 5 indicate that the kidney-recovered variant was significantly more virulent than the sagA/pel mutant from which it was originally selected (p = 0.018) despite not secreting SpeB or SK in culture (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)

Virulence of wild type isolate CS101 (square) and the isogenic β-hemolysis negative sagA/pel mutant, CS101 sagA/pel::Tn917 (circle) and a β-hemolytic kidney recovered CS101 pel::Tn917 KR sagA/pel mutant variant (triangle). Groups of 10 outbred CD1 mice were injected with 1 × 109 cfu into a skin air sack. Time to death was monitored and statistical significance was determined by use of Student's t test (wt vs. sagA/pel::Tn917 p = 0.54; sagA/pel::Tn917 vs. KR p = 0.018).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC64569&req=5

Figure 5: Virulence of wild type isolate CS101 (square) and the isogenic β-hemolysis negative sagA/pel mutant, CS101 sagA/pel::Tn917 (circle) and a β-hemolytic kidney recovered CS101 pel::Tn917 KR sagA/pel mutant variant (triangle). Groups of 10 outbred CD1 mice were injected with 1 × 109 cfu into a skin air sack. Time to death was monitored and statistical significance was determined by use of Student's t test (wt vs. sagA/pel::Tn917 p = 0.54; sagA/pel::Tn917 vs. KR p = 0.018).
Mentions: Based on the M and M-related protein phenotypic characteristics of the β-hemolytic positive kidney-recovered variant of the sagA/pel mutant in vitro, we predicted this variant would be virulent in a mouse skin infection model. To test this prediction the kidney-recovered variant, the wild type and the original sagA/pel mutant were tested for virulence using the skin air sac model. The results present in figure 5 indicate that the kidney-recovered variant was significantly more virulent than the sagA/pel mutant from which it was originally selected (p = 0.018) despite not secreting SpeB or SK in culture (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