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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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Primer extension analysis of sagA/pel from wild type, sagA/pel mutant and kidney-recovered strains. Bacteria were grown overnight at 37°C with 10% CO2 in THY broth. RNA was extracted and sagA/pel was detected using a primer extension assay and sagA/pel-specific primers. Equal amounts of RNA were loaded in each lane. Lane 1, RNA isolated from a wild type CS101 strain. Lane 2, RNA from an isogenic sagA/pel::Tn917 mutant. Lanes 3 and 4, RNA isolated from 2 different KR variants of the sagA/pel mutant. The larger sagA/pel transcript is indicated as t1, the smaller as t2. The DNA sequence in the lower part of the figure is from the region around the sagA/pel promoter. These represent bases 598514–598593 in the S. pyogenes genome [43]. The bases in italics are the putative -10 region of the sagA/pel promoter. The overlined regions are 16, 6 and 6 base inverted repeats. The bold letters are the 5'-ends of the t1 and t2 transcripts as determined by primer extension. The position of the Tn917 insertion is indicated by the ^ symbol. Note that the site of insertion is slightly different from what was previously reported [4]. The t2 transcript was not detected in the KR variants even at longer exposures.
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Figure 3: Primer extension analysis of sagA/pel from wild type, sagA/pel mutant and kidney-recovered strains. Bacteria were grown overnight at 37°C with 10% CO2 in THY broth. RNA was extracted and sagA/pel was detected using a primer extension assay and sagA/pel-specific primers. Equal amounts of RNA were loaded in each lane. Lane 1, RNA isolated from a wild type CS101 strain. Lane 2, RNA from an isogenic sagA/pel::Tn917 mutant. Lanes 3 and 4, RNA isolated from 2 different KR variants of the sagA/pel mutant. The larger sagA/pel transcript is indicated as t1, the smaller as t2. The DNA sequence in the lower part of the figure is from the region around the sagA/pel promoter. These represent bases 598514–598593 in the S. pyogenes genome [43]. The bases in italics are the putative -10 region of the sagA/pel promoter. The overlined regions are 16, 6 and 6 base inverted repeats. The bold letters are the 5'-ends of the t1 and t2 transcripts as determined by primer extension. The position of the Tn917 insertion is indicated by the ^ symbol. Note that the site of insertion is slightly different from what was previously reported [4]. The t2 transcript was not detected in the KR variants even at longer exposures.

Mentions: Primer extension analysis of the wild type and kidney-recovered variants demonstrated that the sagA/pel message expressed in the kidney-recovered variant had an identical transcription start site to the 500 base message present in the wild type strain (Fig. 3). The second transcript, present only in the RNA isolated from the wild type isolate (Fig. 3, lane 1), started 35 bases downstream of the longer transcript. It is not clear whether this is a second transcription start site or a processed form of the larger transcript. It is interesting to note that two 6-base palindromes are located immediately downstream of the 5'-end of the shorter transcript and a 6-base inverted repeat lies just upstream of the 5'-end of the larger transcript (Fig. 3 lower panel).


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)

Primer extension analysis of sagA/pel from wild type, sagA/pel mutant and kidney-recovered strains. Bacteria were grown overnight at 37°C with 10% CO2 in THY broth. RNA was extracted and sagA/pel was detected using a primer extension assay and sagA/pel-specific primers. Equal amounts of RNA were loaded in each lane. Lane 1, RNA isolated from a wild type CS101 strain. Lane 2, RNA from an isogenic sagA/pel::Tn917 mutant. Lanes 3 and 4, RNA isolated from 2 different KR variants of the sagA/pel mutant. The larger sagA/pel transcript is indicated as t1, the smaller as t2. The DNA sequence in the lower part of the figure is from the region around the sagA/pel promoter. These represent bases 598514–598593 in the S. pyogenes genome [43]. The bases in italics are the putative -10 region of the sagA/pel promoter. The overlined regions are 16, 6 and 6 base inverted repeats. The bold letters are the 5'-ends of the t1 and t2 transcripts as determined by primer extension. The position of the Tn917 insertion is indicated by the ^ symbol. Note that the site of insertion is slightly different from what was previously reported [4]. The t2 transcript was not detected in the KR variants even at longer exposures.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC64569&req=5

Figure 3: Primer extension analysis of sagA/pel from wild type, sagA/pel mutant and kidney-recovered strains. Bacteria were grown overnight at 37°C with 10% CO2 in THY broth. RNA was extracted and sagA/pel was detected using a primer extension assay and sagA/pel-specific primers. Equal amounts of RNA were loaded in each lane. Lane 1, RNA isolated from a wild type CS101 strain. Lane 2, RNA from an isogenic sagA/pel::Tn917 mutant. Lanes 3 and 4, RNA isolated from 2 different KR variants of the sagA/pel mutant. The larger sagA/pel transcript is indicated as t1, the smaller as t2. The DNA sequence in the lower part of the figure is from the region around the sagA/pel promoter. These represent bases 598514–598593 in the S. pyogenes genome [43]. The bases in italics are the putative -10 region of the sagA/pel promoter. The overlined regions are 16, 6 and 6 base inverted repeats. The bold letters are the 5'-ends of the t1 and t2 transcripts as determined by primer extension. The position of the Tn917 insertion is indicated by the ^ symbol. Note that the site of insertion is slightly different from what was previously reported [4]. The t2 transcript was not detected in the KR variants even at longer exposures.
Mentions: Primer extension analysis of the wild type and kidney-recovered variants demonstrated that the sagA/pel message expressed in the kidney-recovered variant had an identical transcription start site to the 500 base message present in the wild type strain (Fig. 3). The second transcript, present only in the RNA isolated from the wild type isolate (Fig. 3, lane 1), started 35 bases downstream of the longer transcript. It is not clear whether this is a second transcription start site or a processed form of the larger transcript. It is interesting to note that two 6-base palindromes are located immediately downstream of the 5'-end of the shorter transcript and a 6-base inverted repeat lies just upstream of the 5'-end of the larger transcript (Fig. 3 lower panel).

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