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

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 β-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

Streptococcus pyogenes Infection in Mouse Skin Leads to a Time-Dependent Up-Regulation of Protein H Expression

Streptococcus pyogenes protein H (sph) is an immunoglobulin-binding protein present in the Mga regulon of certain M1 serotype isolates. Although sph is present in many strains, it is frequently not expressed. In this paper we show that protein H was highly expressed after bacteria were injected into the skin of mice and were recovered from the blood, kidney, or spleen at various times postinfection. The percentage of protein H-positive colonies increased with time, reaching 100% in the spleen and kidney within 24 to 72 h postinfection. The up-regulation of sph expression was also observed in a mga mutant

The Hag Protein of Moraxella catarrhalis Strain O35E Is Associated with Adherence to Human Lung and Middle Ear Cells

Previous studies have demonstrated that the Moraxella catarrhalis surface antigen UspA1 is an adhesin for Chang human conjunctival cells. The present report demonstrates that lack of UspA1 expression does not affect the adherence of strain O35E to A549 human lung cells or primary cultures of human middle ear epithelial (HMEE) cells. These results imply that another molecule mediates the adherence of M. catarrhalis to these two cell lines. To identify this adhesin, strain O35E was mutagenized with a transposon and 1,000 mutants were screened in a microcolony formation assay using A549 cells. Nine independent isolates exhibited an 8- to 19-fold reduction in adherence and contained a transposon in the same locus. Nucleotide sequence data and PCR analysis indicated that the transposons were inserted in different locations in the gene encoding the surface protein Hag. Quantitative assays using one representative transposon mutant, O35E.TN2, showed considerably decreased binding to A549 as well as HMEE cells. However, this mutant adhered at wild-type levels to Chang conjunctival cells. These findings suggest that the M. catarrhalis Hag protein is an adhesin for cell lines derived from human lung and middle ear tissues

Activation of the gab Operon in an RpoS-Dependent Manner by Mutations That Truncate the Inner Core of Lipopolysaccharide in Escherichia coli

The gab operon (gabDTPC) in Escherichia coli functions in the conversion of γ-aminobutyrate to succinate. One component of gab operon regulation involves the RpoS sigma factor, which mediates activation at high cell density. Transposon mutagenesis was used to identify new genes that regulate gab operon expression in rich media. A Tn5tmp insertion in the hldD (formerly rfaD) gene increased gabT::lacZ expression 12-fold. The hldD gene product, an ADP-l-glycerol-d-mannoheptose-6-epimerase, catalyzes the conversion of ADP-d-glycerol-d-mannoheptose to ADP-l-glycerol-d-mannoheptose, a precursor for the synthesis of inner-core lipopolysaccharide (LPS). Defined mutations in hldE, required for heptose synthesis, and waaF, required for the addition of the second heptose to the inner core, also resulted in high-level gabT::lacZ expression. The hldD, hldE, and waaF mutants exhibited a mucoid colony phenotype due to production of a colanic acid capsule. However, in the hldD::cat background, the high-level expression of gabT::lacZ was independent of the regulatory components for colanic acid synthesis (rcsA, rcsB, and rcsC) and also independent of manC (cpsB), a structural gene for colanic acid synthesis. Activation of gabT::lacZ in the hldD::cat background was dependent on the RpoS sigma factor. The hldD::cat mutation resulted in a sixfold increase in the levels of a translational RpoS-LacZ fusion and had a marginal effect on a transcriptional fusion. This study reveals a stress-induced pathway, mediated by loss of the LPS inner core, that increases RpoS translation and gab operon expression in E. coli

Moraxella catarrhalis Coaggregates with Streptococcus pyogenes and Modulates Interactions of S. pyogenes with Human Epithelial Cells

The pathogens Streptococcus pyogenes and Moraxella catarrhalis colonize overlapping regions of the human nasopharynx. We have found that M. catarrhalis can dramatically increase S. pyogenes adherence to human epithelial cells and that species-specific coaggregation of these bacteria correlates with this enhanced adherence

Identification of pel, a Streptococcus pyogenes Locus That Affects both Surface and Secreted Proteins

A Tn917 insertion mutant of an M49 serotype, opacity factor-positive Streptococcus pyogenes, was isolated. It had the following phenotypes: decreased β-hemolysis mediated by streptolysin S, reduction in the activity of a secreted cysteine protease and streptokinase, and an altered immunoglobulin and fibrinogen-binding phenotype. The site of insertion of Tn917 into the chromosome and the surrounding sequence, the pel region (pleiotropic effect locus), was determined. Phage A25 transduction confirmed that the pleiotropic changes in phenotype could be cotransduced with Tn917. The pel region was cloned and sequenced, and the transposon was found to be inserted upstream of a single open reading frame which led to a failure to transcribe a 500-base mRNA. The loss of this transcript decreased the transcription of emm and speB genes and reduced the secretion of streptokinase. Enhanced Pel expression from a nisin-inducible plasmid resulted in increased message levels for emm in a wild-type organism. Characterization of the pel mutant provides evidence for the coordinated regulation of secreted and surface proteins and suggests the existence of a new global regulatory factor in S. pyogenes