Regulation of putative extra cellular virulence factors in Staphylococcus epidermidis by the alternative sigma factor sigmaB
Abstract number: o370
Kneschke J.C., Jaeger S., Mack D., Knobloch J.K.
Despite the regulation of PIA synthesis the sigmaB regulon of S. epidermidis is still almost uncharacterized. To investigate the influence of sigmaB activity on secreted proteins we investigated culture supernatants of sigmaB mutants (1457sigB, 8400sigB, 1057sigB) generated in clinical isolates S. epidermidis 1457, 8400, and 1057.
The proteins of culture supernatants were compared using SDS-PAGE between wild type and mutant strains. Proteins identified to be differentially expressed were further characterised using MALDI-TOF mass spectroscopy. Transcription of the genes identified to be differentially expressed was analysed using quantitative RT-PCR.
Two proteins identified as serine protease (SE1543) and cystein protease (SE0184) were significantly upregulated in sigmaB mutants compared to the respective wild types. Transcriptional analysis revealed that the respective genes were repressed in a sigmaB dependent manner. Phenotypic analysis revealed a significantly increased protease activity in all investigated sigmaB mutants. Thereby, strain 1057 displayed the highest proteolytic activity, which was further induced in 1057sigB. Mutants with inactivation of the antisigmafactor RsbW and still intact sigB gene displayed a reduced protease activity compared to the respective wild type strains, corroborating the negative regulation of extra cellular proteases by sigmaB. One protein identified as the (pre) proenzym of the lipase GehD was upregulated in 1457sigB and 8400sigB, whereas in 1057sigB this protein was significantly reduced. However, transcriptional analysis of the gehD gene revealed an increased transcription in all sigmaB mutants. Zymographic and quantitative enzymatic analyses revealed also a differential behaviour of the investigated wild types and mutants. 1457sigB and 8400sigB displayed an increased lipolytic activity, which seem to be caused by increased processing of the proenzym to the fully active lipase. In 1057sigB a reduced lipolytic activity was detected, which resulted from a rapid proteolytic degradation of both, the proenzyme and the active enzyme.
The presented data indicate that sigmaB is a negative transcriptional regulator of proteases SE1543 and SE0184 leading to an increased proteolytic activity of sigmaB mutants. Additionally, sigmaB acts as a negative regulator of lipase GehD. However, the lipolytic activity of individual strains is also determined by their proteolytic activity.
|Session name:||XXIst ISTH Congress|
|Back to top|