Agr‐independent regulation of fibronectin‐binding protein(s) by the regulatory locus sar in Staphylococcus aureus

Abstract

Fibronectin‐binding proteins (FnBPs) are thought to be important for the attachment of Staphylococcus aureus during infection. The regulation of the genes fnbA and fnbB by the global regulatory loci sar and agr was examined using site‐specific regulatory mutants of S. aureus strain Newman. The results from binding assays using both aqueous and solid‐phase fibronectin as well as ligand blotting with biotinylated fibronectin showed that the expression of FnBPA is enhanced in the agr mutant but inhibited in the sar mutant and the sar–agr double mutant. The same regulatory pattern was observed in Northern blot analysis using fnbA‐specific probes. The introduction of sar on a multicopy plasmid increased the already enhanced fnbA transcription of the agr mutant. FnBPB was not detectable by ligand blotting and the fnbB promoter activity in promoter fusion assays was not affected by either sar or agr. The sequence encompassing ORF3 located upstream of sarA was found to be essential for the activation of fnbA transcription. We hypothesize that this sequence may modulate SarA expression and/or activity on the post‐transcriptional level. Gel shift assays demonstrated that SarA binds to the fnbA promoter fragments, probably as a dimer. DNase I footprinting assays with SarA revealed a protected area of 102 bp upstream of fnbA.

SarA ofStaphylococcus aureusBinds to thesarAPromoter To Regulate Gene Expression

“…The SarA binding site on the sarA P3 promoter lies immediately upstream of the Ϫ35 promoter box. This 26-bp binding site shares homology with the 26-bp SarA binding site that we have mapped for the agr, hla, and other target promoters (8,23). Additionally, this site also contains a 7-bp palindrome separated by a 6-bp sequence within the SarA protected region.…”

Influence of a Functional sigB Operon on the Global Regulators sar and agr in Staphylococcus aureus

“…Fibronectin binding activity. Since sar and agr are known to influence the ability of S. aureus to bind to fibronectin (55,60), the effects of RsbU on the fibronectin binding capacity were determined with strain BB255 and its derivatives ( Table 2). Strain GP268, carrying an intact sigB operon, showed a more than 100-fold-lower fibronectin-clumping titer than its rsbUdefective parent, BB255, irrespective of the growth phase, while no difference was apparent between BB255 and its ⌬rsbUVWsigB mutant.…”

“…SarA, the major functional protein encoded by the sar locus, is generally believed to be required for the activation of expression of the agr locus (20,21,46) and influences the regulation of several virulence factors independently from agr (7,11,14,19,41,60). SarA is essentially involved in the capacity of S. aureus to survive inside of polymorphonuclear neutrophils (33), and the ability of S. aureus to enter mammalian cells and induce apoptosis is supposed to be dependent on factors regulated by sar and agr (58).…”

The N-terminal A Domain of Fibronectin-binding Proteins A and B Promotes Adhesion of Staphylococcus aureus to Elastin

“…They are encoded by two closely linked genes, fnbA and fnbB, which are tandemly arrayed but transcribed separately (19,22). Transcription of the fnb genes is restricted to the early exponential phase of growth (19) under the control of Agr and SarA (23,24). Fibronectin binding activity is lost as cultures enter stationary phase due to degradation of both FnBPA and FnBPB by V8 serine protease (25) and masking of the proteins by capsular polysaccharide (26).…”