Expression Of sarA Research Articles

SigmaB modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4.

The accessory sigma factor sigmaB controls a general stress response that is thought to be important for Staphylococcus aureus survival and may contribute to virulence. The strain of choice for genetic studies, 8325-4, carries a small deletion in rsbU, which encodes a positive regulator of sigmaB activity. Consequently, to enable the role of sigmaB in virulence to be addressed, we constructed an rsbU(+) derivative, SH1000, using a method that does not leave behind an antibiotic resistance marker. The phenotypic properties of SH1000 (8325-4 rsbU(+)) were characterized and compared to those of 8325-4, the rsbU mutant, parent strain. A recognition site for sigmaB was located in the promoter region of katA, the gene encoding the sole catalase of S. aureus, by primer extension analysis. However, catalase expression and activity were similar in SH1000 (8325-4 rsbU(+)), suggesting that this promoter may have a minor role in catalase expression under normal conditions. Restoration of sigmaB activity in SH1000 (8325-4 rsbU(+)) resulted in a marked decrease in the levels of the exoproteins SspA and Hla, and this is likely to be mediated by reduced expression of agr in this strain. By using Western blotting and a sarA-lacZ reporter assay, the levels of SarA were found to be similar in strains 8325-4 and SH1000 (8325-4 rsbU(+)) and sigB mutant derivatives of these strains. This finding contrasts with previous reports that suggested that SarA expression levels are altered when they are measured transcriptionally. Inactivation of sarA in each of these strains resulted in an expected decrease in agr expression; however, the relative level of agr in SH1000 (8325-4 rsbU(+)) remained less than the relative levels in 8325-4 and the sigB mutant derivatives. We suggest that SarA is not likely to be the effector in the overall sigmaB-mediated effect on agr expression.

SarA represses agr operon expression in a purified in vitro Staphylococcus aureus transcription system.

Mutation and genetic complementation studies suggested that two chromosomal loci, agr and sar, are involved in the upregulation of several exotoxin genes and the downregulation of a number of surface protein genes in a growth phase-dependent manner in Staphylococcus aureus. We purified recombinant T7-tagged SarA from Escherichia coli and determined its effect on transcription from several S. aureus promoters by using purified RNA polymerase reconstituted with either sigma(A) or sigma(B) from S. aureus. Of the seven sigma(A)-dependent promoters that we tested, SarA repressed transcription from agrP2, agrP3, cna, sarP1, and sea promoters and did not affect sec and znt promoters. Furthermore, SarA had no effect on transcription from the sigma(B)-dependent sarP3 promoter. In vitro experimental data presented in this report suggest that SarA expression is autoregulated.

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

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 level is a determinant of agr activation in Staphylococcus aureus.

The control of virulence determinant expression in Staphylococcus aureus is a complex process involving global regulatory loci such as sar and agr. The sar locus consists of a 372 bp sarA open reading frame (ORF) preceded by a triple promoter region interspersed with two putative smaller ORFs (ORF3 and ORF4). The triple promoter system yields three overlapping sar transcripts (sarA, sarC and sarB of 0.56, 0.8 and 1.2 kb respectively). We have recently shown that the SarA protein binds to the agr promoter region to stimulate the transcription of RNAII and RNAIII, two major transcripts encoded within the agr locus. To assess the role of the region upstream of sarA in agr expression, we evaluated the contribution of ORF3 and ORF4 to SarA protein expression and to agr activation by introducing nonsense mutations into the respective ORFs. Northern analysis of sar mutant clones containing these mutations carried on a shuttle plasmid revealed that all three sar-related transcripts are present. Using anti-SarA monoclonal antibodies with defined epitopes in a competitive ELISA to determine the SarA protein level, we found that the introduction of a stop codon in ORF3 on a shuttle plasmid carrying the intact sar locus in a sar mutant led to a significant decrease in SarA protein level compared with the non-mutated control. The effect of a nonsense mutation in ORF4 on SarA levels is much less. Likewise, an analogous sar mutant clone with a deletion in ORF3 also displayed a lower SarA level than its intact counterpart. The reduction in SarA expression correlated with a lower level of agr activation in the corresponding sar mutant clone. These data suggest that ORF3, and to a lesser degree ORF4, may affect agr expression by modulating SarA protein expression.