Transcription profiling-based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci

Abstract

The advent of transcription profiling technologies has provided researchers with an unprecedented ability to study biological processes. Accordingly, a custom-made Affymetrix GeneChip, constituting >86% of the Staphylococcus aureus genome, was used to identify open reading frames that are regulated by agr and/or SarA, the two best-studied regulators of the organism's virulence response. RNA extracted from wild-type cells and agr, sarA, and agr sarA mutant cells in the early-, mid-, and late-log and stationary phases of growth was analyzed. Open reading frames with transcription patterns expected of genes either up- or downregulated in an agr- and/or SarA-dependent manner were identified. Oligonucleotide microarray and Northern blot analyses confirmed that the transcription of several known virulence genes, including hla (alpha-toxin) and spa (protein A), is regulated by each effector and provided insights about the regulatory cascades involved in both alpha-hemolysin and protein A expression. Several putative virulence factors were also identified as regulated by agr and/or SarA. In addition, genes that are involved in several biological processes but which are difficult to reconcile as playing a direct role in the organism's pathogenesis also appeared to be regulated by each effector, suggesting that products of both the agr and the sarA locus are more-global transcription regulators than previously realized.

FIG. 1

Transcription profiles of known RNAIII and SarA-regulated genes. Transcript levels for wild-type (□), agr mutant (▴), sarA mutant (■), and agr sarA double-mutant (⧫) cells were measured during the early-, mid-, and late-log and stationary phases of growth (x axis). Data points were plotted as relative intensity values (y axis) (as described in Materials and Methods). (A) Average signal intensities for genes constituting RNAII transcripts (agrB, agrD, agrC, and agrA). (B) Signal intensities of RNAIII transcripts. (C) Profiles of alpha-toxin (hla) transcript titers. (Inset) agr and agr sarA mutant results. (D) Profiles of protein A (spa) transcript titers.

FIG. 2

Northern blot analysis of known agr-regulated genes. Shown are levels of RNAIII (A), hla (B), spa (C), and rRNA (D) transcripts during the early-, mid-, and late-log and stationary phases of growth of wild-type (RN27) or agr mutant (RN6911) cells. Signal intensities were determined by densitometry and normalized to rRNA signals. Normalized relative signal intensities are shown below each panel.