Inhibition of Rho Activity Increases Expression of SaeRS-Dependent Virulence Factor Genes in Staphylococcus aureus, Showing a Link between Transcription Termination, Antibiotic Action, and Virulence

Abstract

Staphylococcus aureus causes various diseases ranging from skin and soft tissue infections to life-threatening infections. Adaptation to the different host niches is controlled by a complex network of transcriptional regulators. Global profiling of condition-dependent transcription revealed adaptation of S. aureus HG001 at the levels of transcription initiation and termination. In particular, deletion of the gene encoding the Rho transcription termination factor triggered a remarkable overall increase in antisense transcription and gene expression changes attributable to indirect regulatory effects. The goal of the present study was a detailed comparative analysis of S. aureus HG001 and its isogenic rho deletion mutant. Proteome analysis revealed significant differences in cellular and extracellular protein profiles, most notably increased amounts of the proteins belonging to the SaeR regulon in the Rho-deficient strain. The SaeRS two-component system acts as a major regulator of virulence gene expression in staphylococci. Higher levels of SaeRS-dependent virulence factors such as adhesins, toxins, and immune evasion proteins in the rho mutant resulted in higher virulence in a murine bacteremia model, which was alleviated in a rho complemented strain. Inhibition of Rho activity by bicyclomycin, a specific inhibitor of Rho activity, also induced the expression of SaeRS-dependent genes, at both the mRNA and protein levels, to the same extent as observed in the rho mutant. Taken together, these findings indicate that activation of the Sae system in the absence of Rho is directly linked to Rho's transcription termination activity and establish a new link between antibiotic action and virulence gene expression in S. aureusIMPORTANCE The major human pathogen Staphylococcus aureus is a widespread commensal bacterium but also the most common cause of nosocomial infections. It adapts to the different host niches through a complex gene regulatory network. We show here that the Rho transcription termination factor, which represses pervasive antisense transcription in various bacteria, including S. aureus, plays a role in controlling SaeRS-dependent virulence gene expression. A Rho-deficient strain produces larger amounts of secreted virulence factors in vitro and shows increased virulence in mice. We also show that treatment of S. aureus with the antibiotic bicyclomycin, which inhibits Rho activity and is effective against Gram-negative bacteria, induces the same changes in the proteome as observed in the Rho-deficient strain. Our results reveal for the first time a link between transcription termination and virulence regulation in S. aureus, which implies a novel mechanism by which an antibiotic can modulate the expression of virulence factors.

Keywords: SaeRS TCS; Staphylococcus aureus; antisense transcription; bicyclomycin; proteome; transcription termination.

FIG 1

Heat map of protein abundance differences between the Δrho mutant and the parental strain HG001. By mass spectrometry-based proteome analysis, 1,347 proteins were quantified from S. aureus cells and 999 proteins were quantified from the supernatant of HG001 and the Δrho mutant grown in RPMI and TSB medium. (A) Five hundred forty-one of these proteins could be assigned to 30 different transcription factor regulons when including regulons with at least two quantified proteins. Log₂ ratios (rho mutant versus HG001) of mean protein intensities from three independent experiments are displayed. Significantly changed proteins are marked with a cross. Regulons were extracted from RegPrecise (51) and AureoWiki (52). (B) SaeR-dependent proteins exhibited significantly higher levels in the rho mutant.

FIG 2

Survival analysis of mice infected with the parental strain HG001, a Δrho mutant, or the rho complemented strains. Six- to 8-week-old female mice were infected intravenously with 8 × 10⁷ CFU of HG001 (n = 8), the Δrho mutant (n = 8), and the complemented strain (n = 8). Body weight loss and disease activity index were monitored until day 5 postinfection. Mice with a weight loss of more than 20% of the starting body weight were sacrificed and recorded as dead. The survival rate was significantly different between the parental strain and the Δrho mutant (P = 0.0121) and between the Δrho mutant and the complemented strain (P = 0.0088). Differences were tested for statistical significance by the Gehan-Breslow-Wilcoxon test.

FIG 3

Northern blot analysis of antisense transcription in the absence of active Rho. (A) Transcript levels of asRNA S597 were analyzed by Northern blotting using RNA samples (7 µg per lane) of S. aureus HG001 treated with BCM (40 or 80 µg/ml) and the Δrho mutant. Upon treatment with BCM and in the mutant, a specific band with a size of about 2.3 kb as well as larger transcripts accumulating in the size range of the 23S rRNA were detected. These S597-specific bands were already observed in a previous analysis of Δrho mutant samples (47). Numbers at left are sizes in bases. For the chromosomal location of the S597 gene, see the S. aureus Expression Data Browser at http://genome.jouy.inra.fr/cgi-bin/aeb/viewdetail.py?id=S597_1363050_1363769_1. (B) The methylene blue-stained rRNA bands as control for equal loading of RNA.

FIG 4

Transcriptome analysis of S. aureus treated with bicyclomycin. Box plots of relative expression levels showing 21 asRNAs of S. aureus identified by Mäder et al. (47) (A) and 39 genes belonging to the SaeR regulon (B). Samples from the parental strain (HG001) and the mutant (Δrho) were collected in early exponential growth phase at an OD₆₀₀ of 0.2 (early exp) and 70 min later (exp). At an OD₆₀₀ of 0.2, HG001 cells were treated with 40 or 80 µg/ml BCM, resulting in two additional samples after 70 min (BCM treatment). Expression of antisense RNAs and SaeRS-dependent virulence factor genes increased in the parental strain after treatment with BCM in a dose-dependent manner. Log₂-transformed relative expression levels are shown for two independent experiments.

FIG 5

Heat map of bicyclomycin effects on genes and secreted proteins belonging to the SaeR regulon. (A) Expression levels of SaeR-dependent genes were determined by microarray analysis. (B) The corresponding proteins were quantified by mass spectrometry-based analysis after enrichment of proteins from culture supernatants of S. aureus HG001 treated with BCM and the Δrho mutant. HG001 samples were collected in exponential growth phase 70 min after addition of BCM (0, 40, or 80 µg/ml). Untreated mutant samples were collected at the same time point. Log₂ ratios (BCM-treated HG001 or rho mutant versus untreated HG001) of mean expression levels (A) and mean protein intensities (B) from two independent experiments are shown. Significantly changed proteins are marked with a cross.