Identification of a novel two-component regulatory system that acts in global regulation of virulence factors of Staphylococcus aureus

Abstract

We have previously demonstrated that the presence of oxygen is necessary for the production of toxic shock syndrome toxin 1 (TSST-1) by Staphylococcus aureus in vitro. To investigate the mechanism by which oxygen might regulate toxin production, we identified homologs in S. aureus of the Bacillus subtilis resDE genes. The two-component regulatory system encoded by resDE, ResD-ResE, has been implicated in the global regulation of aerobic and anaerobic respiratory metabolism in B. subtilis. We have designated the S. aureus homologs srrAB (staphylococcal respiratory response). The effects of srrAB expression on expression of RNAIII (the effector molecule of the agr locus) and on production of TSST-1 (an exotoxin) and protein A (a surface-associated virulence factor) were investigated. Expression of RNAIII was inversely related to expression of srrAB. Disruption of srrB resulted in increased levels of RNAIII, while expression of srrAB in trans on a multicopy plasmid resulted in repression of RNAIII transcription, particularly in microaerobic conditions. Disruption of srrB resulted in decreased production of TSST-1 under microaerobic conditions and, to a lesser extent, under aerobic conditions as well. Overexpression of srrAB resulted in nearly complete repression of TSST-1 production in both microaerobic and aerobic conditions. Protein A production by the srrB mutant was upregulated in microaerobic conditions and decreased in aerobic conditions. Protein A production was restored to nearly wild-type levels by complementation of srrAB into the null mutant. These results indicate that the putative two-component system encoded by srrAB, SrrA-SrrB, acts in the global regulation of staphylococcal virulence factors, and may repress virulence factors under low-oxygen conditions. Furthermore, srrAB may provide a mechanistic link between respiratory metabolism, environmental signals, and regulation of virulence factors in S. aureus.

FIG. 1

Coding-strand DNA sequence of the 2.7-kb locus containing the srrAB operon. The putative response regulator (srrA) and histidine kinase (srrB) genes are shown with the translation products given below the nucleotide sequence. The −10 (Pribnow box) consensus sequence of the putative promoter is identified, as are the potential ribosome binding sites (RBSs). Inverted repeats predicted to form stem-loop structures that may function in transcription termination are identified with bolded arrows. Regions predicted to contain transmembrane domains are indicated by underlining of the corresponding amino acids. Boldface indicates highly conserved residues likely to mediate kinase activity and phosphoryl group transfer.

FIG. 2

(A) Disruption of srrAB by a single-crossover event between a fragment (XhoI-BglII) contained in pJMY2 and the corresponding region of srrAB in S. aureus RN4220. Recognition sites for restriction enzymes EcoRI and EcoRV are indicated. Transcripts detected by Northern blotting in the parental strain (MN3050) are indicated. Abbreviations: Em^r, erythromycin resistance cassette; tt, transcription terminator. (B) Southern hybridization of chromosomal digests (EcoRI and EcoRV) of the parental strain (lane 1) and srrB mutant (lane 2) with a probe specific to the XhoI-BglII fragment. Band sizes are indicated at the right.

FIG. 3

Construction of plasmids and strains used in this study for characterization of srrAB. The tstH gene was expressed in both parental and srrB mutant strains via electroporation of pJMY10; the srrAB and tstH genes were expressed in trans in the srrB mutant by electroporation of pJMY11. Abbreviations: Cm^r, chloramphenicol resistance cassette.

FIG. 4

Growth of S. aureus strains in TH broth incubated with shaking at 37°C in either anaerobic or aerobic conditions. OD₆₀₀ was used to determine growth of cultures. Stages of growth at which gene expression were assayed are indicated for the parental strain (MN3050).

FIG. 5

Northern hybridization of total RNA extracted from S. aureus strains at equivalent cell densities (as determined by OD₆₀₀). RNA was harvested from exponential (Exp)-, post exponential (Post-exp)-, and stationary (Stat)-phase cultures incubated in atmospheres containing 2 or 21% (vol/vol) oxygen balanced with nitrogen and 7% carbon dioxide. Equivalent amounts (2.9 μg) of total RNA were loaded onto each lane, processed as described in Materials and Methods, and assayed for expression of srrA, srrB, and RNAIII. Strains: a, MN3050 (parental strain); b, MN4010 (srrB mutant); c, MN4011 (srrB mutant expressing srrAB in trans). (A) Northern hybridizations for srrA, srrB, and RNAIII. Band sizes are indicated at the right. (B) Amount of RNAIII transcript as determined by densitometric analysis. Bars represent percentage of maximum wild-type RNAIII transcript (lane 13) detected.

FIG. 6

TSST-1 concentrations in cultures incubated for 24 h in microaerobic (1% [vol/vol] oxygen or aerobic (21% [vol/vol] oxygen) atmospheres balanced with nitrogen and 7% carbon dioxide. Data are means ± standard errors of the means of triplicate ELISA readings for each culture. Toxin production was measured in three independent sets of cultures with highly similar results.

FIG. 7

Expression of cell surface protein A in various growth stages of cultures incubated in microaerobic (1% [vol/vol] oxygen) or aerobic (21% [vol/vol] oxygen) atmospheres balanced with nitrogen and 7% carbon dioxide. Cells from cultures at equivalent stages of growth were harvested and processed as described in Materials and Methods. Cells exhibiting low forward and side-scatter profiles were gated for analysis to avoid detecting protein A expression on clusters of cells. Protein A production was measured in three independent sets of cultures with highly similar results. ∗, percentage of total events (50,000) detected by the flow cytometer that fall into the range of fluorescence, and thus protein A production, delineated by the marker.