Changes in relative transcript amounts caused by hydrogen sulfide treatment, calprotectin treatment, and deletion of CstR in Staphylococcus Aureus

The Staphylococcus aureus strain Newman uses the dithiol-containing repressor CstR to sense sulfide stress via reactive sulfur species (RSS), allowing transcription of a mitochondrial-like sulfide oxidation system, the core of which is genetically linked to methicillin resistance determinants in MRSA strains. The cytoplasm maintains an excess of reduced relative to oxidized low molecular weight (LMW) thiols that are protective against oxidative stress and transition metal (Zn, Cd and Cu) toxicity, buffering these ions to low “free” concentrations via formation of coordination complexes. We hypothesize that unregulated H2S perturbs the LMW thiol pool by generating RSS; these, in turn, react with CstR cysteines (C31, C60), induce cst derepression, and are cleared by cst-encoded enzymes. These results show that sulfide stress induces the cst operon and a zinc starvation response, represses cysteine biosynthesis, and generally represses genes that are induced by acute oxidative stress; in addition, strong repression of the expression of staphylococcal virulence factors is observed in the ?cstR strain. Overall design: RNA for the sulfide experiment was prepared from cultures of wild-type and DcstR strains of Staphylococcus aureus strain Newman grown exponentially in HHWM media supplemented with thiosulfate and methionine at 37°C to OD600=0.2. At this point Na2S was added to treated cultures at a ?nal concentration of 0.2 mM for a 10 min exposure, then samples were collected from all conditions. For the calprotectin experiment, bacteria were grown in the presence and absence of 960mg/mL calprotectin in growth media containing 38% TSB/ 72% calprotectin buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 3 mM CaCl2, 10 mM ß-mercaptoethanol), supplemented with 1 µM Zn and 1 µM Mn Three independent biological replicates of RNA were prepared for each strain or condition. cDNA libraries were sequenced and the sequencing results were used as the data base for differential expression analysis.