Characterizing the effects of inorganic acid and alkaline shock on the Staphylococcus aureus transcriptome and messenger RNA turnover

Abstract

Staphylococcus aureus pathogenesis can be attributed partially to its ability to adapt to otherwise deleterious host-associated stresses. Here, Affymetrix GeneChips® were used to examine the S. aureus responses to inorganic acid and alkaline shock and to assess whether stress-dependent changes in mRNA turnover are likely to facilitate the organism's ability to tolerate a pH challenge. The results indicate that S. aureus adapts to pH shock by eliciting responses expected of cells coping with pH alteration, including neutralizing cellular pH, DNA repair, amino acid biosynthesis, and virulence factor expression. Further, the S. aureus response to alkaline conditions is strikingly similar to that of stringent response-induced cells. Indeed, we show that alkaline shock stimulates the accumulation of the stringent response activator (p)ppGpp. The results also revealed that pH shock significantly alters the mRNA properties of the cell. A comparison of the mRNA degradation properties of transcripts whose titers either increased or decreased in response to a sudden pH change revealed that alterations in mRNA degradation may, in part, account for the changes in the mRNA levels of factors predicted to mediate pH tolerance. A set of small stable RNA molecules were induced in response to acid- or alkaline-shock conditions and may mediate adaptation to pH stress.

Figure 1. Transcriptional Response to Acid- or Alkaline-shock conditions

Shown are the number of genes predicted to participate in the indicated biological process (X-axis) that exhibit increased (Red; Induced) or decreased (Blue; Repressed) transcript titers in response to acid-shock (Panel A) or alkaline-shock conditions (Panel B).

Figure 2. Growth Characteristics of S. aureus in Neutral, Acidic and Alkaline Medium

Plotted are the numbers of viable colony forming units (CFUs) per milliliter of exponential phase S. aureus following growth for 6 hours after adjusting the medium to pH of 7.4 (diamonds), pH 4.0 (squares) or pH 10.0 (circles). Panel A shows growth properties of wild type S. aureus UAMS-1 cells. Panel B shows growth properties of isogenic capsule mutant cells. Standard deviation is indicated.

Figure 3. Capsule Expression

Panel A shows an immunoblot for capsule detection formed by S. aureus strain UAMS-1 following growth at pH 7.4 (Mock) or induction of the alkaline-shock (pH 10), or acid-shock (pH 4) response. The dilution factor of starting cell-lysate suspensions is shown. Reverse-transcription mediated PCR results of various amounts (0–25 ng) of RNA isolated from wild type (Panel B) or isogenic ΔsigB (Panel C) cells grown at pH 7.4 or following alkaline-shock induction.

Figure 4. mRNA Turnover Properties of Acid- or Alkaline-Shocked Exponential Phase S. aureus

Graphed are the total numbers of S. aureus exponential phase mRNA species (% RNA molecules) exhibiting a half-life of less than 2.5 min (black bar), 2.5–5.0 min (dark-grey), 5.0–15.0 min (white), 15.0–30.0 min (hashed), or greater than 30 min (light-grey) during growth at pH 7.4 or following alkaline-shock (pH 10.0) or acid-shock (pH 4.0) induction.

Figure 5. Comparison of the mRNA Degradation Properties of Acid- or Alkaline-Shock Regulated mRNA Species

Graphed are comparisons of the half-life measurements of mRNA species whose transcript titers increase in response to acid-shock (Panel A) or alkaline-shock (Panel B) conditions. The mRNA turnover properties of these transcripts is shown for unstressed or the indicated stress. Panels C and D show comparisons of the half-life measurements of transcripts that decrease in titer in response to acid- or alkaline- shock conditions, respectively.