Alterations in amino acid metabolism during growth by Staphylococcus aureus following exposure to H2O2 - A multifactorial approach

Abstract

Temperature and pH are known to vary in a wound site due to the immune response and subsequent healing processes. This study used a multifactorial design to examine the cellular responses of Staphylococcus aureus to hydrogen peroxide (0-100 mM) when bacteria were grown in temperatures of 37 ± 2 °C and pH 7 ± 1, conditions potentially encountered in wound sites. A centroid sample was included in the design which represented the mid-point values of all three environmental parameters (37 °C, pH 7, 50 mM H₂O₂). Cytoplasmic extracts and corresponding medium supernatants were analysed for amino acid composition by gas chromatography. Exposures of S. aureus to H₂O₂ during the inoculation process resulted in extended lag phases lasting well after the peroxide had been neutralised by the bacterium's antioxidant systems, after which the bacteria eventually resumed growth at equivalent rates to the controls. Even though the subsequent growth rates appeared normal, the cells exhibited a variant metabolic regime at the mid-exponential phase of growth as a result of the initial exposure to peroxide. The alterations in metabolism were reflected by the differential amino acid profiles measured in the cytoplasmic extracts (P < 0.0001). The data indicated that the metabolic responses to H₂O₂ challenge were uniquely different depending on the variations of temperature and pH. The uptake patterns of amino acids from the media also altered depending on prevailing environmental conditions. From these results, it was proposed that a specific reproducible homeostasis could be induced under a specific set of defined environmental conditions. It was also evident that early toxic insults on the bacterial culture could have lasting impacts on cellular homeostasis after successive generations, even after the offending chemical had been removed and initial cell integrity restored. It was concluded that metabolic homeostasis would be continually adjusting and responding to changing environmental conditions to deploy defensive proteins as well as optimising processes for survival. The powerful ability to continually and rapidly adapt to the environment may represent the key feature supporting the virulence of S. aureus as an opportunistic pathogen invading the wound site.

Keywords: Metabolism; Microbiology.

Fig. 1

The cuboid experiment design whereby each corner of the cube represents a specific variable combination that was tested. The centre sphere (B) represents the centroid experiment where all tested variables directly intersect. The reference control (A) exhibits optimal conditions for S. aureus growth. The other experimental parameters are as follows: 39 °C, pH 8 and 100 mM H₂O₂ (C), 39 °C, pH 8 and 0 mM H₂O₂ (D), 35 °C, pH 8 and 100 mM H₂O₂ (E), 35 °C, pH 8 and 0 mM H₂O₂ (F), 39 °C, pH 6 and 100 mM H₂O₂ (G), 39 °C, pH 6 and 0 mM H₂O₂ (H), 35 °C, pH 6 and 100 mM H₂O₂ (I), 35 °C, pH 6 and 0 mM H₂O₂ (J).

Fig. 2

Dose response curve for the action of H₂O₂ on delaying the growth of S. aureus to reach a target absorbance (A₆₀₀) of 0.23, representing the early exponential phase of growth (n = 3, mean ± SE).

Fig. 3

Comparison of the cytoplasmic amino acids of S. aureus cells grown under optimal control conditions or in the presence of 50 mM H₂O₂ which were harvested at the mid-exponential phase of growth (n = 3, mean ± SE). * = P < 0.05.

Fig. 4

3D canonical plot from the discriminant function analyses applied to the cytoplasmic amino acid profiles from each of the 10 experimental groups. Each case replicate has been colour coded and labelled for the experiments to show grouping.

Fig. 5

4D response contour plots which show the extrapolated impact on cytoplasmic concentrations of proline (PRO top), aspartic acid (ASP middle) and glutamic acid (GLU bottom) by simultaneously adjusting pH and H₂O₂ at 35 °C, 37 °C and 39 °C. The scale for concentration is given in nmoles mg⁻¹ of dried cell mass.

Fig. 6

2D canonical plot from the discriminant function analyses applied to the supernatant amino acid profiles from each of the 10 experimental groups. Each case replicate has been colour coded and labelled for the experiments to show grouping. The contrast between the amino acid uptake profiles for cells grown at either pH 6 or pH 8 at 35 °C without peroxide has been highlighted.