Influence of iron and aeration on Staphylococcus aureus growth, metabolism, and transcription

Abstract

Staphylococcus aureus is a prominent nosocomial pathogen and a major cause of biomaterial-associated infections. The success of S. aureus as a pathogen is due in part to its ability to adapt to stressful environments. As an example, the transition from residing in the nares to residing in the blood or deeper tissues is accompanied by changes in the availability of nutrients and elements such as oxygen and iron. As such, nutrients, oxygen, and iron are important determinants of virulence factor synthesis in S. aureus. In addition to influencing virulence factor synthesis, oxygen and iron are critical cofactors in enzymatic and electron transfer reactions; thus, a change in iron or oxygen availability alters the bacterial metabolome. Changes in metabolism create intracellular signals that alter the activity of metabolite-responsive regulators such as CodY, RpiRc, and CcpA. To assess the extent of metabolomic changes associated with oxygen and iron limitation, S. aureus cells were cultivated in iron-limited medium and/or with decreasing aeration, and the metabolomes were examined by nuclear magnetic resonance (NMR) spectroscopy. As expected, oxygen and iron limitation dramatically decreased tricarboxylic acid (TCA) cycle activity, creating a metabolic block and significantly altering the metabolome. These changes were most prominent during post-exponential-phase growth, when TCA cycle activity was maximal. Importantly, many of the effects of iron limitation were obscured by aeration limitation. Aeration limitation not only obscured the metabolic effects of iron limitation but also overrode the transcription of iron-regulated genes. Finally, in contrast to previous speculation, we confirmed that acidification of the culture medium occurs independent of the availability of iron.

FIG 1

Cultivation of S. aureus strain SA564 under aeration- and/or iron-limiting conditions. (A) Growth curves. (B) Iron concentrations of uninoculated media and cultivation media harvested in the exponential and post-exponential growth phases. (C) Glucose utilization as a function of growth. (D) pH profiles. (E) Acetate accumulation and depletion in culture media. (F) Lactate accumulation and depletion in culture media. Data are representative of experiments performed at least twice. The data in panel B are presented as the means of 3 independent experiments done in triplicate, with error bars representing standard deviations. TSB, tryptic soy broth; DTSB, deferrated TSB. Flask-to-medium ratios were 10:1 (aerobic) and 10:8 (microaerobic/anaerobic).

FIG 2

Temporal and stress-related aconitase activity differences in S. aureus strain SA564 in the exponential (2 h) and post-exponential (6 h) growth phases. (A) Aconitase activity during iron-limited growth. (B) Aconitase activity with decreasing aeration flask-to-medium ratios of 10:8, 10:5, 10:2.5, and 10:1. (C) Addition of iron to DTSB medium restores aconitase activity.

FIG 3

Exponential- and post-exponential-growth-phase metabolomic changes associated with iron- and/or oxygen-limited cultivation. (A and B) PCA score plots of the exponential (A) and post-exponential (B) growth phases of iron- and/or oxygen-limited cultures of S. aureus strain SA564. The ellipses in the PCA score plots correspond to the 95% confidence limits from a normal distribution for each cluster. (C and D) Metabolic tree generated by using the PCA score plot data demonstrating the relationship of iron- and/or oxygen-limited cultures in the exponential growth phase (C) and post-exponential growth phase (D). The statistical significance of each node in the metabolic tree is indicated by a P value. (E and F) Shared and unique structure (SUS) plots generated from the PCA model of the exponential (E) and post-exponential (F) growth phases comparing the classes of DTSB at a 10:1 ratio and TSB at 10:8 to a common reference (TSB, 10:1). The red boxes highlight the chemical shift bins with intensity changes that are shared by both models. The blue boxes highlight the chemical shift bins with intensity changes that are negatively shared by both models (relative intensity changes are in the opposite direction). The brown boxes highlight the chemical shift bins with intensity changes unique to model 1 (TSB at 10:8 versus TSB at 10:1). The green boxes highlight the chemical shift bins with intensity changes unique to model 2 (DTSB at 10:1 versus TSB at 10:1). In the post-exponential growth phase, 73 bins were found to be present in the shared/inversely shared region of the plot, compared to 29 bins found in the same regions in the exponential phase. The flask-to-medium ratios were 10:1 (aerobic) and 10:8 (microaerobic/anaerobic).

FIG 4

Heat maps showing the changes in metabolites as a function of cultivation conditions and growth phase. Culture media were TSB (tryptic soy broth) and DTSB (deferrated TSB). Flask-to-medium ratios were 10:1 (aerobic) and 10:8 (microaerobic/anaerobic). Statistical significance (P < 0.05) is indicated by ** (both oxygen and iron limitation), * (only oxygen limitation), and + (only iron limitation).

FIG 5

Oxygen limitation influences transcription of iron-regulated genes in the exponential (2 h) and post-exponential (6 h) growth phases. Relative mRNA levels for acnA, sbnA, feoB, and sstC were determined by qRT-PCR. The data are the means and standard errors of the means of at least 2 biological replicates, each determined in duplicate. Culture media were TSB (tryptic soy broth) and DTSB (deferrated TSB). Flask-to-medium ratios were 10:1 (aerobic) and 10:8 (microaerobic/anaerobic).

FIG 6

Schematic representation of the central metabolic changes associated with iron or oxygen limitation. The black and blue arrows indicate exponential/post-exponential-growth-phase (EXP/PE) increases or decreases in intracellular concentrations of metabolites in strain SA564 cultivated in TSB with a 10:8 flask-to-medium ratio relative to a control culture grown in TSB with a flask-to-medium ratio of 10:1. The red and orange arrows indicate exponential/post-exponential-growth-phase increases or decreases in intracellular concentrations of metabolites in strain SA564 grown in DTSB with a flask-to-medium ratio of 10:1 relative to a control culture of S. aureus strain SA564 grown in TSB with a flask-to-medium ratio of 10:1. Statistical significance at the 95% confidence level (P < 0.05) is denoted by asterisks above the arrows. Glucose-1-P, glucose-1-phosphate; FBP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde-3-phosphate; 3PG, 3-phosphoglycerate; OAA, oxaloacetate; 2-KG, α-ketoglutarate.