Abstract
Staphylococcus aureus alpha-toxin (Hla) is a potent pore-forming cytotoxin that plays an important role in the pathogenesis of S. aureus infections, including pneumonia. The impact of Hla on the dynamics of the metabolome in eukaryotic host cells has not been investigated comprehensively. Using 1H-NMR, GC-MS and HPLC-MS, we quantified the concentrations of 51 intracellular metabolites and assessed alterations in the amount of 25 extracellular metabolites in the two human bronchial epithelial cell lines S9 and 16HBE14o− under standard culture conditions and after treatment with sub-lethal amounts (2 µg/ml) of recombinant Hla (rHla) in a time-dependent manner. Treatment of cells with rHla caused substantial decreases in the concentrations of intracellular metabolites from different metabolic pathways in both cell lines, including ATP and amino acids. Concomitant increases in the extracellular concentrations were detected for various intracellular compounds, including nucleotides, glutathione disulfide and NAD+. Our results indicate that rHla has a major impact on the metabolome of eukaryotic cells as a consequence of direct rHla-mediated alterations in plasma membrane permeability or indirect effects mediated by cellular signalling. However, cell-specific changes also were observed. Glucose consumption and lactate production rates suggest that the glycolytic activity of S9 cells, but not of 16HBE14o− cells, is increased in response to rHla. This could contribute to the observed higher level of resistance of S9 cells against rHla-induced membrane damage.
Highlights
As a facultative pathogenic bacterium, Staphylococcus aureus is able to compromise the human respiratory tract [1]
The survival rates decreased by more than 20% after 360 min. These results indicate that 2 mg/mL of recombinant Hla (rHla) has marked cytotoxic effects on 16HBE14o2 cells but no or only minor effects on S9 cells during this period of time
Impact of rHla on nucleotide metabolism we investigated the effects of rHla on the balance of the nucleotide pool in the two bronchial epithelial cell lines using an HPLC-MS based approach
Summary
As a facultative pathogenic bacterium, Staphylococcus aureus is able to compromise the human respiratory tract [1]. Upon assembly of a heptameric pre-pore, Hla integrates into the membrane of host cells forming a transmembrane b-barrel pore with an inner diameter of 2.5 nm [9,10]. In different cell types, including keratinocytes, lymphocytes and fibroblasts Hla-mediated poreformation results in a transmembrane flux of monovalent ions and causes a drop in cellular ATP [9,11,12,13]. High amounts of Hla trigger nonspecific integration of Hla molecules into the cell membrane which may result in necrotic cell lysis [13]. Intracellular calcium levels are increased upon treatment of cells with Hla due to influx of Ca2+ ions through the plasma membrane [15,16], but it is still unclear whether this occurs through the Hla-pore or indirectly. A fixable dead cell-stain (Invitrogen; approximately 1,000 g/mol) applied to S9 cells after two hours preincubation with 0.2 mg/ml Hla did not enter the cytosol at higher rates than in untreated control cells [15]
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