Staphylococcus aureus Alpha-Toxin Mediates General and Cell Type-Specific Changes in Metabolite Concentrations of Immortalized Human Airway Epithelial Cells

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.

Staphylococcus aureusα-toxin-mediated cation entry depolarizes membrane potential and activates p38 MAP kinase in airway epithelial cells

“…6C). This clearly indicated that the Ca 2ϩ pumps refilling the intracellular stores were still functional at 1 or 2 h of rHla treatment of cells despite the substantial loss of ATP that had been observed during this time period in rHla-treated cells (17). This result corresponds with previous findings (52) that a cytosolic ATP concentration of 0.1 mmol/l (i.e., only 1/10 of the original concentration) is sufficient to maintain the intracellular calcium stores of cells in a completely filled state.…”

“…Another mechanism potentially explaining the increase in cytosolic calcium in rHla-exposed cells would be that the rHla-induced rapid release of substantial amounts of ATP from the cytosol to the extracellular space, which has been observed in different cell types upon treatment with ␣-toxin (17,27,36), may result in very low cytosolic ATP concentrations. The lack of cellular ATP, in turn, may compromise the activity of Ca 2ϩ -ATPases in the membranes of endoplasmic calcium stores (SERCA) or in the plasma membrane (PMCA) (48).…”

“…The effective diameter of the pore may be even smaller, as indicator molecules such as acridine orange (265 g/mol), BCECF (ϳ600 g/mol), or Indo-1 (ϳ500 g/mol) do not leave the cells upon treatment with Hla (14,58). Many cell types release ATP (507 g/mol) to the external medium upon exposure to Hla (12,17,24,36,41,55,58,59), but it is not clear whether ATP exits the cells through Hla pores or through other pathways. Furthermore, whether the Hla pore is directly permeable to mono-or divalent cations (Na ϩ , Ca 2ϩ ) has been often discussed, but experimental evidence is scarce and in part controversial (2,14,27,31,50,56,58,59).…”

Comparative proteome analysis reveals conserved and specific adaptation patterns of Staphylococcus aureus after internalization by different types of human non-professional phagocytic host cells

“…For this control the pH was set to 6.9, corresponding to the pH of a bacterial culture at OD 600 0.4. Samples of cellular extracts were generated and extracted as described previously (Gierok et al, 2014). In brief, the supernatant was collected separately.…”

A proteomic perspective of the interplay of Staphylococcus aureus and human alveolar epithelial cells during infection

“…Intracellular metabolites were extracted from cells with methanol/water/chloroform in a ratio of 5:5:1. After addition of internal standards, metabolites were lyophilized and identified and quantified by GC/MS and LC/MS as described before [38].…”