Staphylococcus aureus requires cardiolipin for survival under conditions of high salinity

Abstract

Background: The ability of staphylococci to grow in a wide range of salt concentrations is well documented. In this study, we aimed to clarify the role of cardiolipin (CL) in the adaptation of Staphylococcus aureus to high salinity.

Results: Using an improved extraction method, the analysis of phospholipid composition suggested that CL levels increased slightly toward stationary phase, but that this was not induced by high salinity. Deletion of the two CL synthase genes, SA1155 (cls1) and SA1891 (cls2), abolished CL synthesis. The cls2 gene encoded the dominant CL synthase. In a cls2 deletion mutant, Cls1 functioned under stress conditions, including high salinity. Using these mutants, CL was shown to be unnecessary for growth in either basal or high-salt conditions, but it was critical for prolonged survival in high-salt conditions and for generation of the L-form.

Conclusions: CL is not essential for S. aureus growth under conditions of high salinity, but is necessary for survival under prolonged high-salt stress and for the generation of L-form variants.

Figure 1

Effect of lysostaphin treatment on CL extraction efficiency. Prior to lipid extraction, cells (N315) were incubated for 3 min at 37°C in the presence of lysostaphin at the indicated concentrations. CL: Cardiolipin. PG: Phosphatidylglycerol. LPG: Lysyl-phosphatidylglycerol. The means and standard deviations of relative signal intensities are shown at the bottom.

Figure 2

Phospholipid composition of S. aureus N315 under various growth conditions. Cells were grown in LB containing either 0.1% or 15% NaCl, and harvested during the exponential (exp.) or stationary phase. The means and standard deviations of relative signal intensities are shown at the bottom.

Figure 3

Lipid synthesis pathway in S. aureus (modified from the KEGG pathway database). SA1155 (cls1) and SA1891 (cls2) are homologs of the B. subtilis cls gene.

Figure 4

Phospholipid composition of N315 and its isogenic cls mutants. Cells were harvested during stationary phase. The means and standard deviations of relative signal intensities are shown at the bottom.

Figure 5

Growth and stationary-phase survival under low salinity. Cells were grown in 0.1% NaCl LB. A: Growth was monitored by optical density (OD) measurements. N315: filled diamonds; cls1 mutant: filled squares; cls2 mutant: filled triangles; cls1/cls2 double mutant: open circles. Optical densities were checked at least twice, and the means are shown. After 47 h, the OD of only N315 and its cls1/cls2 double mutant were measured. B: Number of CFUs during the long incubation. The means and standard deviations of at least three independent experiments are shown. C: Thin-layer chromatography of phospholipids. Cells were harvested at 23 h. The phospholipid profile was confirmed to be similar up to 153 h (data not shown). The means and standard deviations of relative signal intensities from two independent experiments are shown on the right.

Figure 6

Stationary-phase survival under high salinity. Cells were grown in LB containing either 15% (A, B) or 25% (C, D) NaCl. A, C: ODs were measured at least twice, and the means are shown. B, D: The number of CFUs was determined at least three times. The means and standard deviations are shown. E: Thin-layer chromatography of phospholipids. Note that CL accumulated in the cls2 mutant. The relative signal intensities are shown on the right.

Figure 7

L-form generation in MT01 and its cls mutants. MT01: open squares; cls1 mutant: open triangles; cls2 mutant: filled squares; cls1/cls2 double mutant: filled triangles. L-forms are 'fried-egg-shaped' colonies that appear after prolonged incubation with cell-wall perturbing antimicrobials. The L-form has no cell wall, which we confirmed by disruption at low osmotic pressure. The means of at least two independent determinations are shown.

Figure 8

Summary of the cardiolipin (CL) and phosphatidylglycerol (PG) signal intensities in each strain under distinct NaCl concentrations. Strains cultured in LB containing 0.1% or 15% NaCl were harvested during exponential (3 h for 0.1% NaCl LB, 7 h for 15% NaCl LB) or stationary (23 h for 0.1% NaCl LB, 33 h for 15% NaCl LB) phase. The means and standard deviations of two independent determinations are shown. A: CL. B: PG.

Figure 9

Phospholipid analysis under defined conditions. A: Anaerobic, 37°C, overnight culture (o/n); B: Aerobic, 42°C, o/n; C: Aerobic, 30°C, o/n; D: Aerobic, 37°C, pH 5, exponential-phase culture; E: Aerobic, 37°C, pH 7, exponential-phase culture. Relative signal intensities are shown at the bottom.