Calcium and Magnesium Ions Are Membrane-Active against Stationary-Phase Staphylococcus aureus with High Specificity

Abstract

Staphylococcus aureus (S. aureus) is notorious for its ability to acquire antibiotic-resistance, and antibiotic-resistant S. aureus has become a wide-spread cause of high mortality rate. Novel antimicrobials capable of eradicating S. aureus cells including antibiotic-resistant ones are thus highly desired. Membrane-active bactericides and species-specific antimicrobials are two promising sources of novel anti-infective agents for fighting against bacterial antibiotic-resistance. We herein show that Ca(2+) and Mg(2+), two alkaline-earth-metal ions physiologically essential for diverse living organisms, both disrupt model S. aureus membranes and kill stationary-phase S. aureus cells, indicative of membrane-activity. In contrast to S. aureus, Escherichia coli and Bacillus subtilis exhibit unaffected survival after similar treatment with these two cations, indicative of species-specific activity against S. aureus. Moreover, neither Ca(2+) nor Mg(2+) lyses mouse red blood cells, indicative of hemo-compatibility. This works suggests that Ca(2+) and Mg(2+) may have implications in targeted eradication of S. aureus pathogen including the antibiotic-resistant ones.

Figure 1

(a) Binding of M²⁺ (M = Ca, Mg) with cardiolipin (CL), the major lipid component in S. aureus, converts the originally zero-intrinsic-curvature (C₀~ 0) lipid into M²⁺-CL complexes with negative intrinsic curvature (C₀ < 0). (b–d) Dye leakage assays using large unilamellar vesicles (LUVs) composed of 100% CL as a first-order model for S. aureus membranes. (b) Mg²⁺ at ≥7.2 mM caused significant carboxyl fluorescein (CF) leakage. (c,d) Ca²⁺, though (c) unable to cause detectable CF leakage, caused (d) appreciable quenching in the fluorescence intensity of intravesicular lucigenin. Controls are samples assayed similarly but without M²⁺ additions.

Figure 2

(a,b) Dye leakage assays using DOPG: CL = 58:42 LUVs as a more realistic model for S. aureus membranes. Obviously, both (a) Mg²⁺ and (b) Ca²⁺ caused significant CF leakage from DOPG: CL = 58:42 LUVs and, to do so, their minimum threshold concentrations are 20 and 10 mM, respectively. Controls are samples assayed similarly but without M²⁺ addition.

Figure 3

Plate killing assays against stationary-phase cells of (a) S. aureus, (b) E. coli, and (c) B. subtilis. After 40-min co-incubation with Mg²⁺ (left) and Ca²⁺ (right) at ≤40 mM, S. aureus cells exhibited dose-dependent loss in viability. In contrast, the viability of E. coli and B. subtilis cells is barely impacted in similar assays. Data points are reported as mean ± standard deviation. *and** indicate p < 0.05 and p < 0.01, respectively.

Figure 4. Up to 40 mM, neither Mg²⁺ nor Ca²⁺ caused detectable hemolysis against mouse red blood cells.

Data points are reported as mean ± standard deviation.