Bioenergetic mechanism for nisin resistance, induced by the acid tolerance response of Listeria monocytogenes

Abstract

This study examined the bioenergetics of Listeria monocytogenes, induced to an acid tolerance response (ATR). Changes in bioenergetic parameters were consistent with the increased resistance of ATR-induced (ATR(+)) cells to the antimicrobial peptide nisin. These changes may also explain the increased resistance of L. monocytogenes to other lethal factors. ATR(+) cells had lower transmembrane pH (DeltapH) and electric potential (Deltapsi) than the control (ATR(-)) cells. The decreased proton motive force (PMF) of ATR(+) cells increased their resistance to nisin, the action of which is enhanced by energized membranes. Paradoxically, the intracellular ATP levels of the PMF-depleted ATR(+) cells were approximately 7-fold higher than those in ATR(-) cells. This suggested a role for the F(o)F(1) ATPase enzyme complex, which converts the energy of ATP hydrolysis to PMF. Inhibition of the F(o)F(1) ATPase enzyme complex by N'-N'-1,3-dicyclohexylcarbodiimide increased ATP levels in ATR(-) but not in ATR(+) cells, where ATPase activity was already low. Spectrometric analyses (surface-enhanced laser desorption ionization-time of flight mass spectrometry) suggested that in ATR(+) listeriae, the downregulation of the proton-translocating c subunit of the F(o)F(1) ATPase was responsible for the decreased ATPase activity, thereby sparing vital ATP. These data suggest that regulation of F(o)F(1) ATPase plays an important role in the acid tolerance response of L. monocytogenes and in its induced resistance to nisin.

FIG. 1.

Determination of pH_i of ATR⁺ (thick line) or ATR⁻ (thin line) L. monocytogenes suspended in potassium phosphate buffer at pH_o 5.5 (acidified with lactic acid) containing 0.5% glucose. Cells were exposed to nisin (50 μg ml⁻¹) from time zero. Data are averages of two independent and reproducible spectra.

FIG. 2.

Fluorescence spectra used to assess Δψ of L. monocytogenes. In all cases, the fluorescent probe di-S-C₃ (5) was added at ∼40 s and equilibrated with the cells after ∼200 s. Wide peaks coincident with reagent addition are artifacts from light entering the equipment during the addition. Data represent averages of two independent and reproducible spectra. (a) Assessment of Δψ of L. monocytogenes ATR⁺ cells at pH 5.5 (thick line) or ATR⁻ cells at pH 5.5 (medium line) or 7.0 (thin line) suspended in 50 mM K⁺-HEPES-MES buffer containing 0.5% glucose. (b) Extended assessment of Δψ of live (thick line) and dead (control; thin line) ATR⁺ L. monocytogenes cells suspended in 50 mM K-HEPES-MES buffer at pH 5.5 containing 0.5% glucose. Neither nisin (50 μg ml⁻¹; 300 s) nor valinomycin (added at ∼360 s) depleted the Δψ of the ATR⁺ cells. (c) Extended assessment of Δψ of live (thick line) and dead (thin line) ATR⁻ L. monocytogenes suspended in 50 mM K⁺-HEPES-MES buffer at pH 7.0 containing 0.5% glucose. Nisin (50 μg ml⁻¹; 300 s) rapidly depleted Δψ of the live ATR⁻ cells, and valinomycin (400 s) depleted the residual Δψ. (d) Extended assessment of Δψ of ATR⁺ (thick line) L. monocytogenes suspended in 50 mM K⁺-HEPES-MES buffer at pH 7.0 containing 0.5% glucose. ATR⁺ cells had measurable Δψ values relative to those of the dead cell control (thin line). Nisin (50 μg ml⁻¹; 300 s) rapidly depleted Δψ of the live ATR⁺ cells, and valinomycin (added at ∼360 s) depleted the residual Δψ.

FIG. 3.

Time-dependent intracellular ATP levels of ATR⁺ and ATR⁻ listeriae during treatment with nisin and 0.5% glucose in 50 mM Na⁺-HEPES-MES buffer at pH 5.5. Data are averages of duplicate experiments, and error bars indicate 1 standard error.

FIG. 4.

The intracellular ATP levels of listeriae after ATR induction in the absence or presence of 1 mM DCCD, prior to ATP determinations. ATR⁺ cells (closed symbols) and ATR⁻ cells (open symbols) were suspended in 50 mM K⁺-HEPES-MES buffer at pH 5.5 and 7.0, respectively, in the presence of glucose (triangles) or glucose and nisin (circles). Data are averages of triplicate experiments, and error bars indicate 1 standard deviation.

FIG. 5.

SELDI-TOF MS spectra obtained from lysates of ATR⁺ and ATR⁻ Listeria monocytogenes cells. Vertical axes are relative intensities. Each of the two peak spectra below represents the average of three independent chip spots. See the text for details.