Abstract
Inhibitors of the energy metabolism, such as sodium azide and valinomycin, render yeast cells completely resistant against the killing action of a number of cationic antimicrobial peptides, including the salivary antimicrobial peptide Histatin 5. In this study the Histatin 5-mediated killing of the opportunistic yeast Candida albicans was used as a model system to comprehensively investigate the molecular basis underlying this phenomenon. Using confocal and electron microscopy it was demonstrated that the energy poison azide reversibly blocked the entry of Histatin 5 at the level of the yeast cell wall. Azide treatment hardly induced depolarization of the yeast cell membrane potential, excluding it as a cause of the lowered sensitivity. In contrast, the diminished sensitivity to Histatin 5 of energy-depleted C. albicans was restored by increasing the fluidity of the membrane using the membrane fluidizer benzyl alcohol. Furthermore, rigidification of the membrane by incubation at low temperature or in the presence of the membrane rigidifier Me(2)SO increased the resistance against Histatin 5, while not affecting the energy charge of the cell. In line, azide induced alterations in the physical state of the interior of the lipid bilayer. These data demonstrate that changes in the physical state of the membrane underlie the increased resistance to antimicrobial peptides.
Highlights
In the last few decades an expanding number of antimicrobial peptides have been isolated from virtually all classes of organisms, where they play an important role in the innate defense against microbial and viral infections
Effect of Energy Metabolism on Candidacidal Activities of Antimicrobial Peptides—In previous studies we found that Histatin 5 (Hst5) accumulates in the mitochondria of C. albicans [3, 20]
To identify the step in the killing process that is sensitive to the energy charge of the yeast cell, we examined the effect of azide on the association between F-Hst5 and C. albicans by FACScan analysis (Fig. 3)
Summary
Histatin 5; FITC, fluorescein isothiocyanate; Fmoc, N-(9-fluorenyl)methoxycarbonyl; PPB, potassium phosphate buffer; PI, propidium iodide; FACS, fluorescence-activated cell sorter; DPH, diphenylhexatriene. Azide Rigidifies C. albicans Membrane restoration of the energy charge of the cell. On the other hand, inducing increased membrane rigidity by lowering the temperature or by treatment with a membrane-rigidifying agent led to an increased resistance against Hst. It is hypothesized that the actin cytoskeleton, which is highly sensitive to the energy charge of the cell mediates the effect, because the cytoskeleton inhibitor jasplakinolide induced resistance to Hst
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