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Abstract

Polymyxin B (PMB) is a cationic cyclic peptide that can selectively inhibit the growth of Gram-negative bacteria by disrupting the outer membrane permeability barrier through binding to lipopolysaccharide (LPS). Here, a fluorescent PMB derivative (PMB-Ds) was applied to visually confirm the vacuole as a direct lethal target of PMB against fungal cells, which lack LPS. PMB-Ds could be visualized in the normal rounded vacuolar membrane of cells, suggesting the presence of a molecular ligand assisting the vacuole-targeting mobilization of the peptide in the organism. Vma1p, a cytoplasmic subunit constituent of the yeast vacuolar-type ATPase, was identified as one of the PMB-binding proteins by means of mass spectrometry. Mutant cells carrying a deletion of Vma1p but not those with deletions in two separate PMB-binding proteins were shown to be resistant to the vacuolar membrane disruptive action of PMB. Furthermore, the mutant cells were resistant to PMB lethality even when treated with PMB in combination with allicin, an allyl sulfur compound, which can selectively enhance the vacuole-targeting fungicidal activity of the peptide. In contrast, the parent cells were not made resistant to the vacuolar membrane disruptive action of PMB even if cells were pre-treated with bafilomycin A1, a specific inhibitor of the yeast vacuolar-type H-ATPase. However, the parent cells were rendered more resistant to PMB consequent to Vma1p-GFP localization in the cytoplasm. These findings suggested a role for Vma1p in the vacuole-targeting fungicidal activity of PMB comparable to that of LPS in the outer membrane of Gram-negative bacteria.

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2017-04-01
2019-12-11
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