Elucidation of the Molecular Mechanisms of Action of the Natural Antimicrobial Peptide Subtilosin Against the Bacterial Vaginosis-associated Pathogen Gardnerella vaginalis

Abstract

Subtilosin A is a 35-amino acid long cyclical peptide produced by Bacillus amyloliquefaciens that has potent antimicrobial activity against a variety of human pathogens, including the bacterial vaginosis-related Gardnerella vaginalis. The specific mode of action of subtilosin against G. vaginalis was elucidated by studying its effects on the proton motive force's (PMF) components: transmembrane electric potential (ΔΨ), transmembrane pH gradient (ΔpH), and intracellular ATP levels. The addition of subtilosin to G. vaginalis cells caused an immediate and total depletion of the ΔpH, but had no effect on the ΔΨ. Subtilosin also triggered an instant but partial efflux of intracellular ATP that was twofold higher than that of the positive control bacteriocin, nisin. Taken together, these data suggest that subtilosin inhibits G. vaginalis growth by creating transient pores in the cells' cytoplasmic membrane, leading to an efflux of intracellular ions and ATP and eventually cell death.

Fig. 1

Subtilosin causes an efflux of intracellular ATP from G. vaginalis cells. Closed bars represent the total ATP content (intracellular + extracellular), while open bars represent extracellular ATP. Subtilosin (a) caused an efflux of ATP approximately 1.5-fold higher than that of nisin (b) and 2-fold higher than the negative control. Total ATP levels for nisin (b) were 20% lower than that of subtilosin (a) and both negative controls (a, b), indicating intracellular hydrolysis of ATP

Fig. 2

Subtilosin has no effect on transmembrane electric potential (ΔΨ) in G. vaginalis cells. Two μmol/L of nigericin (Nig) was used to convert the ΔpH portion of the PMF into ΔΨ (a, b). Subtilosin (Sub, a) caused no fluctuation in the fluorescent signal, indicating it has no effect on the ΔΨ. Addition of nisin (Nis, b) caused a corresponding spike in fluorescence, due to release of the DiSC₃ (5) probe and dissipation of the ΔΨ. In both cases, the negative controls (control (−)) subtilosin diluent (a) and nisin diluent (b) had no effect on the ΔΨ. Two μmol/L of valinomycin (Val) was used to dissipate any remaining ΔΨ (a, b). There was no increase in fluorescence in the nisin sample (b), demonstrating that nisin caused a total depletion of the ΔΨ portion of the PMF

Fig. 3

Subtilosin immediately depletes the transmembrane pH gradient (ΔpH) in G. vaginalis cells. Cells were energized with 2.2 mM glucose at start of fluorescence readings. Two μmol/L of valinomycin (Val) was used to transform the ΔΨ of the PMF into ΔpH (a, b). Subtilosin (Sub, a) caused an immediate decrease in the fluorescent signal, indicating a depletion of the ΔpH. Nisin (Nis, b) triggered a slower, gradual decrease in fluorescence. Both negative controls, (control (−)) subtilosin diluent (a) and nisin diluent (b), had no effect on the ΔpH. Two μmol/L of nigericin (Nig) was used to dissipate any remaining ΔpH (a, b). For both subtilosin and nisin, there was no subsequent drop in fluorescence, signifying that both bacteriocins totally depleted the ΔpH portion of the PMF