Mode of action of lactocin 160, a bacteriocin from vaginal Lactobacillus rhamnosus

Abstract

Objectives. To determine the mechanism of antimicrobial action of lactocin 160, a bacteriocin produced by the healthy vaginal strain of Lactobacillus rhamnosus, using an established model, with Micrococcus luteus ATCC 10420 as a test organism. Methods. Sensitivity of M. luteus to lactocin 160 was determined by the diffusion assay. Loss of cellular ATP in the lactocin-treated cells was elucidated using a commercially available ATP determination kit (luciferin-luciferase bioluminescence assay). Luminescence intensity as a reflection of ATP quantity was determined using a luminometer. Dissipation of membrane potential (Δψ) was studied using fluorophore DiSC3(5) with the fluorescence spectrum sensitive to changes in Δψ. Results. Lactocin 160 inhibited growth of M. luteus ATCC 10420 at a concentration of 5 μg/ml. There were no significant changes in the intracellular ATP level of M. luteus upon the addition of 20 μg/ml of lactocin 160. However, the extracellular ATP level increased significantly. This means that the treatment of cells with lactocin 160 resulted in an efflux of ATP from inside the cells. Therefore, a partially purified lactocin 160 preparation (16 μg/ml of the bacteriocin in the sample) killed sensitive cells and dissipated 3.12 ± 0.36% of Δψ. Conclusion. Lactocin 160 has a mode of action typical for bacteriocins. It disturbs the cellular membrane (Δψ dissipation) and induces ATP efflux, most likely because of the pore formation, which is a common mechanism of action for many bacteriocins. © 2005 Taylor & Francis.