A real-time-based in vitro assessment of the oxidative antimicrobial mechanisms of the myeloperoxidase-hydrogen peroxide-halide system

摘要

Mammals have evolved a special cellular mechanism for killing invading microbes, which is called the phagocytosis. Neutrophils are the first phagocytosing cells that migrate into the site of infection. In these cells, hypochlorite (HOCl) and other hypohalites, generated in the myeloperoxidase (MPO)-hydrogen peroxide (H2O2) halide system is primarily responsible for oxidative killing. Here, we present a method for assessing these oxidative mechanisms in an in vitro cell-free system in a kinetical real-time-based manner by utilizing a bioluminescent bacterial probe called Escherichia con-lux. The E. coli-lux method provides a practical tool for assessing the effects of various elementary factors in the MPO-H2O2-halide system. Due to the reported versatile intracellular pH and halide concentration during the formation of the phagolysosome and respiratory burst, the antimicrobial activity of the MPO-H2O2-halide system undergoes extensive alterations. Here, we show that at a physiological pH or lower, the antimicrobial activity of MPO is high, and the system effectively enhances the H2O2-dependent oxidative killing of E. coli by chlorination. The HOCl formed in this reaction is a prominent microbe killer. During the respiratory burst, there is a shift to a more alkaline environment. At pH 7.8, the chlorinating activity of MPO was shown to be absent, and the activity of the HOCl decreased. At this higher pH, the activity of H2O2 is enhanced and high enough to kill E. coil without the participation of MPO, and the lowered chloride concentration seemed still to enhance the H2O2-dependent killing capacity.