MCLA-dependent chemiluminescence suggests that singlet oxygen plays a pivotal role in myeloperoxidase-catalysed bactericidal action in neutrophil phagosomes

Abstract

Bacteria ingested by a neutrophil are located in phagosomes in which H(2)O(2) is produced through the NADPH oxidase-dependent respiratory burst. Myeloperoxidase (MPO) plays important role in the bactericidal action of phagosomes. MPO catalyses the reaction of H(2)O(2) and Cl(-) to produce HClO. The chemical mechanism behind the bactericidal action of the MPO-H(2)O(2)-Cl(-) system is unclear. Bactericidal action may result from (a) the direct reactions of HOCl with biological components (through amine chlorination) or (b) (1)O(2), formed non-enzymatically from HOCl and H(2)O(2), that mainly works to kill microorganisms through bacterial respiratory chain injury. To answer this question, we developed a Cypridina luciferin analogue (MCLA)-dependent chemiluminescence method to determine the rate of formation of (1)O(2) from a (1)O(2) source at pH 4.5-9.0. Using the MCLA-dependent chemiluminescence method, we found that the rate of formation of (1)O(2) from the MPO-H(2)O(2)-Cl(-) system peaked at pH 7.0. Segal et al. (28) reported that almost all Staphylococcus aureus is killed 2 min after phagocytosis by neutrophils where the phagosomal pH is 7.4-7.75. However, amine chlorination by HOCl did not proceed at pH > 7.0. Moreover, the bactericidal activities of the MPO-H(2)O(2)-Cl(-) system with Escherichia coli at pH 4.5 and 8.0 were paralleled by the rate of formation of (1)O(2). Combining these observations and the results reported by Segal et al., we concluded that (1)O(2) is a major chemical species in the killing of bacteria in neutrophil phagosomes.