Oxidative inactivation of pneumolysin by the myeloperoxidase system and stimulated human neutrophils.

Abstract

Pneumolysin, a hemolytic toxin from Streptococcus pneumoniae, is a member of the group of thiol-activated, oxygen-labile cytolysins produced by various Gram-positive bacteria. The toxin activity of pneumolysin, as determined by lysis of 51Cr-labeled human erythrocytes, was destroyed on exposure to the neutrophil enzyme myeloperoxidase, hydrogen peroxide, and a halide (chloride or iodide). Detoxification required each component of the myeloperoxidase system and was prevented by the addition of agents that inhibit heme enzymes (azide, cyanide) or degrade H2O2 (catalase). Reagent H2O2 could be replaced by the peroxide-generating enzyme system glucose oxidase plus glucose. The entire myeloperoxidase system could be replaced by sodium hypochlorite at micromolar concentrations. Toxin inactivation was a function of time of exposure to the myeloperoxidase system (less than 1 min), the rate of formation of H2O2 (0.05 nmol/min), and the concentration of toxin employed. Toxin that had been inactivated by the myeloperoxidase system was reactivated on incubation with the reducing agent dithiothreitol. Pneumolysin was also inactivated when incubated with human neutrophils (10(5)) in the presence of a halide and phorbol myristate acetate, an activator of neutrophil secretion and oxygen metabolism. Toxin inactivation by stimulated neutrophils was blocked by azide, cyanide, or catalase, but not by superoxide dismutase. Neutrophils from patients with impaired oxygen metabolism (chronic granulomatous disease) or absent myeloperoxidase (hereditary deficiency) failed to inactivate the toxin unless they were supplied with an exogenous source of H2O2 or purified myeloperoxidase, respectively. Thus, inactivation of pneumolysin involved the secretion of myeloperoxidase and H2O2, which combined with extracellular halides to form agents (e.g., hypochlorite) capable of oxidizing the toxin. This example of oxidative inactivation of a cytolytic agent may serve as a model for phagocyte-mediated detoxification of microbial products.