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

Pneumolysin, a hemolytic toxin from Streptococcus pneumoniae, is a number 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 (< 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 dihiothreitol. Pneumolysin was also inactivated when incubated with human neurophils (10⁵) in the presence of a halide and phorbol myristate acetate, an activator of neutrophil secretion and oxygen metabolism. Toxin inactivation by stimulated neutorphils 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.