MYCOPLASMA PNEUMONIAE: HYDROGEN PEROXIDE SECRETION AND ITS POSSIBLE ROLE IN VIRULENCE *

Mycoplusrnu pneumoniae secretes a substance which lyses red blood cells. The hemolysin has been identified as hydrogen peroxide (H202). l The toxic actions of H202 in vitro on various enzyme systems and tissues are well known. Cytotoxicity by H202 in vivo is exemplified by its role in primaquine-sensitivity, a genetic disorder characterized by a deficiency of the enzyme glucose-6-phosphate dehydrogenase within erythrocytes.2 Affected individuals develop striking hemolytic responses after ingesting certain antimalarial drugs (the 8-aminoquinolines, e.g. primaquine) or a variety of other substances which can generate H202 within erythro~ytes.3. ~ Similarly, the virulence of M. pneumoniae may be due, in part, to a toxic action of H202 secreted onto the tissues of the respiratory tract. Therefore, we undertook to measure the amounts of H202 secreted by M. pneumoniae. The method employed to measure H202-secretion by M. pneumoniae was one which had been used as a qualitative tool to detect the formation of H202 within erythrocytes3.4 and which subsequently was adapted for quantitative s t u d i e ~ . ~ The method makes use of the enzyme catalase and the catalase-inhibitor 3-amin0, 1,2,4-triazole (FIGURE 1 ). Catalase reacts with H202 to form the enzyme-substrate complex designated by Britton Chance as Complex 1 . Complex 1 can react with another molecule of H202 to liberate oxygen. Alternatively, Complex 1 can react with aminotriazole, in which event the catalase is irreversibly inhibited.6. 7 Since irreversible inhibition of catalase occurs only when H202 is present, this reaction serves as a detection system for H202. The rate of delivery of H202 to the system determines the rate of inhibition of ca t a l a~e . ~ The conversion of an Hp02-production rate into a catalase-inactivation rate and the subsequent measurement of catalase activity provide a highly specific and sensitive technique for studying the rate of formation of H202. The reaction system in the experiments with M. pneumoniae consisted of a solution of 0.25 mg crystalline catalase (Worthington) per ml isotonic saline buffered at pH 7.4; the system contained 50 mM aminotriazole and 1 1 mM glucose. In the prior studies with erythrocytes and hemolytic drugs, endogenous catalase was the detector and the intracellular rate of formation of Hz02 was measured rather than a secretion rate. In the experiments with M. pneumoniae, the H202 secreted into the external catalase-containing medium was measured (M. pneumoniae does not possess catalase) ; the experimental conditions mimicked those which would pertain when M. pneumoniae secretes H202 onto the tissues of the respiratory tract. A model experiment with M. pneumoniae and with glucose oxidase as a comparison standard is presented in TABLE 1. The suspension of M. pneumoniae was prepared from organisms attached to the glass surface of Povitsky bottles.8 The mycoplasma were washed with buffered saline to eliminate growth-medium constituents and then were resuspended in buffered saline. When roughly loB viable mycoplasma were added to 2 ml of the reaction system at 37OC, a steady decline in catalase activity resulted (line 1 ). The rate of inhibition of catalase was