INFECTION AND IMMUNITY, Jan. 2011, p. 474–485 Vol. 79, No. 1 0019-9567/11/$12.00 doi:10.1128/IAI.00910-09 Copyright © 2011, American Society for Microbiology. All Rights Reserved. Myeloperoxidase Selectively Binds and Selectively Kills Microbesᰔ† Robert C. Allen1* and Jackson T. Stephens, Jr.2 Creighton University, School of Medicine, Department of Pathology, Omaha, Nebraska,1 and ExOxEmis, Inc., Little Rock, Arkansas2 Received 11 August 2009/Returned for modification 2 October 2009/Accepted 13 October 2010 Myeloperoxidase (MPO) is reported to selectively bind to bacteria. The present study provides direct evidence of MPO binding selectivity and tests the relationship of selective binding to selective killing. The ؊ microbicidal effectiveness of H2O2 and of OCl was compared to that of MPO plus H2O2. Synergistic microbicidal action was investigated by combining Streptococcus sanguinis,aH2O2-producing microbe showing low MPO binding, with high-MPO-binding Escherichia coli, Staphylococcus aureus,orPseudomonas aeruginosa without exogenous H2O2, with and without MPO, and with and without erythrocytes (red blood cells [RBCs]). Selectivity of MPO microbicidal action was conventionally measured as the MPO MIC and minimal bacteri- cidal concentration (MBC) for 82 bacteria including E. coli, P. aeruginosa, S. aureus, Enterococcus faecalis, ؊ Streptococcus pyogenes, Streptococcus agalactiae, and viridans streptococci. Both H2O2 and OCl destroyed RBCs at submicrobicidal concentrations. Nanomolar concentrations of MPO increased H2O2 microbicidal action 1,000-fold. Streptococci plus MPO produced potent synergistic microbicidal action against all microbes tested, and RBCs caused only a small decrease in potency without erythrocyte damage. MPO directly killed H2O2- producing S. pyogenes but was ineffective against non-H2O2-producing E. faecalis. The MPO MICs and MBCs for E. coli, P. aeruginosa, and S. aureus were significantly lower than those for E. faecalis. The streptococcal studies showed much higher MIC/MBC results, but such testing required lysed horse blood-supplemented medium, thus preventing valid comparison of these results to those for the other microbes. E. faecalis MPO binding is reportedly weak compared to binding of E. coli, P. aeruginosa, and S. aureus but strong compared to binding of streptococci. Selective MPO binding results in selective killing. Ϫ The microbicidal activities of H2O2 and especially OCl are against Gram-positive and Gram-negative bacteria, as well as potent, but such activities are best achieved in the absence of yeast and fungi (23). Of the mammalian peroxidases, MPO is competing substrates. The reactivities of these agents are in- unique in its ability to catalyze the H2O2-dependent oxidation discriminate and capable of damaging mechanisms of immune of ClϪ to OClϪ. Such haloperoxidase activity is required for defense as well as destroying microbes. Alexander Fleming effective microbe killing (22). In addition to the requirement of Ϫ appreciated this relationship, stating that “leukocytes are more H2O2 for OCl production, H2O2 also directly reacts with sensitive to the action of chemical antiseptics than are the Ϫ 1 OCl to produce singlet molecular oxygen ( O2*), a potent bacteria, and, in view of this, it is unlikely that any of these electrophilic oxygenating agent (21). The microbicidal action antiseptics have the power of penetrating into the tissues and of MPO involves highly exergonic oxygenation reactions. destroying the bacteria without first killing the tissues them- These wet combustion reactions yield excited carbonyl prod- selves” (16). ucts that relax with chemiluminescence (2, 3). Healthy human adults produce about 100 billion neutrophil After leaving the blood, neutrophils migrate into cavities leukocytes per day. After a circulating lifetime of about 10 h, such as the mouth (38) and vagina (11). These spaces are these neutrophils leave the blood to enter the body tissues (8). characterized by acidic pH and the presence of flora rich in Myeloperoxidase (MPO), a 145-kDa dimeric alpha-heme halo- lactic acid bacteria (LAB). It is reasonable to assume that the peroxidase present in azurophilic granules, makes up approx- neutrophils that have migrated into the acid milieu of the imately 5% of the dry weight of the neutrophil (33). If a mouth or vagina eventually disintegrate, releasing their MPO neutrophil is assumed to have a cell volume of 450 fl, a specific content into these environments, and that this released MPO is gravity of 1.1, and a cell water content of 84%, healthy human available to interact with microbes of the resident flora. adults synthesize about 2.8 ␮mol (i.e., 0.4 g) of MPO per day. MPO selectively binds to Gram-negative and many Gram- Furthermore, the production of neutrophils and the MPO con- positive bacteria. However, streptococci, especially viridans tent per neutrophil are markedly increased in states of inflam- streptococci, show little MPO binding (4, 6). Viridans strepto- mation and with granulocyte colony-stimulating factor (G- cocci are a major constituent of normal mouth flora (20). As CSF) treatment (7). members of the LAB, streptococci are cytochrome deficient MPO exerts potent and broad-spectrum microbicidal action and catalase negative and produce lactic acid and H2O2 as metabolic products (24). The rates of H2O2 production by * Correspondent author. Mailing address: Department of Pathology, Streptococcus sanguinis and Streptococcus mitis are reported to Creighton University Medical Center, 601 North 30th Street, Omaha, NE be in the range of 12 to 67 nanomoles/min/mg of dry weight 68113. Phone: (402) 280-4326. Fax: (402) 280-5247. E-mail: RobertAllen (10). Another study of Streptococcus oralis and S. sanguinis @creighton.edu. 6 ᰔ reported a range of 1 to 5 nmol/min/10 CFU (18). These rates Published ahead of print on 25 October 2010. † The authors have paid a fee to allow immediate free access to this of H2O2 production are adequate to drive MPO oxidation of Ϫ Ϫ Ϫ article. Cl to OCl and for H2O2 reaction with OCl to produce 474 VOL. 79, 2011 MPO SELECTIVELY BINDS AND KILLS MICROBES 475 1 O2*. At relatively low MPO concentrations, a H2O2-produc- remove remaining leukocytes. The centrifugation and aspiration steps were re- ing LAB showing low MPO binding might escape damage. peated, and the erythrocyte pellet was suspended and diluted with NS to a 8 Furthermore, the streptococcus-generated H O would be concentration of 10 RBCs per ml by hemocytometer count. 2 2 Erythrocytes (0.1 ml containing 107 RBCs) were added to the microbe sus- available to drive combustive reactions against competing mi- pensions where indicated. Human RBCs contain catalase, i.e., H2O2:H2O2 oxi- crobes showing higher MPO binding. doreductase, an enzyme that catalyzes the destruction of H2O2 producing H2O The present report compares the microbicidal capacities of and O2. The mechanism for removal of H2O2 in human erythrocytes is more than Ϫ 90% dependent on catalase action (28). In addition to competitive destruction of H2O2, OCl , and nanomolar MPO plus low concentrations of H2O2, erythrocytes can directly react with oxidizing agents and, thus, compete H2O2. The potency and selectivity of MPO microbicidal action with microbes for available reactants. RBCs are susceptible to damage by high Ϫ are illustrated using either streptococci or glucose oxidase concentrations of H2O2 and lower concentrations of OCl . Introducing RBCs (GO) to generate H2O2. The selectivity of microbicidal action into the reaction mixture provides a means for assessing the specificity of the was conventionally assessed by measuring the MIC and mini- reactants for the microbe relative to the erythrocyte. mal bactericidal concentration (MBC) of MPO against a large Following microbe-RBC incubation and centrifugation, the hemoglobin (Hgb) that remains in the pellet is proportional to the remaining intact erythrocytes, spectrum of clinical bacterial isolates. i.e., to RBC survival. Loss of Hgb from the pellet and its appearance in the (Portions of this research were presented at the 2008 Inter- supernatant indicate hemolysis, i.e., the destruction of RBC membrane integrity national Conference on Gram Positive Pathogens, Omaha, and release of cytoplasmic Hgb into the medium. The degree of hemolysis can be NE, 5 to 8 October 2008, and at the 48th Interscience Confer- assessed by the distribution of Hgb in the pellet and supernatant. Complete hemolysis with Hgb destruction results in the disappearance of Hgb from both ence on Antimicrobial Agents-Infectious Diseases Society of the pellet and supernatant. In addition to the oxidative reactants tested, micro- America 46th Annual Meeting, Washington, DC, 25 to 28 bial toxins can also produce hemolysis and Hgb destruction. October 2008, poster A1-3497). The concentrations of Hgb in the pellet and supernatant were measured by a derivative spectroscopic modification of the hemiglobincyanide (Drabkin) method (27). A suspension of 107 RBCs per ml yielded 0.2 to 0.3 mg of Hgb. The MATERIALS AND METHODS total Hgb of the supernatant and pellet was measured in the control and was Reagents and enzymes. Stock H2O2 and NaOCl solutions were prepared and expressed as unity, i.e., 1.0. The distribution of Hgb between the pellet and quantified. The concentration of the H2O2 stock solution was verified by mea- supernatant was expressed as a proportion of 1.0. This approach provides a ε suring its UV absorbance spectrum using a 240-nm extinction coefficient ( 240)of rough but useful gauge of hemolytic activity. Small losses of Hgb from the pellet Ϫ1 ⅐ Ϫ1 ε Ϫ1 ⅐ Ϫ1 43.6 M cm and a 300-nm extinction coefficient ( 300) of 1.0 M cm . to the supernatant are an expected consequence of the preparation and exper- The concentration of the NaOCl stock solution was verified by measuring its imental treatment, but large losses to the supernatant indicate active hemolysis. ε absorbance spectrum using a 292-nm extinction coefficient ( 292)of350 Complete destruction of Hgb is indicated by a total loss from the pellet and MϪ1 ⅐ cmϪ1. supernatant. ؊ Porcine MPO, EC 1.11.1.7, was produced by ExOxEmis, Inc.