Lactic acid bacteria (LAB) are incapable of cytochrome synthesis and lack the heme electron transport mechanisms required for efficient oxygen-based metabolism. Consequently, LAB redox activity is flavoenzyme-based and metabolism is fermentative, producing lactic acid, and in many cases, hydrogen peroxide (H2O2). Despite this seeming metabolic limitation, LAB dominate in the normal flora of the mouth, vagina and lower gastrointestinal tract in man. Myeloperoxidase (MPO) is produced by the neutrophil leukocytes and monocytes that provide the innate phagocyte defense against infecting pathogens. MPO is unique in its ability to catalyze the H2O2-dependent oxidation of chloride (Cl-) to hypochlorite (OCl-). In turn, this OCl- directly reacts with a second H2O2 to produce singlet molecular oxygen (
In 1892 Döderlein suggested that fermentative lactic acid producing vaginal bacteria protected the host from pathogenic microbes [
The lactic acid bacteria (LAB) are Gram-positive, nonspore forming microbes incapable of cytochrome synthesis and lacking the heme electron transport mechanisms required for efficient respiration. As such, LAB metabolism is fermentative. The LAB are acid-tolerant, aerotolerant and lack heme catalase activity [
Many LAB produce H2O2 in the presence of oxygen [7,8]. The viridans streptococci that comprise the normal mouth flora [
Neutrophil leukocytes and monocytes provide the innate phagocyte defense against pathogenic infection and are the microbicidal effector cells of the acute inflammatory response. Large quantities of neutrophils and monocytes are produced by the myelopoietic bone marrow and released into the circulating blood daily. Cytokines and chemotactic molecules direct phagocyte migration from the circulating blood to sites of infection where contact with opsonified microbes results in phagocytosis and formation of a phagosome. Fusion of the phagosome with lysosomal azurophilic granules containing cationic enzymes produces the phagolysosome. Both neutrophils and monocytes contain relatively large quantities of a cationic haloperoxidase, i.e., myeloperoxidase (MPO) [
As depicted in the schematic of
rial oxygen consumption. These metabolic activities are necessary for phagolysosomal acidification and H2O2 production, and provide an optimal milieu for H2O2- dependent MPO oxidization of Cl− to OCl− [15-18]. The reaction of OCl− with a second H2O2 yields electronically excited singlet molecular oxygen, a highly reactive but metastable electrophilic oxygenating agent with a microsecond lifetime. Both OCl− and * are potent microbicidal agents.
Each day the bone marrow of a healthy human adult releases about a hundred billion neutrophils and monocytes into the circulating blood [
The circulating lifetime of neutrophils in the blood is less that twelve hours, and is followed by a less well characterized tissue phase that can last for a few days [
Migrating tissue phase neutrophils transport sufficient MPO to body flora spaces to have an effect on the microbial flora. The presence of MPO might be expected to exert microbicidal action against the resident H2O2-producing LAB [26,27], but the opposite is observed.
Direct MPO binding to bacteria can be visually demonstrated by contacting bacteria in suspension with MPO, and then pelleting the bacteria by centrifugation. The degree of bacterial pellet staining is proportional to the MPO bound [
Furthermore, MPO binding correlates with MPO killing. Bacteria showing strong MPO binding were rapidly and effectively killed when MPO was present in nanomolar quantities with about 100 micromolar (µM) H2O2 (i.e., a greater than thousandfold dilution of 3% pharmacy H2O2). Streptococci and lactobacilli show relatively weak MPO-binding, and as such, these LAB are relatively protected from MPO microbicidal action.
In the acidic milieu of the neutrophil’s phagolysosome, or in acidic body spaces populated by LAB flora, MPO catalyzes the oxidation of chloride (Cl−) to hypochlorous acid (HOCl). Hypochlorite, the conjugate base of hypochlorous acid, is the active ingredient in household bleach, and has been employed as a disinfectant and deodorizing agent since Claude Louis Berthollet introduced it as Eau de Javel in 1789 [31-33]. Hypochlorous acid is a weak acid with a pKa of 7.5, and as such, the acid predominates in phagolysosomal and fermentation milieux.
The chloronium (Cl+) character of HOCl allows it to participate in a variety of reactions resulting in dehydrogenations and chloramine formation. The bactericidal action of hypochlorite is broad and complete at a concentration of 6 µM. However, a thousandfold higher hypochlorite concentration, i.e., 6 mM, is required for the same level of microbicidal action when human erythrocytes are added at a ratio of about five erythrocytes per bacterium [
The critical importance of microbe-specific MPO binding is best understood relative to the reactive lifetime of and its restricted radius of reactivity [35,36]. Like chlorine gas (Cl2), has singlet spin multiplicity, is a potent electrophilic reactant, and participates in highly exergonic reactions [18,37]. Unlike Cl2, is a metastable electronically excited state with a relatively short lifetime. Singlet spin multiplicity is critically important for reactivity. Understanding the role of multiplicity in reactivity is best approached by considering the Wigner spin conservation rules [38,39]. In essence, the neutrophil leukocyte changes the spin quantum number of oxygen, thus removing the barrier to spin-allowed reaction with singlet multiplicity biological molecules. The wet combustive oxygenations that follow produce electronically excited oxygenation products. As shown in
The short lifetime of restricts reactivity to within a space of less than a micron from its point of generation [28,40,41]. MPO microbicidal effectiveness is limited by its proximity to the target microbe. For successful microbicidal action, primary production of HOCl, and especially, secondary production of, must occur sufficiently close to the target microbe for adequate oxygenation activity. The concentration of HOCl decreases with the distance from the MPO production site. Once HOCl reacts with a second H2O2 molecule to generate, the reactive radius is restricted to within about 0.2 µm. Chloramines produced by direct reaction of HOCl with amine components of the microbe, also react with additional H2O2 to produce, and such reactions are facilitated by an acidic milieu.
The results of
In this experiment S. sanguinis metabolism is the only source of H2O2. E. coli are catalase positive and relatively well protected from low concentrations of H2O2. Erythrocytes show no significant MPO binding and contain abundant catalase. Introducing 107 erythrocytes with their several magnitudes greater mass than E. coli also provides competitive substrate for reaction with available
oxidants. The absence of measurable hemolysis confirmed the absence of erythrocyte damage during the course of the experiment [
LAB-MPO microbicidal action in the presence of erythrocytes demonstrates the highly selective nature of MPO-mediated oxidative attack. Only the MPO-binding E. coli were killed. The absence of significant MPObinding to S. sanguinis and erythrocytes, and the proximity requirement imposed by the lifetime of allow LAB-MPO synergistic microbicidal action against E. coli. In the presence of relatively low H2O2 concentrations and low MPO concentrations, selectivity of MPO binding results in selectivity of killing.
LAB are incapable of cytochrome synthesis, and conesquently, metabolism is fermentative. Nonetheless, these LAB play dominant roles in the flora of the mouth, vagina and lower gastrointestinal tract. In contradistinction to other Gram-positive and to all Gram-negative bacteria, LAB show very low MPO binding. LAB fermentative metabolism provides the acidic milieu and sufficient H2O2 for MPO microbicidal action, as demonstrated by the results shown in
LAB show poor MPO binding. Thus, the proximity requirement necessary for effective MPO microbicidal action spares LAB. At low MPO concentrations LAB are protected from their metabolic product H2O2. Instead, LAB-MPO synergistic microbicidal action is focused on competing MPO-binding microbes. The presence MPO in milieux containing LAB favors the killing of microbes showing significant MPO binding. As such, LAB-MPO synergy provides a mechanism for establishing and maintaining LAB as the dominant microbes of the normal flora of man.
The support of ExOxEmis, Inc. is gratefully acknowledged.