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Most noncytoplasmic bacterial proteins are exported through the SecYEG channel in the cytoplasmic membrane. This channel and its associated proteins, collectively referred to as the Sec pathway, have strong appeal as a possible antibiotic drug target, yet progress toward new drugs targeting this pathway has been slow, perhaps due partly to many researchers' focus on a single component, the SecA ATPase. Here we report on a pathway-based screen in which beta-galactosidase (β-gal) activity is trapped in the cytoplasm of Escherichia coli cells if translocation through SecYEG is impaired. Several hit compounds passed a counterscreen distinguishing between β-gal overexpression and impaired β-gal export. However, the most extensively characterized hit gave limited E. coli growth inhibition (EC(50) ≥ 400 µM), and growth inhibition could not be unambiguously linked to the compound's effect on the Sec pathway. Our study and others underscore the challenges of finding potent druglike hits against this otherwise promising drug target.

Rationale: Progress has been made in understanding how the cystic fibrosis (CF) basic defect produces lung infection susceptibility. However, it remains unclear why CF exclusively leads to chronic infections that are noninvasive and highly resistant to eradication. Although biofilm formation has been suggested as a mechanism, recent work raises questions about the role of biofilms in CF. Objectives: To learn how airway conditions attributed to CF transmembrane regulator dysfunction could lead to chronic infection, and to determine if biofilm-inhibiting genetic adaptations that are common in CF isolates affect the capacity of Pseudomonas aeruginosa to develop chronic infection phenotypes. Methods: We studied P. aeruginosa isolates grown in agar and mucus gels containing sputum from patients with CF and measured their susceptibility to killing by antibiotics and host defenses. We also measured the invasive virulence of P. aeruginosa grown in sputum gels using airway epithelial cells and a murine infection model. Measurements and Main Results: We found that conditions likely to result from increased mucus density, hyperinflammation, and defective bacterial killing could all cause P. aeruginosa to grow in bacterial aggregates. Aggregated growth markedly increased the resistance of bacteria to killing by host defenses and antibiotics, and reduced their invasiveness. In addition, we found that biofilm-inhibiting mutations do not impede aggregate formation in gel growth environments. Conclusions: Our findings suggest that conditions associated with several CF pathogenesis hypotheses could cause the noninvasive and resistant infection phenotype, independently of the bacterial functions needed for biofilm formation.

More than 20% of bacterial proteins are noncytoplasmic, and most of these pass through the SecYEG channel en route to the periplasm, cell membrane, or surrounding environment. The Sec pathway, encompassing SecYEG and several associated proteins (SecA, SecB, YidC, SecDFYajC), is of interest as a potential drug target because it is distinct from targets of current drugs, is essential for bacterial growth, and exhibits dissimilarities in eukaryotes and bacteria that increase the likelihood of selectively inhibiting the microbial pathway. As a step toward validating the pathway as a drug target, we have adapted a mechanism-based whole-cell assay in a manner suitable for high-throughput screening (HTS). The assay uses an engineered strain of Escherichia coli that accumulates beta-galactosidase (β-gal) in its cytoplasm if translocation through SecYEG is blocked. The assay should facilitate rapid identification of compounds that specifically block the Sec pathway because widely, toxic compounds and nonspecific protein synthesis inhibitors prevent β-gal production and thus do not register as hits. Testing of current antibiotics confirmed that they do not generally act through the Sec pathway. A mini-screen of 800 compounds indicated the assay's readiness for larger screening projects.

Reactive oxygen intermediates generated by neutrophils kill bacteria and are implicated in inflammatory tissue injury, but precise molecular targets are undefined. We demonstrate that neutrophils use myeloperoxidase (MPO) to convert methionine residues of ingested Escherichia coli to methionine sulfoxide in high yield. Neutrophils deficient in individual components of the MPO system (MPO, H 2 O 2 , chloride) exhibited impaired bactericidal activity and impaired capacity to oxidize methionine. HOCl, the principal physiologic product of the MPO system, is a highly efficient oxidant for methionine, and its microbicidal effects were found to correspond linearly with oxidation of methionine residues in bacterial cytosolic and inner membrane proteins. In contrast, outer envelope proteins were initially oxidized without associated microbicidal effect. Disruption of bacterial methionine sulfoxide repair systems rendered E. coli more susceptible to killing by HOCl, whereas over-expression of a repair enzyme, methionine sulfoxide reductase A, rendered them resistant, suggesting a direct role for methionine oxidation in bactericidal activity. Prominent among oxidized bacterial proteins were those engaged in synthesis and translocation of peptides to the cell envelope, an essential physiological function. Moreover, HOCl impaired protein translocation early in the course of bacterial killing. Together, our findings indicate that MPO-mediated methionine oxidation contributes to bacterial killing by neutrophils. The findings further suggest that protein translocation to the cell envelope is one important pathway targeted for damage.

An imbalance between the proteolytic activity of matrix metalloproteinases (MMPs) and the activity of tissue inhibitors of metalloproteinases (TIMPs) is implicated in tissue injury during inflammation. The N-terminal cysteine of TIMP-1 plays a key role in the inhibitory activity of the protein because it coordinates the essential catalytic Zn2+ of the MMP, preventing the metal ion from functioning. An important mechanism for controlling the interaction of TIMPs with MMPs might involve hypochlorous acid (HOCl), a potent oxidant produced by the myeloperoxidase (MPO) system of phagocytes. Here, we show that HOCl generated by the MPO-H2O2-chloride system inactivates TIMP-1 by oxidizing its N-terminal cysteine. The product is a novel 2-oxo acid. Liquid chromatography-mass spectrometry and tandem mass spectrometry analyses demonstrated that methionine and N-terminal cysteine residues were rapidly oxidized by MPO-derived HOCl but only oxidation of the N-terminal cysteine of TIMP-1 correlated well with loss of inhibitory activity. Importantly, we detected the signature 2-oxo-acid N-terminal peptide in tryptic digests of bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome, demonstrating that TIMP-1 oxidation occurs in vivo. Loss of the N-terminal amino group and disulfide structure are crucial for preventing TIMP-1 from inhibiting MMPs. Our findings suggest that pericellular production of HOCl by phagocytes is a pathogenic mechanism for impairing TIMP-1 activity during inflammation.

It is increasingly clear that there are caspase-dependent and -independent mechanisms for the execution of cell death and that the utilization of these mechanisms is stimulus- and cell type–dependent. Intriguingly, broad-spectrum caspase inhibition enhances death receptor agonist-induced cell death in a few transformed cell lines. Endogenously produced oxidants are causally linked to necroticlike cell death in these instances. We report here that broad-spectrum caspase inhibitors effectively attenuated apoptosis induced in human neutrophils by incubation with agonistic anti-Fas antibody or by coincubation with tumor necrosis factor-α (TNF-α) and cycloheximide ex vivo. In contrast, the same caspase inhibitors could augment cell death upon stimulation by TNF-α alone during the 6-hour time course examined. Caspase inhibitor–sensitized, TNF-α–stimulated, dying neutrophils exhibit apoptoticlike and necroticlike features. This occurred without apparent alteration in nuclear factor–κB (NF-κB) activation. Nevertheless, intracellular oxidant production was enhanced and sustained in caspase inhibitor-sensitized, TNF-α–stimulated neutrophils obtained from healthy subjects. However, despite reduced or absent intracellular oxidant production following TNF-α stimulation, cell death was also augmented in neutrophils isolated from patients with chronic granulomatous disease incubated with a caspase inhibitor and TNF-α. These results demonstrate that, in human neutrophils, TNF-α induces a caspase-independent but protein synthesis–dependent cell death signal. Furthermore, they suggest that TNF-α activates a caspase-dependent pathway that negatively regulates reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity.

The generation of extracellular oxidants by neutrophils has been widely investigated, but knowledge about the chemical reactions that occur in the phagolysosome, the cellular compartment that kills pathogens, is more limited. One important pathway may involve the production of potent halogenating agents such as hypochlorous acid (HOCl) by the myeloperoxidase-hydrogen peroxide-halide system. However, explorations of the oxidation chemistry of phagolysosomes have been hampered by the organelle's inaccessibility. To overcome this limitation, we recovered Escherichia coli that had been internalized by human neutrophils. We then analyzed the bacterial proteins for 3-chlorotyrosine, a stable marker of damage by HOCl. Mass spectrometric analysis revealed that levels of 3-chlorotyrosine in E. coli proteins increased markedly after the bacteria were internalized by human neutrophils. This increase failed to occur in E. coli exposed to neutrophils deficient in NADPH oxidase or myeloperoxidase, implicating H(2)O(2) and myeloperoxidase in the halogenation reaction. The extent of protein chlorination by normal neutrophils paralleled bacterial killing. Our observations support the view that the phagolysosome of human neutrophils uses the myeloperoxidase-hydrogen peroxide-chloride system to chlorinate bacterial proteins. In striking contrast, human neutrophils failed to nitrate bacterial proteins unless the medium was supplemented with 1 mm nitrite, and the level of nitration was low. Protein chlorination associated with bacterial killing was unaffected by the presence of nitrite in the medium. Nitration required NADPH oxidase but appeared to be independent of myeloperoxidase, suggesting that neutrophils can nitrate proteins through a pathway that requires nitrite but is independent of myeloperoxidase.

Review of myeloperoxidase supporting optimal microbicidal activity in the phagosomes of human neutrophils. Successful immune defense requires integration of multiple effector systems to match the diverse virulence properties that members of the microbial world might express as they initiate and promote infection. Human neutrophils—the first cellular responders to invading microbes—exert most of their antimicrobial activity in phagosomes, specialized membrane-bound intracellular compartments formed by ingestion of microorganisms. The toxins generated de novo by the phagocyte NADPH oxidase and delivered by fusion of neutrophil granules with nascent phagosomes create conditions that kill and degrade ingested microbes. Antimicrobial activity reflects multiple and complex synergies among the phagosomal contents, and optimal action relies on oxidants generated in the presence of MPO. The absence of life-threatening infectious complications in individuals with MPO deficiency is frequently offered as evidence that the MPO oxidant system is ancillary rather than essential for neutrophil-mediated antimicrobial activity. However, that argument fails to consider observations from humans and KO mice that demonstrate that microbial killing by MPO-deficient cells is less efficient than that of normal neutrophils. We present evidence in support of MPO as a major arm of oxidative killing by neutrophils and propose that the essential contribution of MPO to normal innate host defense is manifest only when exposure to pathogens overwhelms the capacity of other host defense mechanisms.

In this report we show that interferon gamma treatment of U937 cells induces increased expression of the gamma-subunit of the high-affinity Fc receptor for IgG (Fc gamma RI). Interferon treatment results in a 10-fold increased expression of the gamma-subunit and induces expression of a phosphorylated form (gamma 1). The increased expression of the gamma-subunit correlates with its ability to transmit a signal via Fc gamma R, as measured by activation of the respiratory burst using insoluble immune complexes. During Fc gamma R activation, a mobility shift occurs in the phosphorylated form of this gamma 1-subunit. Phosphoamino acid analysis demonstrates that this gamma 1 subunit is threonine phosphorylated in resting differentiated U937 cells and becomes predominantly serine phosphorylated on Fc receptor activation. The mobility shift in the gamma-subunit can be induced by treating U937 cells with phorbol 12-myristate 13-acetate or by monoclonal antibody cross-linking of Fc gamma RI. Hence the gamma-subunit is serine phosphorylated in response to Fc gamma RI and protein kinase C activation. Therefore the gamma-subunit, initially described as a subunit of Fc epsilon RI, now appears to be involved in signal transduction via Fc gamma RI. The data also suggest that the gamma-subunit, in contrast with the zeta-subunit of the T-cell receptor-CD3 complex, is a substrate for serine/threonine kinase(s) in the cell. The serine phosphorylation of the gamma-subunit suggests a divergence of structure and function between the gamma-subunit and its homologue, the zeta-subunit of the T-cell receptor. Phosphorylation of the gamma-subunit on serine may play some regulatory role in Fc gamma RI signal transduction in myeloid cells.

The resonance Raman spectra of neutrophil cytochrome b558 obtained upon Soret excitation indicate that the heme is low spin six-coordinate in both ferric and ferrous oxidation states; comparison with the spectra of bis-imidazole hemin suggests imidazole or imidazolate axial ligation. Minor bands attributable to vibrational motions of ring-conjugated vinyl substituents were also observed, consistent with a heme assignment of protoporphyrin IX. The spectra of deoxycholate-solubilized cytochrome b558 were indistinguishable from neutrophil plasma membranes or specific granules, as were spectra from unstimulated and phorbol myristate acetate-stimulated cells, indicating that the hemes are structurally identical in various subcellular environments and cellular physiological states. However, structural complexity was suggested by biphasic ferric-ferrous photoreduction under 413-nm illumination and the absence of an EPR spectrum for the ferric heme under conditions where simple bis-imidazole heme-containing cytochromes are expected to give detectable signals. Midpoint reduction potentials and resonance Raman spectra of the soluble cytochrome b558 from an individual with cytochrome b558 positive (type IA.2) chronic granulomatous disease were nearly identical to normal oxidase, with the exception that the deficient oxidase did not undergo heme photoreduction. Possible structural models are discussed in relation to other physical properties (ligand binding, thermodynamic potentials) exhibited by the cytochrome.

Neutrophils and monocytes employ a diverse array of antimicrobial effector systems to support their host defense functions. The mechanisms of action of most of these systems are incompletely understood. The present report indicates that microbicidal activity by a neutrophil-derived antimicrobial system, consisting of myeloperoxidase, enzymatically generated hydrogen peroxide, and chloride ion, is accompanied by prompt cessation of DNA synthesis in Escherichia coli, as determined by markedly reduced incorporation of [3H]thymidine into trichloracetic acid-precipitable material. Simultaneously, the myeloperoxidase system mediates a decline in the ability of E. coli membranes to bind hemimethylated DNA sequences containing the E. coli chromosomal origin of replication (oriC). Binding of oriC to the E. coli membrane is an essential element of orderly chromosomal DNA replication. Comparable early changes in DNA synthesis and DNA-membrane interactions were not observed with alternative oxidant or antibiotic-mediated microbicidal systems. It is proposed that oxidants generated by the myeloperoxidase system modify the E. coli membrane in such a fashion that oriC binding is markedly impaired. As a consequence chromosomal DNA replication is impaired and organisms can no longer replicate.

Neutrophil myeloperoxidase, hydrogen peroxide, and chloride constitute a potent antimicrobial system with multiple effects on microbial cytoplasmic membranes. Among these is inhibition of succinate-dependent respiration mediated, principally, through inactivation of succinate dehydrogenase. Succinate-dependent respiration is inhibited at rates that correlate with loss of microbial viability, suggesting that loss of respiration might contribute to the microbicidal event. Because respiration in Escherichia coli can be mediated by dehydrogenases other than succinate dehydrogenase, the effects of the myeloperoxidase system on other membrane dehydrogenases were evaluated by histochemical activity stains of electrophoretically separated membrane proteins. Two bands of succinate dehydrogenase activity proved the most susceptible to inactivation with complete loss of staining activity within 20 min, under the conditions employed. A group with intermediate susceptibility, consisting of lactate, malate, glycerol-3-phosphate, and dihydroorotate dehydrogenases as well as three bands of glucose-6-phosphate dehydrogenase, was almost completely inactivated within 30 min. The relatively resistant group, including the dehydrogenases for glutamate, NADH, and NADPH and the remaining bands of glucose-6-phosphate dehydrogenase, retained substantial amounts of diaphorase activity for up to 60 min of incubation with the myeloperoxidase system. The differential effects of myeloperoxidase on dehydrogenase inactivation could not be correlated with published enzyme contents of flavin or iron-sulfur centers, potential targets of myeloperoxidase-derived oxidants. Despite the relative resistance of NADH dehydrogenase/diaphorase activity to myeloperoxidase-mediated inactivation, electron transport particles prepared from E. coli incubated for 20 min with the myeloperoxidase system lost 55% of their NADH oxidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract —Superoxide, the reduced form of molecular oxygen, has been implicated in the genesis of vascular disease. One potential mechanism involves oxidation of low density lipoprotein into an atherogenic particle. A second involves reaction with nitric oxide to generate peroxynitrite, a highly oxidizing intermediate. A third involves regulation of signal transduction in artery wall cells. One well-characterized pathway for superoxide production resides in macrophages, the cellular hallmark of the early atherosclerotic lesion. Macrophages contain a membrane-bound NADPH oxidase that reduces oxygen to superoxide. In the current studies, we used mice that are deficient in the gp91-phox subunit of the NADPH oxidase–a model of chronic granulomatous disease (CGD)–to explore the role of superoxide in atherosclerotic vascular disease. Wild-type and CGD mice on the C57BL/6 background received a high-fat diet for 20 weeks to induce hypercholesterolemia. At the end of this period, the 2 strains of mice had comparable plasma lipid levels, and their atherosclerotic lesions were similar in size. We also crossed CGD mice with apolipoprotein E–deficient (apoE−/−) mice to generate spontaneously hypercholesterolemic animals that lacked functional NADPH oxidase. After 24 weeks, the CGD-apoE−/− animals had lower plasma cholesterol and triglyceride levels than did the apoE−/− animals, but there was no difference in the extent of atherosclerotic plaque. Our findings suggest that superoxide generated by the NADPH oxidase of phagocytes does not promote atherosclerosis in mice with either diet-induced or genetic forms of hypercholesterolemia.

Myeloperoxidase (MPO) is an abundant constituent (up to 5% of dry weight) of polymorphonuclear leukocytes [1]. In conjunction with phagocyte-generated hydrogen peroxide and any of several cofactors, the enzyme catalyzes the formation of a broad array of microbicidal products. The number of potentially microbicidal products exceeds the several effective cofactors implicated, including the halides chloride, bromide, iodide, the pseudohalide thiocyanate, and nitrite [1, 2]. The in vivo relevance of the cofactors based on their anticipated physiologic concentrations is open to debate. Chloride, however, is clearly available at the greater than to mM concentrations required for it to be an effective MPO cofactor.

Nitration of tyrosine residues in proteins occurs in a wide range of inflammatory diseases involving neutrophil and macrophage activation. We report that both myeloperoxidase (MPO) and horseradish peroxidase (HRP) utilize nitrite (NO 2 − ) and hydrogen peroxide (H 2 O 2 ) as substrates to catalyze tyrosine nitration in proteins. MPO was approximately 10 times more effective than HRP as a nitration catalyst of bovine serum albumin (BSA). Nitration of BSA by MPO did not require chloride as a cofactor. Physiologic levels of chloride did not significantly inhibit nitration by MPO. Oxidation of chloride to hypochlorous acid (HOCl) is catalyzed by MPO but not by HRP, yet HRP also catalyzed nitration from hydrogen peroxide plus nitrite. Therefore, HOCl formation was not obligatory for tyrosine nitration. Although HOCl plus nitrite can nitrate the amino acid tyrosine in simple solutions, protein nitration by HOCl plus nitrite was not observed in heart homogenates, probably due to the presence of multiple alternative targets of both HOCl and HOCl plus nitrite. In contrast, MPO catalyzed nitration of many proteins in rat heart homogenates using NO 2 − plus H 2 O 2 , suggesting that peroxidase-catalyzed nitration of tyrosine could occur in the presence of competing substrates in vivo. HOCl could substitute for H 2 O 2 as the oxidizing substrate for nitration of either BSA or tissue homogenates catalyzed by either peroxidase. Activated neutrophils may generate nitrotyrosine by several mechanisms, including peroxynitrite, HOCl plus nitrite, and a chloride-independent mechanism involving MPO, nitrite, and hydrogen peroxide.

Neutrophil microbicidal activity is a consequence of overlapping antimicrobial systems that vary in prominence according to the conditions of the neutrophil-microbe interaction, the nature of the microbe, and its metabolic state. In this study, normal, myeloperoxidase-deficient, and respiratory burst-deficient (chronic granulomatous disease [CGD]) neutrophils killed Escherichia coli with equivalent, high efficiencies. Killing by CGD and myeloperoxidase-deficient neutrophils was not augmented by supplements, such as exogenous H2O2 and myeloperoxidase, directed at ameliorating their metabolic defects, suggesting that nonoxidative microbicidal systems were sufficient for a full microbicidal effect. Neutrophils with an intact myeloperoxidase antimicrobial system (normal or appropriately supplemented deficient cells) were capable of rapidly suppressing E. coli DNA synthesis, while unsupplemented CGD or myeloperoxidase-deficient cells were far less effective, indicating that the myeloperoxidase system was active in normal neutrophils. The degree of DNA synthesis inhibition by myeloperoxidase-sufficient neutrophils could account, in a cell-free system, for most of the observed microbicidal activity. While the myeloperoxidase system was active and probably bactericidal, it was not rate limiting for microbicidal activity and appears to have been redundant with other microbicidal systems in the cell. Rapid and extensive inhibition of bacterial DNA synthesis appears to be an indicator of myeloperoxidase activity in neutrophils.

Publisher Summary This chapter discusses the peroxynitrite-dependent tyrosine nitration catalyzed by superoxide dismutase, myeloperoxidase, and horseradish peroxidase. Peroxynitrite is a potent nitrating agent formed by the near diffusion limited reaction of superoxide and nitric oxide (NO). Peroxynitrite is more cytotoxic to Escherichia coli and Trypanosoma cruzi than NO, superoxide, or hydrogen peroxide. Peroxynitrite is a strong oxidant capable of yielding the chemistry commonly attributed to hydroxyl radical damage. The peroxynitrite anion can exist in two stable conformers. The cis conformer, which is ring-like because of overlapping of the terminal oxygen orbitals, is slightly more stable and is the only conformer found in alkaline solution, cis-Peroxynitrite cannot directly isomerize to nitrate. The simplest means to observe tyrosine nitration is to mix rapidly a small volume of peroxynitrite with a solution containing the target phenol and measure the resulting yellow absorbance from the nitrophenolic. Because peroxynitrite rapidly decomposes at neutral pH, the secret to reproducibility involves rapid mixing and accurate pipetting.

Fc-receptor stimulation in certain myeloid cells results in an increase in oxygen consumption termed the respiratory burst. In this report we examine the effects of protein tyrosine phosphatase inhibitors on the Fc gamma receptor-induced myeloid respiratory burst. Antiphosphotyrosine immunoblotting of neutrophils stimulated with opsonized oil particles shows that Fc-receptor stimulation is associated with the tyrosine phosphorylation of cellular proteins. Pretreatment of neutrophils for 10 min with vanadate or phenylarsine oxide (PAO), protein tyrosine phosphatase inhibitors, augments tyrosine phosphorylation in response to Fc-receptor stimulation. Vanadate and PAO inhibit the respiratory burst in a dose-dependent fashion, but have no effect on Fc gamma receptor-mediated phagocytosis, suggesting that the inhibition of the respiratory burst is not due to a general inhibition of Fc gamma-receptor signaling. Neutrophil phagolysosomal membranes were isolated from vanadate-treated and control neutrophils after Fc-receptor stimulation show a reduction in protein tyrosine phosphatase activity and a reduction in the NADPH-dependent oxidase activity and contain greater amounts of phosphotyrosine, relative to control membranes. Vanadate did not inhibit the NADPH-oxidase directly or interfere with the superoxide assay. Vanadate and PAO also inhibited the respiratory burst of interferon-differentiated U937 cells in response to immune complex and Fc gamma RI crosslinking. Pretreatment of U937 cells with PAO completely blocks the serine phosphorylation of the gamma subunit of the Fc gamma R, a response that is associated with Fc gamma RI-receptor activation. The data supports the recent observation that CD45 modulates signal transduction through the Fc gamma RI receptor, suggesting that protein tyrosine phosphatases play a positive modulatory role in the signal relay pathway(s) involving the myeloid Fc gamma RI receptor, resulting in the phosphorylation of the gamma subunit and the activation of the NADPH-oxidase complex.

Hypochlorous acid and related oxidants derived from myeloperoxidase-catalyzed reactions contribute to the microbicidal activities of phagocytosing neutrophils and monocytes. Microbial iron-sulfur (Fe/S) clusters have been suggested as general targets of myeloperoxidase-derived oxidations, but no susceptible Fe/S site has yet been identified. In this study, the effects of HOCl and myeloperoxidase-catalyzed peroxidation of chloride ion upon EPR-detectable Fe/S clusters in Escherichia coli and Pseudomonas aeruginosa were examined. Increasing amounts of oxidant produced progressive loss of signal amplitudes from the S-1 and S-3 Fe/S clusters of succinate:ubiquinone oxidoreductase in respiring membrane fragments. These changes were compared to loss of microbial viability, succinate uptake rates, succinate dehydrogenase activity and succinate-dependent respiration. The amounts of oxidant required to destroy Fe/S clusters exceeded the amounts required to kill organisms or inhibit respiratory function by factors of four or five. Power saturation characteristics of the S-1 signal indicated that the S-2 signal was also resistant to modification, even in highly oxidized membranes. Loss of succinate-dependent respiration was closely associated with HOCl and myeloperoxidase-mediated microbicidal activity against P. aeruginosa and was also an early event in the oxidant-mediated metabolic dysfunctions of E. coli. However, these effects were not caused by the destruction of the Fe/S clusters within the succinate:ubiquinone oxidoreductase. Rather, the major respiration-inhibiting lesion(s) appeared to reside at points in the respiratory chain between the Fe/S clusters and the ubiquinone reductase site.

A previously described assay for neutrophil phagocytosis of oil droplets labeled with the lipophilic red dye, oil red O, has been adapted to a microtiter plate format. Oil laden neutrophils are fixed to the plate with glutaraldehyde, washed free of uningested oil, and evaluated for degree of oil red O uptake by direct determination of light absorption at 540 nm in a microtiter plate reader. Advantages of the modified procedure include the ability to determine rates of phagocytosis (multiple determinations) under diverse conditions, simultaneously and with greater facility. Multiple centrifugation steps are eliminated and the requirement for extraction of oil red O from each cell pellet prior to quantitation becomes optional. The system has been evaluated for oil droplets emulsified with lipopolysaccharide alone or with lipopolysaccharide together with bovine serum albumin. For droplets emulsified with lipopolysaccharide alone, efficient opsonization was accomplished with fresh serum alone, through heat labile, presumably complement-dependent, pathways. For droplets emulsified with lipopolysaccharide and bovine serum albumin, specific antiserum to bovine serum albumin was a considerably more efficient opsonin than non-immune fresh serum. A third system is described in which tetanus toxoid was introduced into oil droplets that had been previously emulsified with lipopolysaccharide. Inclusion of toxoid antigens rendered the oil droplets susceptible to opsonization with tetanus immune globulin, making the assay system possibly applicable to evaluation of immunoglobulin function in humans.

Myeloperoxidase (MPO), H2O2, and chloride comprise a potent antimicrobial system believed to contribute to the antimicrobial functions of neutrophils and monocytes. The mechanisms of microbicidal action are complex and not fully defined. This report describes the MPO-mediated inactivation, in Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, of a class of cytoplasmic membrane enzymes (penicillin-binding proteins, PBPs) found in all eubacteria, that covalently bind beta-lactam antibiotics to their active sites with loss of enzymatic activity. Inactivation of "essential" PBPs, including PBP1-PBP3 of E. coli, leads to unbalanced bacterial growth and cell death. MPO treatment of bacteria was associated with loss of penicillin binding by PBPs, strongly suggesting PBP inactivation. In E. coli, PBP inactivation was most rapid with PBP3, where the rate of decline in binding activity approximated but did not equal loss of viability. Changes in E. coli morphology (elongation), observed just before bacteriolysis, were consistent with early predominant inactivation of PBP3. We conclude that inactivation of essential PBPs is sufficient to account for an important fraction of MPO-mediated bacterial action. This feature of MPO action interestingly recapitulates an antibacterial strategy evolved by beta-lactam-producing molds that must compete with bacteria for limited ecologic niches.

Two of the cytosolic NADPH oxidase components, p47-phox and p67-phox, translocate to the plasma membrane in normal neutrophils stimulated with phorbol myristate acetate (PMA). We have now studied the translocation process in neutrophils of patients with chronic granulomatous disease (CGD), an inherited syndrome in which the oxidase system fails to produce superoxide due to lesions affecting any one of its four known components: the gp91-phox and p22-phox subunits of cytochrome b558 (the membrane-bound terminal electron transporter of the oxidase), p47-phox, and p67-phox. In contrast to normal cells, neither p47-phox nor p67-phox translocated to the membrane in PMA-stimulated CGD neutrophils which lack cytochrome b558. In one patient with a rare X-linked form of CGD caused by a Pro----His substitution in gp91-phox, but whose neutrophils have normal levels of this mutant cytochrome b558, translocation was normal. In two patients with p47-phox deficiency, p67-phox failed to translocate, whereas p47-phox was detected in the particulate fraction of PMA-stimulated neutrophils from two patients deficient in p67-phox. Our data suggest that cytochrome b558 or a closely linked factor provides an essential membrane docking site for the cytosolic oxidase components and that it is p47-phox that mediates the assembly of these components on the membrane.

Extracellular superoxide was detected in cultures of monkey and human arterial smooth muscle cells as indicated by superoxide dismutase inhibitable reduction of cytochrome c. Superoxide production by these cells in the presence of Fe or Cu resulted in modification of low density lipoprotein (LDL). The degree of LDL modification was directly proportional to the rate of superoxide production by cells. Superoxide dismutase (100 micrograms/ml), and the general free radical scavengers butylated hydroxytoluene and butylated hydroxyanisole (50 microM), inhibited Fe- and Cu-mediated modification of LDL by monkey smooth muscle cells, while catalase (100 micrograms/ml) and mannitol (25 mM) had no effect. The chelators desferrioxamine and diethylenetriamine pentaacetic acid completely inhibited Fe- and Cu-promoted modification of LDL, while EGTA had no inhibitory effect. EDTA stimulated Fe-promoted modification in the 1-100 microM range while inhibiting Cu-mediated modification of LDL. LDL modified by smooth muscle cells in the presence of 10 microM Fe or Cu stimulated [14C]oleate incorporation into cholesteryl ester by human macrophages and murine J774 cells to a degree comparable to that produced by acetylated LDL. LDL incubated with smooth muscle cells and metal ions in the presence of superoxide dismutase failed to enhance macrophage cholesteryl ester accumulation. Thus, arterial smooth muscle cells in culture generate superoxide and modify LDL by a superoxide-dependent, Fe or Cu catalyzed free radical process, resulting in enhanced uptake of the modified LDL by macrophages. Neither hydroxyl radicals nor H2O2 are likely to be involved. Superoxide-dependent lipid peroxidation may contribute to biological modification of LDL, resulting in foam cell formation and atherogenesis.

In an earlier study, an enzymic superoxide anion-generating system consisting of acetaldehyde plus xanthine oxidase was found to be toxic to Staphylococcus aureus. Both superoxide anion (O ) and its dismutation product hydrogen peroxide (H2O2) were required and it was proposed that (O ) and H2O2 interact to form the more powerful bactericidal agent(s), hydroxyl radical (OH·) and/or singlet oxygen. Iron chelated by EDTA appears to be a heretofore unrecognized requirement for the xanthine oxidase bactericidal system. The evidence is as follows: (1) the addition of iron salts to the xanthine oxidase system increased bactericidal activity whereas the iron chelators diethylenetriaminepentaacetic acid (DTPA) and desferrioxamine were inhibitory; (2) dialysis of the EDTA-containing xanthine oxidase preparation abolished bactericidal activity which was restored on the addition of EDTA; (3) removal of trace amounts of iron by passage of salt solutions through a Chelex-100 column abolished bactericidal activity which was restored on the addition of iron. Iron and EDTA were most effective when present at 1:1 stoichiometry and they could not be replaced by a variety of other metals or chelators. The bactericidal activity of the acetaldehyde-xanthine oxidase-iron-EDTA system was inhibited by superoxide dismutase, catalase, and the OH · scavengers ethanol and mannitol, suggesting that the complex served as a catalyst of the reaction between (O ) and H2O2 to form OH· (Haber-Weiss reaction). Possible reasons for the relative catalytic specificity of iron-EDTA are considered.

Myeloperoxidase, a granule-associated enzyme of neutrophils and monocytes, combines with H2O2 and chloride to form a potent microbicidal system that contributes to phagocyte antimicrobial activity. The nature of the lesion or lesions induced by the myeloperoxidase system which are responsible for the loss of microbial replicative activity (viability) remains unknown. Using Escherichia coli grown to late log or stationary phase under conditions of low aeration with succinate as the sole carbon source, we found that myeloperoxidase-induced loss of microbial viability could be correlated with a decrease in succinate-dependent respiration (succinate oxidase activity). Succinate dehydrogenase activity fell rapidly to undetectable levels during incubation with the myeloperoxidase system, suggesting that damage to the dehydrogenase was a major factor in the loss of oxidase activity. Other components of the succinate oxidase system were resistant to the actions of myeloperoxidase. The ubiquinone-8 and cytochrome components of the respiratory chain remained nearly constant in amount despite reduction of respiration to undetectable levels. However, as expected from the loss of succinate dehydrogenase activity, succinate-ubiquinone reductase and succinate-cytochrome reductase activities were markedly impaired. We propose that the loss of E. coli viability induced by the myeloperoxidase-H2O2-chloride system is due in part to the loss of electron transport function consequent to the oxidation of critical catalytic centers in susceptible dehydrogenases.

Extracellular superoxide (O2-.) was detected in cultures of monkey arterial smooth muscle cells as measured by the superoxide dismutase-inhibitable reduction of cytochrome c and acetylated cytochrome c. Reduction of cytochrome c by these cells required L-cystine in the incubation medium. A variety of other sulfur-containing amino acids, including D-cystine, L-cystathionine, L-methionine, and djenkolic acid did not support O2-. generation when present at concentrations equimolar to L-cystine. At millimolar concentrations, the chelators EDTA and diethylene triamine penta-acetic acid inhibited O2-. production by smooth muscle cells. This effect was maximal when the chelator was present at the same concentration as the sum of the Ca2+ and Mg2+ in the medium, suggesting a role for these cations in O2-. generation by cells. Modification of low density lipoprotein (LDL) by arterial smooth muscle cells, as assessed by changes in lipid peroxide content, mobility on agarose gel electrophoresis, and apoprotein B fragmentation, was also L-cystine-dependent. LDL modification also required micromolar concentrations of the transition metal ion Cu(II) or Fe(III) and was inhibited by superoxide dismutase. LDL modified by smooth muscle cells in the presence of L-cystine and Cu(II) was taken up and degraded less well than native LDL by human skin fibroblasts, suggesting that recognition by the LDL receptor was lost. In contrast, LDL modified by smooth muscle cells was taken up and degraded to a greater degree than native LDL by mouse peritoneal macrophages, consistent with recognition by the scavenger receptor. These results indicate that monkey arterial smooth muscle cells produce O2-. and modify LDL by an L-cystine-dependent process. This may involve reduction of cystine to a thiol, possibly cysteine or a cysteine-containing peptide such as glutathione. Sulfur-containing amino acids may play a role in atherogenesis by supporting cell-mediated generation of reactive oxygen species and modification of lipoprotein to a form recognized by the scavenger receptor.

Deferoxamine accelerates the autooxidation of iron as measured by the rapid disappearance of Fe2+, the associated appearance of Fe3+, and the uptake of oxygen. Protons are released in the reaction. The formation of H2O2 was detected by the horseradish peroxidase-catalyzed oxidation of scopoletin, and the formation of hydroxyl radicals (OH·) was suggested by the formation of the OH· spin trap adduct (DMPO/OH)· with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and the generation of the methyl radical adduct on the further addition of dimethyl sulfoxide. (DMPO/OH)· adduct formation was inhibited by catalase but not by superoxide dismutase. The oxidant formed converted iodide to a trichloroacetic acid-precipitable form (iodination) and was bactericidal to logarithmic phase Escherichia coli. Both iodination and bactericidal activity was inhibited by catalase and by OH· scavengers, but not by superoxide dismutase. Iodination was optimal in 5 × 10-4 M acetate buffer, pH 5.0, and when the Fe2+ and deferoxamine concentrations were equimolar at 10-4 M. Fe2+ could not be replaced by Fe3+, Co2+, Zn2+, Ca2+, Mg2+, or Mn2+, or deferoxamine by EDTA, diethylenetriaminepentaacetic acid, or bathophenanthroline. These findings indicate that Fe2+ and deferoxamine can act as an oxygen radical generating system, which may contribute to its biological effects in vitro and in vivo.

Hypochlorous acid (HOC1) rapidly chlorinates fluorescein compounds forming, sequentially, the corresponding 4'-chlorofluorescein and 4',5'-dichlorofluoresceins. Chlorination by cell-free myeloperoxidase-catalyzed chloride peroxidation systems gives rise to these compounds as well as variable amounts of isomeric compounds chlorinated in the 2'- and 2',7'-positions. The fluorescence intensity of the dianionic form of the dye is partially quenched upon chlorination, and its proton equilibrium constants are shifted to more acidic values. Fluorescein covalently bound to zymosan (5-isothiocyanatofluorescein-zymosan) also formed these products when the unopsonized particles were incubated with phorbol myristate acetate- or N-formyl-methionyl-leucyl-phenylalanine-stimulated human neutrophils. This reaction was associated with a fall in fluorescence intensity, which was not observed when cells from individuals with chronic granulomatous disease or myeloperoxidase deficiency were used or when azide or catalase were added to the reaction medium. Fluorescent changes accompanying phagocytosis of serum-opsonized 5-isothiocyanatofluorescein-zymosan were also consistent with chlorination of the label; the changes were shown to be myeloperoxidase-dependent by use of myeloperoxidase-deficient or azide-treated cells. Oxidative bleaching of the structurally similar sulfonphthalein dyes by HOCl also occurs at rates which parallel the dye basicities. Results are discussed in relation to the use of fluoresceinated particles and sulfonphthalein dyes in the measurement of intraphagosomal acidification.

Human mononuclear cells were used to evaluate the role of superoxide in the oxidation of low density lipoprotein (LDL). Unstimulated cells produced little superoxide or LDL oxidation as assayed by lipid peroxide content. Stimulation of the cells with phorbol myristate acetate (PMA) resulted in an increase both in superoxide production and in LDL oxidation. Mononuclear cell-mediated LDL oxidation was time- and cell number-dependent and was markedly enhanced by the presence of Fe (10 microM). Superoxide was required for the initiation of LDL oxidation as indicated by inhibition of the reaction by early addition of superoxide dismutase (SOD). Propagation of LDL oxidation was superoxide-independent, since the later addition of SOD resulted in progressively less inhibition of LDL oxidation. Propagation of LDL oxidation also was, in part, cell-independent as indicated by continued oxidation of LDL when mononuclear cells were removed following a 1 to 8 hour period with cells. Optimal LDL oxidation required the presence of mononuclear cells throughout the incubation period, suggesting that cellular factors in addition to superoxide play a role in LDL oxidation. Further evidence for the role of superoxide in the oxidation of LDL by mononuclear cells was obtained with cells from patients with genetic deficiencies of either superoxide generation (chronic granulomatous disease) or myeloperoxidase. PMA-stimulated cells from a patient with chronic granulomatous disease neither generated superoxide nor modified LDL. Incubation of LDL with cells from a patient with myeloperoxidase deficiency (in which superoxide production is normal or increased) resulted in oxidation of the lipoprotein equivalent to that observed with normal cells. Other inhibitors of oxidation reactions also were tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of 32P-loaded neutrophils with phorbol myristate acetate causes the labeling of a family of three 48K proteins that focus near neutral pH. The relationship between these phosphoproteins and the activation of the respiratory burst has been supported by the previous finding that phosphorylation was defective in the two most common types of chronic granulomatous disease (CGD): X-linked cytochrome-negative (X/-) and autosomal cytochrome-positive (A/+). In this report, these studies have now been extended to the rare A/- and X/+ forms of the disease. In all three patients with A/- CGD examined, the two most acidic 48K proteins failed to undergo enhanced phosphorylation in response to phorbol stimulation, a finding similar to that seen in X/- patients. In contrast, neutrophils from two patients with X/+ CGD appeared to phosphorylate the neutral 48K proteins in a normal fashion. It thus appears that the different phosphorylation patterns seen in chronic granulomatous disease are a reflection of the genetic heterogeneity of this disorder. These findings lend further support to the conclusion that the 48K phosphoprotein family is related to the respiratory burst, although not necessarily in a straightforward manner.

A microbicidal system, mediated by neutrophil myeloperoxidase, inhibits succinate-dependent respiration in Escherichia coli at rates that correlate with loss of microbial viability. Succinate dehydrogenase, the initial enzyme of the succinate oxidase respiratory pathway, catalyzes the reduction of ubiquinone to ubiquinol, which is reoxidized by terminal oxidase complexes. The steady-state ratio of ubiquinol to total quinone (ubiquinol + ubiquinone) reflects the balance between dehydrogenase-dependent ubiquinone reduction and terminal oxidase-dependent ubiquinol oxidation. Myeloperoxidase had no effect on total quinone content of E. coli but altered the steady-state ratio of ubiquinol to total quinone. The ratio doubled for organisms incubated with the myeloperoxidase system for 10 min, suggesting decreased ubiquinol oxidase activity, which was confirmed by observation of a 50% decrease in oxidation of the ubiquinol analogue 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol. Despite inhibition of ubiquinol oxidase, overall succinate oxidase activity remained unchanged, suggesting that succinate dehydrogenase activity was preserved and that the dehydrogenase was rate limiting. Microbial viability was unaffected by early changes in ubiquinol oxidase activity. Longer (60 min) exposure of E. coli to the myeloperoxidase system resulted in only modest further inhibition of the ubiquinol oxidase, but the ubiquinol to total quinone ratio fell to 0%, reflecting complete loss of succinate dehydrogenase activity. Succinate oxidase activity was abolished, and there was extensive loss of microbial viability. Early myeloperoxidase-mediated injury to ubiquinol oxidase appeared to be compensated for by higher steady-state levels of ubiquinol which sustained electron turnover by mass effect. Later myeloperoxidase-mediated injuries eliminated succinate-dependent ubiquinone reduction, through inhibition of succinate dehydrogenase, with loss of succinate oxidase activity, effects which were associated with, although not clearly causal for, microbicidal activity.

Publisher Summary Peroxidases when combined with H 2 O 2 and a halide (chloride, bromide, iodide, and pseudohalide thiocyanate) form a potent cytotoxic system, which contributes to the host defense against invading microorganisms and possibly tumor cells. Neutrophils and monocytes contain the same peroxidase (myeloperoxidase, MPO), and eosinophils a different peroxidase (eosinophil peroxidase, EPO), in cytoplasmic granules, and these enzymes are discharged into the phagosome following particle ingestion. Phagocytosis also is associated with a respiratory burst and much of the added oxygen consumed is converted to H 2 O 2 . Peroxidase, H 2 O 2 , and a halide interact in the phagosome to destroy the ingested organism. The components of the peroxidase system can also be released extracellularly where they may attack adjacent normal or malignant cells, uningested organisms, or soluble mediators. A variety of methods has been employed for the measurement of the toxicity of the peroxidase system. These methods depend on the nature of the target cell and include the measurement of replication in growth medium, Cr release, metabolic activity, and morphologic changes. This chapter focuses on bactericidal activity as measured by decrease in colony-forming units, using Escherichia coli as the target, MPO as the peroxidase, and chloride as the halide.

The acetaldehyde-xanthine oxidase system in the presence and absence of myeloperoxidase (MPO) and chloride has been employed as a model of the oxygen-dependent antimicrobial systems of the PMN. The unsupplemented xanthine oxidase system was bactericidal at relatively high acetaldehyde concentrations. The bactericidal activity was inhibited by superoxide dismutase (SOD), catalase, the hydroxyl radical (OH.) scavengers, mannitol and benzoate, the singlet oxygen (1O2) quenchers, azide, histidine, and 1,4-diazabicyclo[2,2,2]octane (DABCO) and by the purines, xanthine, hypoxanthine, and uric acid. The latter effect may account for the relatively weak bactericidal activity of the xanthine oxidase system when purines are employed as substrate. A white, carotenoid-negative mutant strain of Sarcina lutea was more susceptible to the acetaldehyde-xanthine oxidase system than was the yellow, carotenoid-positive parent strain. Carotenoid pigments are potent 1O2 quenchers. The xanthine oxidase system catalyzes the conversion of 2,5-diphenylfuran to cis-dibenzoylethylene, a reaction which can occur by a 1O2 mechanism. This conversion is inhibited by SOD, catalase, azide, histidine, DABCO, xanthine, hypoxanthine, and uric acid but is only slightly inhibited by mannitol and benzoate. The addition of MPO and chloride to the acetaldehyde-xanthine oxidase system greatly increases bactericidal activity; the minimal effective acetaldehyde concentration is decreased 100-fold and the rate and extent of bacterial killing is increased. The bactericidal activity of the MPO-supplemented system is inhibited by catalase, benzoate, azide, DABCO, and histidine but not by SOD or mannitol. Thus, the acetaldehyde-xanthine oxidase system which like phagocytosing PMNs generates superoxide (O.2-) and hydrogen peroxide, is bactericidal both in the presence and absence of MPO and chloride. The MPO-supplemented system is considerably more potent; however, when MPO is absent, bactericidal activity is observed which may be mediated by the interaction of H2O2 and O.2- to form OH. and 1O2.

An 8-year-old boy with severe recurrent bacterial infections was found to lack the cell-surface multimeric polypeptide complex defined by murine monoclonal antibody 60.3. This complex is expressed on the surface of all normal polymorphonuclear leucocytes (PMN), monocytes, and lymphocytes. Incubation of normal lymphocytes or PMN with antibody 60.3 produced functional abnormalities comparable with those observed in the patient's cells, suggesting that the polypeptide complex plays a significant part in several membrane-dependent leucocyte functions.