Cathepsin G Degrades Staphylococcus aureus Biofilms.

Polymorphonuclear leukocytes (PMN) phagocytose and kill individual bacteria but are far less efficient when challenged with bacterial aggregates. Consequently, growth within a biofilm affords Staphylococcus aureus some protection but PMN penetrate S. aureus biofilms and phagocytose bacteria, suggesting that enzymes released through neutrophil degranulation degrade biofilms into fragments small enough for phagocytosis. Here we show that the capacity of PMN to invade biofilms depended largely on the activity of secreted cathepsin G.

A structurally dynamic N-terminal region drives function of the staphylococcal peroxidase inhibitor (SPIN).

The heme-containing enzyme myeloperoxidase (MPO) is critical for optimal antimicrobial activity of human neutrophils. We recently discovered that the bacterium Staphylococcus aureus expresses a novel immune evasion protein, called SPIN, that binds tightly to MPO, inhibits MPO activity, and contributes to bacterial survival following phagocytosis. A co-crystal structure of SPIN bound to MPO suggested that SPIN blocks substrate access to the catalytic heme by inserting an N-terminal ß-hairpin into the MPO active-site channel. Here, we describe a series of experiments that more completely define the structure/function relationships of SPIN. Whereas the SPIN N terminus adopts a ß-hairpin confirmation upon binding to MPO, the solution NMR studies presented here are consistent with this region of SPIN being dynamically structured in the unbound state. Curiously, whereas the N-terminal ß-hairpin of SPIN accounts for ∼55% of the buried surface area in the SPIN-MPO complex, its deletion did not significantly change the affinity of SPIN for MPO but did eliminate the ability of SPIN to inhibit MPO. The flexible nature of the SPIN N terminus rendered it susceptible to proteolytic degradation by a series of chymotrypsin-like proteases found within neutrophil granules, thereby abrogating SPIN activity. Degradation of SPIN was prevented by the S. aureus immune evasion protein Eap, which acts as a selective inhibitor of neutrophil serine proteases. Together, these studies provide insight into MPO inhibition by SPIN and suggest possible functional synergy between two distinct classes of S. aureus immune evasion proteins.

Contribution of liver alcohol dehydrogenase to metabolism of alcohols in rats.

The kinetics of oxidation of various alcohols by purified rat liver alcohol dehydrogenase (ADH) were compared with the kinetics of elimination of the alcohols in rats in order to investigate the roles of ADH and other factors that contribute to the rates of metabolism of alcohols. Primary alcohols (ethanol, 1-propanol, 1-butanol, 2-methyl-1-propanol, 3-methyl-1-butanol) and diols (1,3-propanediol, 1,3-butanediol, 1,4-butanediol, 1,5-pentanediol) were eliminated in rats with zero-order kinetics at doses of 5-20 mmol/kg. Ethanol was eliminated most rapidly, at 7.9 mmol/kgh. Secondary alcohols (2-propanol-d7, 2-propanol, 2-butanol, 3-pentanol, cyclopentanol, cyclohexanol) were eliminated with first order kinetics at doses of 5-10 mmol/kg, and the corresponding ketones were formed and slowly eliminated with zero or first order kinetics. The rates of elimination of various alcohols were inhibited on average 73% (55% for 2-propanol to 90% for ethanol) by 1 mmol/kg of 4-methylpyrazole, a good inhibitor of ADH, indicating a major role for ADH in the metabolism of the alcohols. The Michaelis kinetic constants from in vitro studies (pH 7.3, 37 °C) with isolated rat liver enzyme were used to calculate the expected relative rates of metabolism in rats. The rates of elimination generally increased with increased activity of ADH, but a maximum rate of 6±1 mmol/kg h was observed for the best substrates, suggesting that ADH activity is not solely rate-limiting. Because secondary alcohols only require one NAD(+) for the conversion to ketones whereas primary alcohols require two equivalents of NAD(+) for oxidation to the carboxylic acids, it appears that the rate of oxidation of NADH to NAD(+) is not a major limiting factor for metabolism of these alcohols, but the rate-limiting factors are yet to be identified.

Functional consequence of positive selection revealed through rational mutagenesis of human myeloperoxidase.

Myeloperoxidase (MPO) is a member of the mammalian heme peroxidase (MHP) multigene family. Whereas all MHPs oxidize specific halides to generate the corresponding hypohalous acid, MPO is unique in its capacity to oxidize chloride at physiologic pH to produce hypochlorous acid (HOCl), a potent microbicide that contributes to neutrophil-mediated host defense against infection. We have previously resolved the evolutionary relationships in this functionally diverse multigene family and predicted in silico that positive Darwinian selection played a major role in the observed functional diversities (Loughran NB, O'Connor B, O'Fagain C, O'Connell MJ. 2008. The phylogeny of the mammalian heme peroxidases and the evolution of their diverse functions. BMC Evol Biol. 8:101). In this work, we have replaced positively selected residues asparagine 496 (N496), tyrosine 500 (Y500), and leucine 504 (L504) with the amino acids present in the ancestral MHP and have examined the effects on the structure, biosynthesis, and activity of MPO. Analysis in silico predicted that N496F, Y500F, or L504T would perturb hydrogen bonding in the heme pocket of MPO and thus disrupt the structural integrity of the enzyme. Biosynthesis of the mutants stably expressed in human embryonic kidney 293 cells yielded apoproMPO, the heme-free, enzymatically inactive precursor of MPO, that failed to undergo normal maturation or proteolytic processing. As a consequence of the maturational arrest at the apoproMPO stage of development, cells expressing MPO with mutations N496F, Y500F, L504T, individually or in combination, lacked normal peroxidase or chlorinating activity. Taken together, our data provide further support for the in silico predictions of positive selection and highlight the correlation between positive selection and functional divergence. Our data demonstrate that directly probing the functional importance of positive selection can provide important insights into understanding protein evolution.

NADPH oxidase (NOX) isoforms are inhibited by celastrol with a dual mode of action.

BACKGROUND: Celastrol is one of several bioactive compounds extracted from the medicinal plant Tripterygium wilfordii. Celastrol is used to treat inflammatory conditions, and shows benefits in models of neurodegenerative disease, cancer and arthritis, although its mechanism of action is incompletely understood. EXPERIMENTAL APPROACH: Celastrol was tested on human NADPH oxidases (NOXs) using a panel of experiments: production of reactive oxygen species and oxygen consumption by NOX enzymes, xanthine oxidase activity, cell toxicity, phagocyte oxidase subunit translocation, and binding to cytosolic subunits of NOX enzymes. The effect of celastrol was compared with diphenyleneiodonium, an established inhibitor of flavoproteins. KEY RESULTS: Low concentrations of celastrol completely inhibited NOX1, NOX2, NOX4 and NOX5 within minutes with concentration-response curves exhibiting higher Hill coefficients and lower IC50 values for NOX1 and NOX2 compared with NOX4 and NOX5, suggesting differences in their mode of action. In a cell-free system, celastrol had an IC50 of 1.24 and 8.4 µM for NOX2 and NOX5, respectively. Cytotoxicity, oxidant scavenging, and inhibition of p47(phox) translocation could not account for NOX inhibition. Celastrol bound to a recombinant p47(phox) and disrupted the binding of the proline rich region of p22(phox) to the tandem SH3 domain of p47(phox) and NOXO1, the cytosolic subunits of NOX2 and NOX1, respectively. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that celastrol is a potent inhibitor of NOX enzymes in general with increased potency against NOX1 and NOX2. Furthermore, inhibition of NOX1 and NOX2 was mediated via a novel mode of action, namely inhibition of a functional association between cytosolic subunits and the membrane flavocytochrome.

Neutrophil bleaching of GFP-expressing staphylococci: probing the intraphagosomal fate of individual bacteria.

Successful host defense against bacteria such as Staphylococcus aureus (SA) depends on a prompt response by circulating polymorphonuclear leukocytes (PMN). Stimulated PMN create in their phagosomes an environment inhospitable to most ingested bacteria. Granules that fuse with the phagosome deliver an array of catalytic and noncatalytic antimicrobial peptides, while activation of the NADPH oxidase at the phagosomal membrane generates reactive oxygen species within the phagosome, including hypochlorous acid (HOCl), formed by the oxidation of chloride by the granule protein myeloperoxidase in the presence of H(2)O(2). In this study, we used SA-expressing cytosolic GFP to provide a novel probe of the fate of SA in human PMN. PMN bleaching of GFP in SA required phagocytosis, active myeloperoxidase, H(2)O(2) from the NADPH oxidase, and chloride. Not all ingested SA were bleached, and the number of cocci within PMN-retaining fluorescent GFP closely correlated with the number of viable bacteria remaining intracellularly. The percent of intracellular fluorescent and viable SA increased at higher multiplicity of infection and when SA presented to PMN had been harvested from the stationary phase of growth. These studies demonstrate that the loss of GFP fluorescence in ingested SA provides a sensitive experimental probe for monitoring biochemical events within individual phagosomes and for identifying subpopulations of SA that resist intracellular PMN cytotoxicity. Defining the molecular basis of SA survival within PMN should provide important insights into bacterial and host properties that limit PMN antistaphylococcal action and thus contribute to the pathogenesis of staphylococcal infection.

TNF-related apoptosis-inducing ligand (TRAIL) is expressed throughout myeloid development, resulting in a broad distribution among neutrophil granules.

TRAIL induces apoptosis in a variety of tumor cells. Our laboratory found that human neutrophils contain an intracellular reservoir of prefabricated TRAIL that is released after stimulation with Mycobacterium bovis bacillus Calmette-Guérin. In this study, we examined the subcellular distribution of TRAIL in freshly isolated neutrophils. Neutrophil granules, secretory vesicles (SV), and plasma membrane vesicles were isolated by subcellular fractionation, followed by free-flow electrophoresis, and examined by ELISA and immunoblot. TRAIL was found in all membrane-bound fractions with the highest amounts in the fractions enriched in azurophilic granule (AG) and SV. Immunofluorescence confocal microscopy showed that TRAIL colocalized independently with myeloperoxidase (MPO), lactoferrin (LF), and albumin, respective markers of AG, specific granules, and SV. Furthermore, immunotransmission electron microscopy demonstrated that TRAIL colocalized intracellularly with MPO and albumin. We examined TRAIL expression in PLB-985 cells induced with dimethylformamide and in CD34-positive stem cells treated with G-CSF. Quantitative RT-PCR analysis showed that TRAIL was expressed in each stage of development, whereas MPO and LF were only expressed at distinct times during differentiation. Collectively, these findings suggest that TRAIL is expressed throughout neutrophil development, resulting in a broad distribution among different granule subtypes.

Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils.

BACKGROUND: Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods. RESULTS: To identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting) and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP) and dysferlin were further validated by immunoblot analysis. CONCLUSION: Our data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli.

Impact of two novel mutations on the structure and function of human myeloperoxidase.

The heme protein myeloperoxidase (MPO) contributes critically to O(2)-dependent neutrophil antimicrobial activity. Two Japanese adults were identified with inherited MPO deficiency because of mutations at Arg-499 or Gly-501, conserved residues near the proximal histidine in the heme pocket. Because of the proximity of these residues to a critical histidine in the heme pocket, we examined the biosynthesis, function, and spectral properties of the peroxidase stably expressed in human embryonic kidney cells. Biosynthesis of normal MPO by human embryonic kidney cells faithfully mirrored events previously identified in cells expressing endogenous MPO. Mutant apopro-MPO was 90 kDa and interacted normally with the molecular chaperones ERp57, calreticulin, and calnexin in the endoplasmic reticulum. However, mutant precursors were not proteolytically processed into subunits of MPO, although secretion of the unprocessed precursors occurred normally. Although delta-[(14)C]aminolevulinic acid incorporation demonstrated formation of pro-MPO in both mutants, neither protein was enzymatically active. The Soret band for each mutant was shifted from the normal 430 to approximately 412 nm, confirming that heme was incorporated but suggesting that the number of covalent bonds or other structural aspects of the heme pocket were disrupted by the mutations. These studies demonstrate that despite heme incorporation, mutations in the heme environs compromised the oxidizing potential of MPO.

Monocyte p110alpha phosphatidylinositol 3-kinase regulates phagocytosis, the phagocyte oxidase, and cytokine production.

Mononuclear phagocytes are critical modulators and effectors of innate and adaptive immune responses, and PI-3Ks have been shown to be multifunctional monocyte regulators. The PI-3K family includes eight catalytic isoforms, and only limited information is available about how these contribute to fine specificity in monocyte cell regulation. We examined the regulation of phagocytosis, the phagocyte oxidative burst, and LPS-induced cytokine production by human monocytic cells deficient in p110alpha PI-3K. We observed that p110alpha PI-3K was required for phagocytosis of IgG-opsonized and nonopsonized zymosan in differentiated THP-1 cells, and the latter was inhibitable by mannose. In contrast, p110alpha PI-3K was not required for ingestion serum-opsonized zymosan. Taken together, these results suggest that FcgammaR- and mannose receptor-mediated phagocytosis are p110alpha-dependent, whereas CR3-mediated phagocytosis involves a distinct isoform. It is notable that the phagocyte oxidative burst induced in response to PMA or opsonized zymosan was also found to be dependent on p110alpha in THP-1 cells. Furthermore, p110alpha was observed to exert selective and bidirectional effects on the secretion of pivotal cytokines. Incubation of p110alpha-deficient THP-1 cells with LPS showed that p110alpha was required for IL-12p40 and IL-6 production, whereas it negatively regulated the production of TNF-alpha and IL-10. Cells deficient in p110alpha also exhibited enhanced p38 MAPK, JNK, and NF-kappaB phosphorylation. Thus, p110alpha PI-3K appears to uniquely regulate important monocyte functions, where other PI-3K isoforms are uninvolved or unable to fully compensate.

CFTR Expression in human neutrophils and the phagolysosomal chlorination defect in cystic fibrosis.

Production of hypochlorous acid (HOCl) in neutrophils, a critical oxidant involved in bacterial killing, requires chloride anions. Because the primary defect of cystic fibrosis (CF) is the loss of chloride transport function of the CF transmembrane conductance regulator (CFTR), we hypothesized that CF neutrophils may be deficient in chlorination of bacterial components due to a limited chloride supply to the phagolysosomal compartment. Multiple approaches, including RT-PCR, immunofluorescence staining, and immunoblotting, were used to demonstrate that CFTR is expressed in resting neutrophils at the mRNA and protein levels. Probing fractions of resting neutrophils isolated by Percoll gradient fractionation and free flow electrophoresis for CFTR revealed its presence exclusively in secretory vesicles. The CFTR chloride channel was also detected in phagolysosomes, a special organelle formed after phagocytosis. Interestingly, HL-60 cells, a human promyelocytic leukemia cell line, upregulated CFTR expresssion when induced to differentiate into neutrophils with DMSO, strongly suggesting its potential role in mature neutrophil function. Analyses by gas chromatography and mass spectrometry (GC-MS) revealed that neutrophils from CF patients had a defect in their ability to chlorinate bacterial proteins from Pseudomonas aeruginosa metabolically prelabeled with [(13)C]-l-tyrosine, unveiling defective intraphagolysosomal HOCl production. In contrast, both normal and CF neutrophils exhibited normal extracellular production of HOCl when stimulated with phorbol ester, indicating that CF neutrophils had the normal ability to produce this oxidant in the extracellular medium. This report provides evidence which suggests that CFTR channel expression in neutrophils and its dysfunction affect neutrophil chlorination of phagocytosed bacteria.

Neutrophil stimulation with Mycobacterium bovis bacillus Calmette-Guerin (BCG) results in the release of functional soluble TRAIL/Apo-2L.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been used to treat bladder cancer for almost 30 years; however, the effector mechanism of the BCG-induced antitumor response remains enigmatic. Most BCG research has focused on the mononuclear-cell infiltrate, but growing evidence supports a role for neutrophils in the antitumor response. Previously, we demonstrated increased urinary tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo-2L) levels from BCG-responsive patients compared to nonresponders. Interestingly, neutrophils isolated from the urine expressed TRAIL/Apo-2L, leading us to investigate the neutrophil response to BCG. BCG-stimulated neutrophils expressed surface-bound and released functional soluble TRAIL/Apo-2L. Whereas neither interferon alpha (IFN-alpha) nor IFN-gamma directly induced TRAIL/Apo2L expression by neutrophils, IFN-alpha did stimulate TRAIL gene transcription, and IFN-primed neutrophils contained and released more TRAIL/Apo-2L after BCG stimulation than did unprimed neutrophils. In unstimulated neutrophils TRAIL/Apo-2L was present predominantly in the azurophilic granules and plasma-membrane-enriched/secretory-granule fraction. Finally, we observed that killed BCG, Toll-like receptor 2 (TLR2) and TLR4 agonists, and an M tuberculosis cell-wall fraction were each capable of inducing the release of soluble TRAIL/Apo-2L from neutrophils. These results further characterize the potential role neutrophils may play in initiating the antitumor response described with BCG treatment for superficial bladder cancer.

Metalloproteases from Pseudomonas aeruginosa degrade human RANTES, MCP-1, and ENA-78.

The gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen associated with both an acute lung disease in patients with hospital-acquired pneumonia and a chronic, progressive lung disease in individuals with cystic fibrosis. A unique characteristic of this bacterium in its natural environment is the secretion of a wide variety of factors designed to ensure its growth and survival. Evidence suggests, however, that when present in the human host, these same factors may contribute to disease. In the course of studying the effect of P. aeruginosa secretory factors on airway epithelial cells, we observed that metalloproteases in bacterial-conditioned medium, as well as purified alkaline protease and elastase, degraded human RANTES, monocyte chemotactic protein-1 (MCP-1), and epithelial neutrophil-activating protein-78 (ENA-78). Under identical conditions, interleukin-8 (IL-8) was significantly more resistant to proteolysis. Degradation was accompanied by a loss of chemotactic activity. These data suggest that metalloproteases from P. aeruginosa could alter the relative amounts of critical immunomodulatory cytokines in the airway and, thus, could contribute to the pathophysiology observed in P. aeruginosa-associated lung disease.

Regulation of calreticulin expression during induction of differentiation in human myeloid cells. Evidence for remodeling of the endoplasmic reticulum.

Induction of differentiation of HL-60 human myeloid cells profoundly affected expression of calreticulin, a Ca(2+)-binding endoplasmic reticulum chaperone. Induction with Me(2)SO or retinoic acid reduced levels of calreticulin protein by approximately 60% within 4 days. Pulse-chase studies indicated that labeled calreticulin decayed at similar rates in differentiated and undifferentiated cells (t(12) approximately 4.6 days), but the biosynthetic rate was <10% of control after 4 days. Differentiation also induced a rapid decline in calreticulin mRNA levels (90% reduction after 1 day) without a decrease in transcript stability (t(12) approximately 5 h). Nuclear run-on analysis demonstrated rapid down-regulation of gene transcription (21% of control at 2 h). Differentiation also greatly reduced the Ca(2+) content of the cells (25% of control), although residual Ca(2+) pools remained sensitive to thapsigargin, ionomycin, and inositol trisphosphate. Progressive decreases were also observed in levels of calnexin and ERp57, whereas BiP/GRP78 and protein disulfide isomerase were only modestly affected. Ultrastructural studies showed a substantial reduction in endoplasmic reticulum content of the cells. Thus, terminal differentiation of myeloid cells was associated with decreased endoplasmic reticulum content, selective reductions in molecular chaperones, and diminished intracellular Ca(2+) stores, perhaps reflecting an endoplasmic reticulum remodeling program as a prominent feature of granulocytic differentiation.

Biosynthesis of the major surface protease GP63 of Leishmania chagasi.

The protozoan Leishmania chagasi expresses a surface metalloprotease, GP63, whose abundance increases 14-fold as parasites grow from logarithmic to stationary phase. L. chagasi GP63 is encoded by three classes of MSP genes that are differentially expressed during parasite growth. Using metabolic labeling and immunoprecipitation, we found L. chagasi GP63 first appeared as a 66-kDa band that was replaced by a 63-kDa protein. This pattern also occurred in transfected L. donovani harboring detectable products of only one MSP gene, suggesting a precursor-product relationship. The half-life of GP63 increased from 29 h in logarithmic phase to >72 h in stationary phase promastigotes. GP63 loss from the cell was complemented by the appearance of a 63-kDa GP63 in extracellular medium in both membrane-associated and -free forms. Calculations suggested that the long and lengthening T(1/2) of cell-associated GP63 accounts in part for its progressive accumulation in the cell during promastigote growth. The current findings add yet another level of complexity to post-transcriptionally regulated expression of an abundant surface molecule in a trypanosomatid protozoan.