Characterization of Staphylococcus aureus cardiolipin synthases 1 and 2 and their contribution to accumulation of cardiolipin in stationary phase and within phagocytes.

In many bacteria, including Staphylococcus aureus, progression from the logarithmic to the stationary phase is accompanied by conversion of most of bacterial membrane phosphatidylglycerol (PG) to cardiolipin (CL). Phagocytosis of S. aureus by human neutrophils also induces the conversion of most bacterial PG to CL. The genome of all sequenced strains of S. aureus contains two open reading frames (ORFs) predicting proteins encoded with ∼30% identity to the principal CL synthase (cls) of Escherichia coli. To test whether these ORFs (cls1 and cls2) encode cardiolipin synthases and contribute to CL accumulation in S. aureus, we expressed these proteins in a cls strain of E. coli and created isogenic single and double mutants in S. aureus. The expression of either Cls1 or Cls2 in CL-deficient E. coli resulted in CL accumulation in the stationary phase. S. aureus with deletion of both cls1 and cls2 showed no detectable CL accumulation in the stationary phase or after phagocytosis by neutrophils. CL accumulation in the stationary phase was due almost solely to Cls2, whereas both Cls1 and Cls2 contributed to CL accumulation following phagocytosis by neutrophils. Differences in the relative contributions of Cls1 and Cls2 to CL accumulation under different triggering conditions suggest differences in the role and regulation of these two enzymes.

Sorting for storage in myeloid cells of nonmyeloid proteins and chimeras with the propeptide of myeloperoxidase precursor.

During formation of polymorphonuclear neutrophils, proteins are synthesized for storage in granules. Whereas sorting of proteins into distinct subtypes of cytoplasmic granules may reflect the coordinated expression of the proteins contained in them, still the mechanism(s) for the retrieval of proteins from the constitutive secretion is unknown. To investigate the mechanisms of retrieval, nonmyeloid secretory proteins were expressed in myeloid cell lines, and their subcellular fate was assessed. The contribution of the propeptide (MPOpro) of the myeloperoxidase (MPO) precursor was investigated by determining the fate of chimeras containing MPOpro. The nonmyeloid protein alpha(1)-microglobulin (alpha(1)-m) was targeted to storage organelles in 32D cells and colocalized with the lysosomal marker LAMP-1, whereas soluble TNF receptor 1 (sTNFR1) was secreted without granule targeting. Fusion of MPOpro to alpha(1)-m delayed exit from endoplasmic reticulum (ER), but subsequent targeting to dense organelles was indistinguishable from that of alpha(1)-m alone. Fusion proteins between MPOpro and sTNFR1 or green fluorescent protein expressed in myeloid 32D, K562, or PLB-985 cells did not associate stably with calreticulin or calnexin, molecular chaperones that normally interact transiently with the MPO precursor, but were still efficiently retained in the ER followed by degradation. We conclude that normally secreted, nonmyeloid proteins can be targeted efficiently to storage organelles in myeloid cells, that myeloid cells selectively target some proteins for storage but not others, and that MPOpro may contribute to the prolonged ER retention of the MPO precursor independent of the ER-molecular chaperones calreticulin and calnexin.

Identification and cloning of the SNARE proteins VAMP-2 and syntaxin-4 from HL-60 cells and human neutrophils.

Degranulation and membrane fusion by neutrophils are essential to host defense. We sought homologues of neuron-specific fusion proteins in human neutrophils and in their precursors, the promyelocytic cell line HL-60. We screened a differentiated HL-60 library and obtained an 848 bp sequence with a 351 bp open reading frame, identical to that published for human VAMP-2 and including 5' and 3' untranslated regions. RNA from HL-60 cells during differentiation into the neutrophil lineage was subjected to Northern blot analysis. which revealed a transcript of approximately 1050 bp at all stages of differentiation. The amount of these transcripts increased approximately threefold during differentiation, a finding confirmed by quantitative RT-PCR. We also detected mRNA for VAMP-2 in human neutrophils and monocytes using RT-PCR. In like fashion, transcripts of syntaxin-4, another fusion protein, were recovered from a neutrophil cDNA library. As with VAMP-2, expression of syntaxin-4 (determined by Northern blots) also increased, but by only 50%, during differentiation of HL-60 cells. These studies demonstrate that neutrophils and their progenitors possess mRNA for the fusion proteins VAMP-2 and syntaxin-4, and that their transcription increases during differentiation, concurrent with the functional maturation of myeloid cells.

Contributions of myeloperoxidase to proinflammatory events: more than an antimicrobial system.

Optimal oxygen-dependent antimicrobial activity of circulating polymorphonuclear leukocytes reflects the synergistic effects of the myeloperoxidase (MPO)-hydrogen peroxide-halide system. Delivered from its storage compartment to the phagolysosome during fusion of the azurophilic granules, MPO catalyzes the oxidation of chloride in the presence of H2O2, chemistry unique to MPO, and thereby generates an array of highly reactive oxidants. Recent investigations of a wide range of inflammatory disorders have identified biochemical markers of MPO-dependent reactions, thus indirectly implicating MPO in their pathogenesis, progression, or perpetuation. The implied involvement of MPO-dependent events in diseases such as atherosclerosis forces reexamination of several fundamental tenets about MPO that are derived from studies of myeloid cells, most notably factors important in the regulated expression of MPO gene transcription. The evidence supporting a role for MPO in the pathogenesis of atherosclerosis, demyelinating diseases of the central nervous system, and specific cancers is reviewed and some of the new questions raised by these studies are discussed. Lastly, an appreciation for the existence of a broad family of proteins structurally related to MPO and the functional diversity implied by the corresponding structures may provide insights into novel ways in which MPO can function as more than an important antimicrobial component.

1alpha,25-Dihydroxyvitamin D3-induced monocyte antimycobacterial activity is regulated by phosphatidylinositol 3-kinase and mediated by the NADPH-dependent phagocyte oxidase.

We investigated the basis for the induction of monocyte antimycobacterial activity by 1alpha,25-dihydroxyvitamin D(3) (D(3)). As expected, incubation of Mycobacterium tuberculosis-infected THP-1 cells or human peripheral blood, monocyte-derived macrophages with hormone resulted in the induction of antimycobacterial activity. This effect was significantly abrogated by pretreatment of cells with either of the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, wortmannin or LY294002, or with antisense oligonucleotides to the p110 subunit of PI 3-Kalpha. Cells infected with M. tuberculosis alone or incubated with D(3) alone produced little or undetectable amounts of superoxide anion (O(2)). In contrast, exposure of M. tuberculosis-infected cells to D(3) led to significant production of O(2), and this response was eliminated by either wortmannin, LY294002, or p110 antisense oligonucleotides. As was observed for PI 3-K inactivation, the reactive oxygen intermediate scavenger, 4-hydroxy-TEMPO, and degradative enzymes, polyethylene glycol coupled to either superoxide dismutase or catalase, also abrogated D(3)-induced antimycobacterial activity. Superoxide production by THP-1 cells in response to D(3) required prior infection with live M. tuberculosis, since exposure of cells to either killed M. tuberculosis or latex beads did not prime for an oxidative burst in response to subsequent hormone treatment. Consistent with these findings, redistribution of the cytosolic oxidase components p47(phox) and p67(phox) to the membrane fraction was observed in cells incubated with live M. tuberculosis and D(3) but not in response to combined treatment with heat-killed M. tuberculosis followed by D(3). Redistribution of p47(phox) and p67(phox) to the membrane fraction in response to live M. tuberculosis and D(3) was also abrogated under conditions where PI 3-K was inactivated. Taken together, these results indicate that D(3)-induced, human monocyte antimycobacterial activity is regulated by PI 3-K and mediated by the NADPH-dependent phagocyte oxidase.

Heme-ligating histidines in flavocytochrome b(558): identification of specific histidines in gp91(phox).

The phagocyte NADPH-dependent oxidase generates superoxide (O(2)) by reducing molecular oxygen through flavocytochrome b(558) (flavocytochrome b), a heterodimeric oxidoreductase composed of gp91(phox) and p22(phox) subunits. Although each flavocytochrome b molecule contains two heme groups, their precise distribution within the heterodimer is unknown. Among functionally and/or structurally related oxidoreductases, histidines at codons 101, 111, 115, 119, 209, 210, and 222 of gp91(phox) are conserved and potential candidates to ligate heme. We compared biochemical and functional features of normal flavocytochrome b with those in cells expressing gp91(phox) harboring amino acid substitutions at each of these histidines. Surface expression of flavocytochrome b and heterodimer formation were relatively unaffected in cells expressing gp91(phox) H111L, H119L, or H210L. These mutations also had no effect on the flavocytochrome b heme spectrum, although NADPH oxidase activity was decreased in cells expressing gp91(phox) H119L or H210L. In contrast, gp65 was not processed to gp91(phox), heterodimers did not form, and flavocytochrome b was not expressed on the surface of cells expressing gp91(phox) H101L, H115L, H115D, H209C, H209Y, H222L, H222C, or H222R. Similarly, this subset of mutants lacked detectable O(2)-generating activity, and flavocytochrome b purified from these cells contained little or no heme. These findings demonstrate that His(101), His(115), His(209), and His(222) of gp91(phox) are critical for heme binding and biosynthetic maturation of flavocytochrome b.

Salmonella pathogenicity island 2-encoded type III secretion system mediates exclusion of NADPH oxidase assembly from the phagosomal membrane.

Salmonella typhimurium requires a type III secretion system encoded by pathogenicity island (SPI)-2 to survive and proliferate within macrophages. This survival implies that S. typhimurium avoids or withstands bactericidal events targeted to the microbe-containing vacuole, which include intraphagosomal production of reactive oxygen species (ROS), phagosomal acidification, and delivery of hydrolytic enzymes to the phagosome via fusion with lysosomes. Recent evidence suggests that S. typhimurium alters ROS production by murine macrophages in an SPI-2-dependent manner. To gain insights into the mechanism by which S. typhimurium inhibits intraphagosomal ROS production, we analyzed the subcellular distribution of NADPH oxidase components during infection of human monocyte-derived macrophages by wild-type (WT) or several SPI-2 mutant strains of S. typhimurium. We found that the membrane component of the NADPH oxidase, flavocytochrome b(558), was actively excluded or rapidly removed from the phagosomal membrane of WT-infected monocyte-derived macrophages, thereby preventing assembly of the NADPH oxidase complex and intraphagosomal production of superoxide anion. In contrast, the NADPH oxidase assembled on and generated ROS in phagosomes containing SPI-2 mutant S. typhimurium. Subversion of NADPH oxidase assembly by S. typhimurium was accompanied by increased bacterial replication relative to that of SPI-2 mutant strains, suggesting that the ability of WT S. typhimurium to prevent NADPH oxidase assembly at the phagosomal membrane represents an important virulence factor influencing its intracellular survival.

Phage display epitope mapping of human neutrophil flavocytochrome b558. Identification of two juxtaposed extracellular domains.

Despite extensive experimental and clinical evidence demonstrating the critical role of flavocytochrome b558 (Cyt b) in the NADPH-dependent oxidase, there is a paucity of direct structural data defining its topology in the phagocyte membrane. Unlike other Cyt b-specific monoclonal antibodies, 7D5 binds exclusively to an extracellular domain, and identification of its epitope should provide novel insight into the membrane topology of Cyt b. To that end, we examined biochemical features of 7D5-Cyt b binding and used the J404 phage display nonapeptide library to identify the bound epitope. 7D5 precipitated only heterodimeric gp91-p22phox and not individual or denatured Cyt b subunits from detergent extracts of human neutrophils and promyelocytic leukemia cells (gp91-PLB). Moreover, 7D5 precipitated precursor gp65-p22phox complexes from detergent extracts of the biosynthetically active gp91-PLB cells, demonstrating that complex carbohydrates were not required for epitope recognition. Epitope mimetics selected from the J404 phage display library by 7D5 demonstrated that (226)RIVRG(230) and (160)IKNP(163) regions of gp91phox were both bound by 7D5. These studies reveal specific information about Cyt b membrane topology and structure, namely that gp91phox residues (226)RIVRG(230) and (160)IKNP(163) are closely juxtaposed on extracytoplasmic domains and that predicted helices containing residues Gly(165)-Ile(190) and Ser(200)-Glu(225) are adjacent to each other in the membrane.

Impact of missense mutations on biosynthesis of myeloperoxidase.

We have examined the biosynthesis of normal and mutant forms of myeloperoxidase (MPO) in order to gain insights into the critical features of normal biogenesis of MPO. The expression of wild-type and mutant forms of MPO in a stably transfected cell line devoid of endogenous MPO as well as in established human promyelocytic cell lines has allowed understanding of several features of MPO biosynthesis. It is clear that heme insertion into apoproMPO is necessary for proper folding, egress from the endoplasmic reticulum (ER), and eventual entry into the maturation pathway. In addition, molecular chaperones calreticulin and calnexin interact with normal MPO precursors in a sequential and regulated fashion. Studies of naturally occurring mutants, specifically missense mutations underlying inherited MPO deficiency, and mutations in putatively important residues in MPO have highlighted special features of the ER quality control system in the context of MPO biosynthesis. With identification of additional genotypes of MPO deficiency and the recent solution of MPO crystal structure at 1.8 A, this approach provides a powerful technique to assess structure-function relationships in MPO that are likely applicable to other members of the family of animal peroxidases.

Processing and maturation of flavocytochrome b558 include incorporation of heme as a prerequisite for heterodimer assembly.

The phagocyte NADPH-dependent oxidase generates superoxide by reducing molecular oxygen through a transmembrane heterodimer known as flavocytochrome b(558) (flavocytochrome b). We investigated the biosynthesis of flavocytochrome b subunits gp91(phox) and p22(phox) to elucidate features of flavocytochrome b processing in myeloid cells. Although the gp91(phox) precursor, gp65, was processed to gp91(phox) within 4-8 h of chase, unassembled gp65 and p22(phox) monomers were degraded by the cytosolic proteasome. gp65 associated with p22(phox) post-translationally, within 1-4 h of chase, but prior to its modification in the Golgi complex. Moreover, p22(phox) coprecipitated with unglycosylated gp91(phox) primary translation product made in the presence of tunicamycin, suggesting that heterodimer formation does not require glycosylation. Blocking heme synthesis with succinyl acetone completely inhibited heterodimer formation, although biogenesis of gp65 and p22(phox) was unaffected. In succinyl acetone-treated cells, p22(phox) and gp65 were degraded completely by 8 h of chase, a process mediated by the cytosolic proteasome. Taken together, these data suggest that the formation of the gp65-p22(phox) heterodimer is relatively inefficient and that acquisition of heme by gp65 precedes and is required for its association with p22(phox), a process that requires neither the addition of N-linked oligosaccharides nor modification in the Golgi complex.

NADPH oxidase activation and assembly during phagocytosis.

Generation of superoxide (O2-) by the NADPH-dependent oxidase of polymorphonuclear leukocytes is an essential component of the innate immune response to invading microorganisms. To examine NADPH oxidase function during phagocytosis, we evaluated its activation and assembly following ingestion of serum-opsonized Neisseria meningitidis, serogroup B (NMB), and compared it with that elicited by serum-opsonized zymosan (OPZ). Opsonized N. meningitidis- and OPZ-dependent generation of reactive oxygen species by polymorphonuclear leukocytes peaked early and then terminated. Phosphorylation of p47phox coincided with peak generation of reactive oxygen species by either stimulus, consistent with a role for p47phox phosphorylation during NADPH oxidase activation, and correlated with phagosomal colocalization of flavocytochrome b558 (flavocytochrome b) and p47phox and p67phox (p47/67phox). Termination of respiratory burst activity did not reflect dephosphorylation of plasma membrane- and/or phagosome-associated p47phox; in contrast, the specific activity of phosphorylated p47phox at the phagosomal membrane increased. Most significantly, termination of oxidase activity paralleled the loss of p47/67phox from both NMB and OPZ phagosomes despite the continued presence of flavocytochrome b. These data suggest that 1) the onset of respiratory burst activity during phagocytosis is linked to the phosphorylation of p47phox and its translocation to the phagosome; and 2) termination of oxidase activity correlates with loss of p47/67phox from flavocytochrome b-enriched phagosomes and additional phosphorylation of membrane-associated p47phox.

The NADPH-dependent oxidase of phagocytes.

Polymorphonuclear leukocytes (PMNs) represent a prominent cellular element in the innate immune system, serving to ingest exogenous particles and microbes and to kill phagocytosed microorganisms. The microbicidal activity of PMNs depends on the interactions of a broad array of potent systems, including relatively stable degradative proteins as well as labile reactive radicals. These systems can be categorized as oxygen-dependent and nonoxidative mechanisms, although the physiologically relative activity depends on the precisely orchestrated interplay between both systems. The enzyme complex responsible for the activity of the oxygen-dependent system is the respiratory burst oxidase and its important contribution to host defense is best illustrated by the frequent and severe infections seen in individuals whose PMNs lack oxidase activity, namely patients with chronic granulomatous disease (CGD). Multiple elements comprise the oxygen-dependent system, and significant advances have been made in the past decade in understanding the protein components of the respiratory burst oxidase, their subcellular distribution in resting PMNs, and their agonist-dependent assembly into a functional system at phagosomal and plasma membranes. In parallel, substantial insights into the molecular bases of CGD have likewise been made. Nonetheless there remain significant gaps in our understanding of the precise functional contributions of particular components of the system, the molecular mechanisms that regulate their coordinated assembly, and the role of related proteins in nonphagocytic cells.

Quality control in the endoplasmic reticulum: lessons from hereditary myeloperoxidase deficiency.

The optimal level of oxygen-dependent microbicidal activity in human neutrophils depends on the generation of highly toxic products, including hypochlorous acid, by hydrogen peroxide in the presence of chloride anion and the neutrophil granule protein myeloperoxidase (MPO). The biosynthesis of MPO is normally restricted to the promyelocytic stage of myeloid development and includes N-linked glycosylation, heme insertion, proteolytic processing, subunit dimerization, and eventual targeting to the azurophilic granule. In the endoplasmic reticulum, MPO precursors interact transiently with calreticulin and calnexin, presumably in their capacity as molecular chaperones. In light of the important role of the MPO-H2O2-chloride system in human host defense, the relatively high prevalence of inherited MPO deficiency was an unanticipated insight provided by the widespread use of automated flow cytometry for the enumeration of leukocytes in clinical specimens. In many cases of inherited MPO deficiency, affected neutrophils have immunochemical evidence of precursor protein but lack the subunits of mature MPO, peroxidase activity, or the ability to chlorinate target proteins. To date, four genotypes have been reported to cause inherited MPO deficiency, each of which results in missense mutations. In the genotype Y173C, the mutant precursor is retained in the endoplasmic reticulum by virtue of its prolonged interaction with calnexin, and it eventually undergoes degradation in the 20S proteasome. In this way, the quality control system operating in the endoplasmic reticulum retrieves malfolded MPO precursors from the biosynthetic pathway and creates the biochemical phenotype of MPO deficiency. Thus MPO deficiency caused by Y173C joins the ranks of cystic fibrosis, protein C deficiency, and other genetic disorders that reflect abnormalities in protein folding.

Despite structural similarities between gp91phox and FRE1, flavocytochrome b558 does not mediate iron uptake by myeloid cells.

Superoxide (O2-) generated by the phagocyte reduced nicotinamide adenine dinucleotide phosphate oxidase is dependent on electron transfer by flavocytochrome b558 (flavocytochrome b), a transmembrane heterodimer that forms the redox center of the oxidase at the plasma or phagosomal membrane. The larger of its two subunits, gp91phox, is homologous to the yeast iron reductase subunit FRE1, and these two proteins share many structural and functional characteristics. Because FRE1 is required for iron uptake in yeast, we hypothesized that flavocytochrome b might serve a similar function in human phagocytes and thus provide a mechanism for the transferrin-independent iron acquisition observed in myeloid cells. To determine whether flavocytochrome b was required for iron uptake, we compared iron acquisition by polymorphonuclear neutrophils (PMNs) or Epstein-Barr virus (EBV)-transformed B lymphocytes derived from individuals with X-linked chronic granulomatous disease (CGD) with iron acquisition by normal cells. Our results indicate that all cells acquired iron to the same extent and that uptake could be significantly enhanced in the presence of the trivalent metal gallium. The gallium enhancement of iron uptake observed in PMNs or in EBV-transformed B lymphocytes derived from healthy individuals was mirrored by those derived from individuals deficient in flavocytochrome b. Furthermore, both normal and CGD-derived EBV-transformed B lymphocytes had similar iron reductase activity, suggesting that flavocytochrome b is not a biologically significant iron reductase. In contrast to previously suggested hypotheses, these results show conclusively that flavocytochrome b is not necessary for cellular iron acquisition, despite structural and functional similarities between yeast iron reductases and flavocytochrome b.

Transient association of the nicotinamide adenine dinucleotide phosphate oxidase subunits p47phox and p67phox with phagosomes in neutrophils from patients with X-linked chronic granulomatous disease.

Optimal microbicidal activity of polymorphonuclear leukocytes (PMNs) requires recruitment of a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to the phagosome. In this study, we used a synchronized phagocytosis assay and immunofluorescence microscopy (IFM) to examine the association of cytosolic NADPH oxidase subunits with phagosomes containing opsonized zymosan (OpZ). Ingestion of OpZ began within 30 seconds of particle binding and forming phagosomes were enriched for both F-actin and the actin-binding protein p57. NADPH oxidase subunits p47phox and p67phox were also recruited to forming phagosomes and were retained on mature phagosomes for at least 15 minutes. Colocalization of F-actin, p57, and p47phox on phagosomes was confirmed by immunoblotting. Translocation of p67phox, but not p57, to forming phagosomes was deficient in PMNs lacking p47phox. Surprisingly, we found that in PMNs from six individuals with X-linked chronic granulomatous disease (CGD), p47phox and p67phox accumulated in the periphagosomal area during ingestion of OpZ. However, in marked contrast to normal PMNs, p47phox and p67phox were shed from nascent phagosomes along with F-actin and p57 once OpZ was internalized (approximately 5 minutes). These data support a model in which flavocytochrome b is required for stable membrane binding of p47phox and p67phox, but not their association with the cytoskeleton or transport to the cell periphery.

Biosynthesis of flavocytochrome b558 . gp91(phox) is synthesized as a 65-kDa precursor (p65) in the endoplasmic reticulum.

The redox center of the phagocyte NADPH oxidase is flavocytochrome b558, a transmembrane protein with two subunits, gp91(phox) and p22(phox). In this study we investigated the identity, subcellular localization, and maturation of a putative 65-kDa gp91(phox) precursor (p65). Expressing the gp91(phox) cDNA in an in vitro transcription and translation system, we found that synthesis of p65 required endoplasmic reticulum (ER) microsomes. Sucrose density gradient centrifugation of postnuclear supernatants obtained from a PLB-985 derived cell line with a constitutively expressed gp91(phox) transgene demonstrated that p65 co-sedimented with the ER marker protein calreticulin and myeloperoxidase precursors. Unexpectedly, the majority of p22(phox) was found in subcellular compartments containing the mature 91-kDa form of gp91(phox) and not with p65, suggesting that heterodimer formation may occur in a post-ER compartment. The heme synthesis inhibitor, succinyl acetone, reduced the abundance of mature gp91(phox) and p22(phox) but had little or no impact on p65. These studies demonstrate (a) gp91(phox) is synthesized as a glycosylated 65-kDa precursor in the ER, (b) heterodimer formation is not a co-translational process, and (c) heme insertion is a determinant in the formation of a stable heterodimer but does not appear to affect the stability of p65.