Introduction

Staphylococcus aureus is a common human pathogen capable of causing a wide range of infection in normal and compromised hosts (Lowy, 1998). It is characterized by an exceptional ability to exploit host immune functions (Foster, 2005), and several clinically important interactions are mediated by protein A, a surface virulence factor that is highly conserved and abundantly expressed in the lung (Lowy, 1998; Goerke et al, 2000). Protein A can impede phagocytosis by binding the Fc component of immunoglobulin (Uhlen et al, 1984; Foster, 2005), activate clotting by binding von Willebrand factor (Hartleib et al, 2000), act as a superantigen for B cells by binding the Fab region of VH3 bearing IgM (Moks et al, 1986; Sasso et al, 1989; Roben et al, 1995; Jansson et al, 1998) and through its activation of TNFR1, initiate staphylococcal pneumonia (Gómez et al, 2004).

Activation and regulation of the TNF- signaling cascade is critically important in the host response to infection (Chen and Goeddel, 2002; Aggarwal, 2003; Saunders et al, 2005; Rahman and McFadden, 2006). Whereas the production of TNF- is predominantly by immune cells, mucosal epithelial cells express TNFR1 and are highly responsive to local concentrations of TNF-. During staphylococcal pneumonia, TNFR1 is specifically mobilized to the apical surface of the airway epithelium, providing access to inhaled staphylococci (Gómez et al, 2004). The abundance of TNFR1 is controlled by mobilization from intracellular stores and cleavage from the cell surface (Bradley et al, 1995; Peschon et al, 1998; Jones et al, 1999; Reddy et al, 2000; Wang et al, 2003a; Xanthoulea et al, 2004; Garton et al, 2006). Release of the TNFR1 ectodomain prevents ongoing signaling and serves to neutralize free TNF- in the airway.

The interactions of protein A and TNFR1 are essential for the pathogenesis of staphylococcal pneumonia. TNFR1-null mice have decreased rates of infection as protein A-defective mutants of S. aureus did in wild-type animals (Gómez et al, 2004). TNFR1 recognition is mediated by the same domain of protein A that binds IgG (Gómez et al, 2006) and mimics TNF- proinflammatory signaling (Gómez et al, 2004). In addition, we noted that protein A also induces the cleavage of TNFR1 and its shedding from the epithelial surface. Cleavage of TNFR1 is mediated by TACE, the TNF- converting enzyme, a metalloprotease of the ADAM family that is a central regulator of TNF- signaling (Peschon et al, 1998; Reddy et al, 2000). Such metalloproteases are important in many aspects of lung defense, carcinogenesis and repair (Peschon et al, 1998; Reddy et al, 2000; Garton et al, 2003; Mezyk et al, 2003; Tsakadze et al, 2006). However, bacterial activation of TACE has not been previously characterized. As protein A induces TNFR1 shedding, we postulated that this virulence factor must activate TACE, and in the experiments detailed, outline the pathway through which this occurs.

Results

S. aureus protein A-induces TNFR1 mobilization and receptor shedding

Protein A rapidly induced recruitment of TNFR1 to the cell surface (Figure 1A) and sTNFR1 was detected as soon as 2 h after epithelial stimulation and continued to increase for at least 24 h (Figure 1B). As sTNFR1 accumulated in the culture supernatant, the total amount of cell-associated TNFR1 decreased (Figure 1C). TNFR1 shedding was dependent upon receptor mobilization from Golgi stores as Brefeldin A prevented both TNFR1 mobilization and shedding in Protein A-stimulated cells (Figure 1A and B). In contrast, cycloheximide treatment did not have any significant effect on TNFR1 mobilization (Figure 1D) or shedding even at later time points (data not shown). TNFR1 transcription was not affected by protein A stimulation (data not shown). TNFR1 mobilization from Golgi stores in response to protein A was confirmed by confocal imaging. TNFR1 colocalized with a Golgi probe in the unstimulated but not in the Protein A stimulated cells (Figure 1E). The natural TNFR1 ligand, TNF-, did not induce mobilization of TNFR1 (Figure 1A) or stimulate receptor shedding (Figure 1B). A small amount of sTNFR1 was detectable 24 h after TNF- stimulation, equivalent to 30% of the response induced by protein A.

To determine whether TNFR1 shedding is limited to the airway epithelial cells, the effects of protein A on superficial TNFR1 on macrophages was examined. Whereas TNFR1 is abundant on the macrophage surface, additional receptor was mobilized and shed in response to protein A but not TNF- (Figure 1F and G). By 24 h the amount of shed TNFR1 in response to TNF- was equivalent to only 53% of that induced by protein A (Figure 1G). To verify that basal levels of TNFR1 on the cell surface of unstimulated airway epithelial cells are sufficient to mediate TNF- signaling, IL-8 production in response to TNF- was determined and found to be comparable to that induced by protein A (Figure 1H).

Distribution of TACE and TNFR1 on the surface of airway epithelial cells

TACE is the major metalloprotease that cleaves TNFR1 from the surface of airway epithelial cells in response to protein A, as demonstrated by RNA interference (Figure 2A). Depletion of TACE expression significantly reduced protein A-induced TNFR1 shedding. For TACE to efficiently cleave TNFR1, both the protease and its substrate must be available on the apical surface of the airway cell. TACE is abundant on the surface of the airway epithelial cells (Figure 2B). As TACE transcription is not inducible by protein A (data not shown); nor is the distribution of TACE changed following cell stimulation (Figure 2C), we postulated that the substrate TNFR1 must be actively mobilized to the cell surface. Using confocal imaging we found that TNFR1, which is mobilized in response to protein A stimulation (Figure 1A), colocalizes with TACE on the apical surface of the cells (Figure 2D). This finding was confirmed using human airway epithelial cells in primary culture (Figure 2D).

Protein A induces TACE phosphorylation

Previous reports suggest that metalloproteinases are activated by reactive oxygen species (ROS) generated in response to bacterial infection (Hino et al, 1999; Fang, 2004). However, protein A activation of TACE was not inhibited by the ROS scavenger n-propyl gallete (data not shown), suggesting that other pathways are involved. TACE is phosphorylated at both ser 819 and thr 735 by erk1/2 MAPK in transfected cell lines stimulated with PMA or growth factors (Diaz-Rodriguez et al, 2002; Fan et al, 2003). The airway epithelial cells express the pro-form and the mature form (devoid of the pro-domain) of TACE as it has been described for other cell lines. We detected threonine phosphorylation of the mature form of TACE as early as 5–10 min and maximal amounts at 30 min following protein A stimulation (Figure 3A). TACE phosphorylation persisted for up to 24 h (Figure 3B). Phosphorylation was not observed in cells treated with the MEK inhibitor UO126 (Figure 3A). No serine phosphorylation of TACE was detected (data not shown). Association of phosphorylated erk1/2 with the mature form of TACE was detected at 5 min following protein A stimulation (Figure 3C), whereas phospho-erk1/2, in complex with TACE, was not present in cells treated with the MEK inhibitor (Figure 3C). When erk1/2 phosphorylation was prevented with the MEK inhibitor UO126, airway epithelial cells did not release sTNFR1 in response to protein A (Figure 3D). Expression of an MEK dominant-negative mutation resulted in a dose-dependent inhibition of sTNFR1 release (Figure 3E), confirming the role of erk1/2 in TACE activation. TNF-, which failed to induce TNFR1 shedding, did not induce phosphorylation of TACE or erk1/2 (Figure 3F).

Protein A interacts with EGFR

Both TNF- and protein A signal proinflammatory responses through TNFR1 and the expected TRADD–TRAF2 cascade (Gómez et al, 2004). To determine if the protein A–TNFR1–TRAF2 cascade is involved in receptor shedding, we monitored sTNFR1 in cells expressing a TRAF2 dominant-negative mutant (DN) (Figure 4A). Whereas IL-8 production was inhibited (data not shown), shedding was unaffected, indicating that an epithelial receptor distinct from TNFR1 must be involved. In the original analysis of the interactions between protein A and epithelial cells we identified a protein with a molecular weight of approximately 170 kDa, the size of the epidermal growth factor receptor (EGFR), in addition to TNFR1 (Gómez et al, 2004). EGFR is apically expressed in the airway epithelial cells, and the signaling cascade that leads to TACE activation resembles EGFR signaling (Zwick et al, 1999). Co-immunoprecipitation studies were performed to determine if EGFR directly interacts with protein A (Figure 4B). EGFR was detected by immunoblot in protein A stimulated lysates captured with antibody to protein A but not in control lysates stimulated with BSA. Protein A–EGFR interaction on the apical surface of polarized airway epithelial cells was then demonstrated by confocal imaging (Figure 4C).

A discrete region of protein A, domain D, is sufficient for TNFR1 recognition and IL-8 induction, as well as TNFR1 shedding (Gómez et al, 2006). We screened a collection of point mutations in domain D to determine if this region recognizes EGFR. Two mutants (L17A and F5A) that retained the ability to induce IL-8 production (Gómez et al, 2006) but failed to induce TNFR1 shedding were identified (Figure 4D). Neither mutant was able interact with EGFR, as demonstrated by co-immunoprecipitation and confocal imaging, indicating that discrete binding domains recognize TNFR1 and EGFR independently (Figure 4B and C). Both mutants stimulated TNFR1 mobilization (Figure 4E) and colocalization with TACE (Figure 4F), indicating that receptor mobilization induced by protein A is independent of EGFR binding, and that the protein A–EGFR interaction is required for TACE activation (Figure 4D).

Protein A–EGFR signaling mediates TACE activation

Activation of EGFR involves both Src-dependent phosphorylation and autophosphorylation of multiple tyrosines (Schlessinger, 2000). EGFR phosphorylation at Tyr1173 was detected as soon as 5 min after protein A stimulation and was blocked by the EGFR tyrosine kinase inhibitors AG1478 and C56 (Figure 5A). Protein A-induced erk1/2 phosphorylation was also blocked by AG1478 and C56, indicating that erk1/2 activation is mediated by EGFR (Figure 5A). The EGFR tyrosine kinase inhibitor C56 significantly blocked TNFR1 shedding (Figure 5B). As expected, the L17A mutant did not induce EGFR phosphorylation (Figure 5C). Erk1/2 was not phosphorylated in response to protein A in the presence of the c-Src inhibitor PP1 (Figure 5D), and both PP1 and PP2, another c-Src inhibitor, decreased sTNFR1 shedding in a dose-dependent manner (Figure 5E). These observations are consistent with the requirement for c-Src in EGFR-mediated TACE activation.

Protein A-induced TNFR1 shedding is not mediated by EGFR ligands

EGFR signaling in the airway cells can be activated through autocrine pathways that involve the induction of TGF- and other EGFR ligands (Shao and Nadel, 2005; Janes et al, 2006; Zhao et al, 2006). Protein A binds directly to EGFR and also induces TGF- production (Figure 6A). However, it is very unlikely that EGFR activation was secondary to TGF- production because the amount of TGF- produced by the airway epithelial cells (1 ng/ml) in response to protein A was not sufficient to induce EGFR phosphorylation (data not shown). Moreover, stimulation of the airway epithelial cells with a 10-fold increased concentration of TGF- (10 ng/ml) did not induce TNFR1 shedding (Figure 6B). We also excluded autocrine stimulation by other EGFR ligands by demonstrating that protein A induced EGFR phosphorylation in the presence of TAPI-1, a metalloprotease inhibitor that prevents the release of EGFR ligands by TACE (Figure 6C).

Discussion

Staphylococci are common human pathogens, that target a broad range of eukaryotic tissues. While S. aureus interactions with many host matrix components facilitate the attachment stage of pathogenesis, their ability to exploit the immune system enables them to persist and thrive, even in the presence of a normal immune response. Protein A expression appears to play an important role in the success of S. aureus as a human pathogen. Most bacterial pathogens elicit proinflammatory signaling that stimulates the influx of neutrophils to eradicate infection. The host, then, must regulate chemokine and cytokine responses appropriately. However, in addition to its potent immunostimulatory activity, S. aureus, through the expression of protein A, has also evolved complex mechanisms to regulate TNFR1 signaling. Protein A effectively activates TACE, which is targeted to newly mobilized TNFR1, inducing its release from the cell surface.

The binding of protein A to the Fc region of IgG, the Fab region of immunoglobulin of the VH3 subclass (Moks et al, 1986; Sasso et al, 1989; Roben et al, 1995; Jansson et al, 1998), the von Willebrand factor (Hartleib et al, 2000) and TNFR1 (Gómez et al, 2006) have been mapped. The protein A–TNFR1 interaction mimics the pathway stimulated by the natural ligand TNF-. However, the same IgG-binding domain of protein A also has novel interactions with epithelial cells that are not analogous to those of the endogenous ligand TNF-. Although TNF-–TNFR1 activation and protein A–TNFR1 ligation both stimulate an identical proinflammatory signaling cascade (Gómez et al, 2004), protein A also interacts with EGFR to induce phosphorylation and activation of TACE.

The involvement of EGFR, ubiquitously expressed on the airway cells, in mediating TNFR1 shedding via TACE was unanticipated. Although there are several reports detailing TACE activation to cleave ligands involved in subsequent autocrine stimulation of EGFR (Shao and Nadel, 2005; Janes et al, 2006; Zhao et al, 2006), we show instead, that a protein A–EGFR interaction stimulates phosphorylation of both EGFR and TACE itself. Although the specific EGFR locus that is recognized by protein A was not mapped, we show by co-immunoprecipation and confocal imaging that protein A and EGFR are closely associated. Moreover, we identified protein A mutants capable of activating IL-8 signaling that are unable to activate EGFR, indicating a discrete interaction, separate from the proinflammatory signal. This protein A–EGFR interaction is required for receptor shedding, as mutants unable to bind EGFR fail to induce EGFR activation and TNFR1 cleavage. EGFR is also a target of an autocrine system stimulated by the epithelial production of TGF-. Although protein A induces TGF- production, this cytokine by itself failed to induce receptor shedding, and the interaction of protein A with EGFR is sufficient to activate TACE in the presence of a metalloprotease inhibitor that prevents TGF- release.

Several pathways that initiate TACE activity have been identified in the airway epithelial cells (Shao et al, 2004; Shao and Nadel, 2005; Kuwahara et al, 2006). The phosphorylation of TACE by erk1/2, induced by protein A, is also induced by phorbol esters (Doedens et al, 2003) or ROS (Hino et al, 1999; Zhang et al, 2001; Shao et al, 2004) in other cell systems. The signaling pathways initiated by the activation of TNFR1, however, are insufficient to activate TACE and induce TNFR1 shedding, as shown in cells where TNFR1 signaling was blocked through the expression of a TRAF2 DN mutant. Moreover, TNF- alone is an insufficient stimulus to induce TACE activation. Alternative routes of TACE activation, such as TGF-–EGFR signaling were also insufficient to induce TNFR1 shedding. It appears that mobilization of TNFR1, induced by protein A but not by TNF-, is necessary to deliver the receptor to the cell surface, where it becomes a substrate for the activated TACE. As TACE has many potential substrates, the mobilization of TNFR1 in conjunction with TACE phosphorylation may serve to target activated TACE to this specific substrate. The domains of protein A that are involved in receptor mobilization are yet to be identified.

The interaction between protein A, a single bacterial virulence factor, and EGFR, provides a novel mechanism to regulate TNFR1 availability. Other human pathogens also exploit EGFR signaling to evade immune responses. Members of the Herpes virus family have several interactions with EGFR that contribute to pathogenesis. Binding of the cytomegalovirus glycoprotein B to EGFR induces receptor phosphorylation and contributes to viral invasion (Wang et al, 2003b; Compton, 2004). Similarly, the Epstein-Barr virus latent membrane protein 1 (LMP1) activates EGFR phosphorylation to induce cell proliferation (Miller et al, 1997; Brinkmann and Schulz, 2006). Of note, protein A shares conserved sequence domains with LMP1, indicating that Staphylococci may be using a similar strategy to exploit host signaling cascades without inducing responses that would be deleterious to either the host cell or the organism.

These studies provide further evidence to demonstrate the contribution of protein A to the success of S. aureus as a human pathogen. It is an exceptional virulence factor, a single protein that can target multiple immunologically important eukaryotic receptors. It is probably not a coincidence that protein A is among the most highly conserved staphylococcal virulence factors expressed, nor that its levels of expression are significantly increased in staphylococci isolated from invasive human infections.

Materials and methods

Cell lines, bacterial strains and reagents

1HAEo- and 16HBE cells (human airway epithelial lines) (D Gruenert, Pacific Medical Center Research Institute, San Francisco, California) were grown as previously described (Rajan et al, 2000; Ratner et al, 2001) Primary airway epithelial cells isolated from human nasal polyps (HNP) were grown on Transwell-clear filters (Corning Costar) in M3 medium as previously described (DiMango et al, 1998). RAW cells (a murine macrophage line) were grown in RPMI medium supplemented with 10% fetal calf serum (Invitrogen). Protein A from S. aureus Newman and the IgG binding domain D were cloned and purified as a GST-fusion protein, resuspended in PBS and used at a concentration of 2.5 M for stimulation. Mutations were introduced into SpA domain D using a PCR-based mutagenesis strategy. The amino-acid substitutions F5A and L17A were selected for this study among 10 constructed (Gómez et al, 2006). TNF- and TGF- (Calbiochem) were used for stimulation at 100 ng/ml and 10 ng/ml, respectively. Brefeldin A (Sigma-Fluka) was used at 10 g/ml and cycloheximide (Sigma) was used at 10 g/ml. TAPI-1 (Calbiochem) was used at 50 M.

Flow cytometry

Cells were stimulated with protein A or TNF-, washed three times and stained with anti-TNFR1 (H-271) or anti-TACE (C-15) polyclonal antibodies (Santa Cruz Biotech). Alexa Fluor 488-conjugated secondary antibody (Molecular Probes) was used. Cells were then washed, fixed in 1% paraformaldehyde and analyzed with a Becton Dickinson FACS Calibur. Data were collected using Cell Quest software and analyzed with Winmdi.

IL-8, TGF- and sTNFR1 ELISA

Cells were weaned from serum for 24 h and exposed to protein A, TNF- or TGF- for 4 h unless indicated. IL-8 (BD pharmingen), TGF- and sTNFR1 (R&D Systems) in the supernatant were measured by ELISA. The effect of MEK, c-Src and EGFR tyrosine kinase inhibitors was tested by pretreating the cells for 30 min with 10 M UO126 (MEK, Calbiochem), 50 M PP1 (c-Src, Biomol), 50 M PP2 (c-Src, Calbiochem), 10 M AG1478 (EGFR tyrosine kinase, Calbiochem) or 50 M compound 56 (EGFR tyrosine kinase, Calbiochem) and adding fresh inhibitors during stimulation. The effect of MEK and TRAF2 DN mutants was tested by transfecting 1HAEo- cells grown to 50–70% confluence with HMEK (K97R) (Adamo et al, 2004) TRAF2 DN (Gómez et al, 2004) or a vector control using FuGene6.0 (Roche). After 16 h, cells were weaned from serum for 24 h and stimulated with protein A.

Immunoprecipitation and Western Blot

Cells were lysed using 60 mM n-octyl--D-glucopyranoside in TBS (0.1 M Tris–HCl and 0.15 M NaCl (pH 7.8)) containing Complete Mini protease inhibitor tablets (Roche), 1 mM sodium orthovanadate, 100 mM sodium fluoride and 20 M GM6001. For protein A–EGFR co-immunoprecipitation, lysates (500 g of protein) from cells stimulated with bovine serum albumin (control), protein A, protein A domain D or the L17A and F5A mutants were incubated with the monoclonal antibody to protein A (Sigma) overnight at 4°C with shaking. For TACE immunoprecipitations, cell lysates (300 g of protein) were incubated with 1 g of goat anti-TACE (C-15) antibody (Santa Cruz Biotech) overnight at 4°C with shaking. Protein G agarose beads were then added for 1 h at 4°C with shaking. Beads were washed twice with 500 mM NaCl, 50 mM Tris and 1% NP-40, followed by a wash with 20 mM Tris and resuspended in NUPAGE sample buffer and reducing agent (Invitrogen). Proteins were separated on 4–12% bis–tris NUPAGE gels (Invitrogen), transferred to PVDF Immobilon P membrane (Millipore) and blocked with 5% milk in TBST (50 mM Tris pH 7.5, 150 mM NaCl and 0.05% Tween) for 1 h at room temperature. Immunodetection was performed using anti-phospho-Threonine (Cell signaling), anti-phospho-erk1/2, anti-erk1/2, anti-phospho EGFR (Tyr 1173), anti-EGFR (1005) or anti-TACE (C-15) (Santa Cruz Biotech) antibodies, followed by secondary antibodies conjugated to horseradish peroxidase (Santa Cruz). Anti-TACE antibody C-15 recognized both the pro-form and the mature form (devoid of the pro-domain) of TACE in the airway epithelial cells. The identity of these bands in the airway cell lines used was confirmed by using the anti-TACE antibody H-300 that only recognizes the pro-form of TACE.

RNA interference

Inhibition of TACE expression in the 16HBE airway epithelial cells was previously described (Gómez et al, 2005). Briefly, two cell lines were constructed by using two pairs of oligonucleotides containing 19 bp of human TACE were generated as follows: pair 1: 5'gatccccGTAAGGCCCAGGAGTGTTTttcaaga
gaAAACACTCCTGGGCCTTACttttggaaa3' and 5'agcttttccaaaaGTAAGGCCCAGGAGTGTTTt
ctcttgaaAAACACTCCTGGGCCTTACggg3'; pair 2: 5'gatccccCATAGAGCCACTTTGGAGAttcaaga
gaTCTCCAAAGTGGCTCTATGttttggaaa3' and 5'agcttttccaaaaCATAGAGCCACTTTGGAGAt
ctcttgaaTCTCCAAAGTGGCTCTATGggg3'. To construct pRS-TACE-1 and pRS-TACE-2, the oligos were annealed and ligated into BglII and HindIII sites of the pRetroSuper vector (pRS) (Brummelkamp et al, 2002). Construct integrity was confirmed by direct sequencing of the plasmid. Packaging of retroviral constructs was carried out in HEK293T cells (Pear et al, 1993). 16HBE cells were infected for 18 h in the presence of 4 mg/ml polybrene (Sigma). pBabe-puro-EGFP was used to monitor the efficiency of transfection to 293T cells and infection. A pRS-scramble plasmid (pRS-sc) was used as a control by cloning the sequence ggcagttccaccccagtgc into pRS as described for pRS–TACE.

Confocal microscopy

1HAEo-, 16HBE or primary airway epithelial cells isolated from human nasal polyps (HNP) were grown on Transwell-Clear filters (Corning-Costar) with an air–liquid interface to form polarized monolayers. Protein A stimulated and control unstimulated cells were fixed with 4% paraformaldehyde and after blocking with 5% normal serum, rabbit polyclonal anti-TNFR1 or goat polyclonal anti-TACE antibodies (Santa Cruz Biotech) were added for 1 h. For protein A binding experiments, cells were fixed as above and incubated with the full-length protein A, protein A domain D or the mutant L17A for 1 h at room temperature. After washing, rabbit polyclonal anti-EGFR and monoclonal antibody to protein A were added for 1 h. For Golgi colocalization experiments, cells were incubated prior to stimulation with 5 uM of the Golgi probe BODIPY TR (Molecular Probes) for 30 min at 4°C. Cells were preincubated with the media or brefeldin for 30 min and stimulated in the presence or absence of brefeldin for 2 h. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-100 and then blocked and stained for TNFR1 as described above. Alexa Fluor 488- and 594-conjugated secondary antibodies (Molecular Probes) were used. After washing, filters were removed from transwells and mounted with Vectashield (Vector Laboratories Inc.) onto glass slides.

Acknowledgements

This work was funded by the NIH grant HL079395 to ASP, a US Cystic Fibrosis Foundation postdoctoral fellowship to MIG and the Health Research Board of Ireland (MO'S). Confocal microscopy was performed at the Herbert Irving Optical Microscopy facility at Columbia University.

References

Adamo R, Sokol S, Soong G, Gómez MI, Prince A (2004) Pseudomonas aeruginosa flagella activate airway epithelial cells through asialoGM1 and toll-like receptor 2 as well as toll-like receptor 5. Am J Respir Cell Mol Biol 30: 627–634 | Article | PubMed | ISI | ChemPort |

Aggarwal BB (2003) Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol 3: 745–756 | Article | PubMed | ISI | ChemPort |

Bradley JR, Thiru S, Pober JS (1995) Disparate localization of 55-kd and 75-kd tumor necrosis factor receptors in human endothelial cells. Am J Pathol 146: 27–32 | PubMed | ISI | ChemPort |

Brinkmann MM, Schulz TF (2006) Regulation of intracellular signalling by the terminal membrane proteins of members of the Gammaherpesvirinae. J Gen Virol 87: 1047–1074 | Article | PubMed | ChemPort |

Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550–553 | Article | PubMed | ISI | ChemPort |

Chen G, Goeddel DV (2002) TNF-R1 signaling: a beautiful pathway. Science 296: 1634–1635 | Article | PubMed | ISI | ChemPort |

Compton T (2004) Receptors and immune sensors: the complex entry path of human cytomegalovirus. Trends Cell Biol 14: 5–8 | Article | PubMed | ChemPort |

Diaz-Rodriguez E, Montero JC, Esparis-Ogando A, Yuste L, Pandiella A (2002) Extracellular signal-regulated kinase phosphorylates tumor necrosis factor alpha-converting enzyme at threonine 735: a potential role in regulated shedding. Mol Biol Cell 13: 2031–2044 | Article | PubMed | ISI | ChemPort |

DiMango E, Ratner AJ, Bryan R, Tabibi S, Prince A (1998) Activation of NF-kappaB by adherent Pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells. J Clin Invest 101: 2598–2605 | PubMed | ChemPort |

Doedens JR, Mahimkar RM, Black RA (2003) TACE/ADAM-17 enzymatic activity is increased in response to cellular stimulation. Biochem Biophys Res Commun 308: 331–338 | Article | PubMed | ISI | ChemPort |

Fan H, Turck CW, Derynck R (2003) Characterization of growth factor-induced serine phosphorylation of tumor necrosis factor-alpha converting enzyme and of an alternatively translated polypeptide. J Biol Chem 278: 18617–18627 | Article | PubMed | ChemPort |

Fang FC (2004) Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat Rev Microbiol 2: 820–832 | Article | PubMed | ISI | ChemPort |

Foster TJ (2005) Immune evasion by staphylococci. Nat Rev Microbiol 3: 948–958 | Article | PubMed | ChemPort |

Garton KJ, Gough PJ, Philalay J, Wille PT, Blobel CP, Whitehead RH, Dempsey PJ, Raines EW (2003) Stimulated shedding of vascular cell adhesion molecule 1 (VCAM-1) is mediated by tumor necrosis factor-alpha-converting enzyme (ADAM 17). J Biol Chem 278: 37459–37464 | Article | PubMed | ChemPort |

Garton KJ, Gough PJ, Raines EW (2006) Emerging roles for ectodomain shedding in the regulation of inflammatory responses. J Leukoc Biol 79: 1105–1116 | Article | PubMed | ChemPort |

Goerke C, Campana S, Bayer MG, Doring G, Botzenhart K, Wolz C (2000) Direct quantitative transcript analysis of the agr regulon of Staphylococcus aureus during human infection in comparison to the expression profile in vitro. Infect Immun 68: 1304–1311 | Article | PubMed | ISI | ChemPort |

Gómez MI, Lee A, Reddy B, Muir A, Soong G, Pitt A, Cheung A, Prince A (2004) Staphylococcus aureus protein A induces airway epithelial inflammatory responses by activating TNFR1. Nat Med 10: 842–848 | Article | PubMed | ChemPort |

Gómez MI, O'Seaghdha M, Magargee M, Foster TJ, Prince AS (2006) Staphylococcus aureus protein A activates TNFR1 signaling through conserved IgG binding domains. J Biol Chem 29: 20190–20196

Gómez MI, Sokol SH, Muir AB, Soong G, Bastien J, Prince AS (2005) Bacterial induction of TNF-alpha converting enzyme expression and IL-6 receptor alpha shedding regulates airway inflammatory signaling. J Immunol 175: 1930–1936 | PubMed |

Hartleib J, Kohler N, Dickinson RB, Chhatwal GS, Sixma JJ, Hartford OM, Foster TJ, Peters G, Kehrel BE, Herrmann M (2000) Protein A is the von Willebrand factor binding protein on Staphylococcus aureus. Blood 96: 2149–2156 | PubMed | ISI | ChemPort |

Hino T, Nakamura H, Abe S, Saito H, Inage M, Terashita K, Kato S, Tomoike H (1999) Hydrogen peroxide enhances shedding of type I soluble tumor necrosis factor receptor from pulmonary epithelial cells. Am J Respir Cell Mol Biol 20: 122–128 | PubMed | ISI | ChemPort |

Janes KA, Gaudet S, Albeck JG, Nielsen UB, Lauffenburger DA, Sorger PK (2006) The response of human epithelial cells to TNF involves an inducible autocrine cascade. Cell 124: 1225–1239 | Article | PubMed | ChemPort |

Jansson B, Uhlen M, Nygren PA (1998) All individual domains of staphylococcal protein A show Fab binding. FEMS Immunol Med Microbiol 20: 69–78 | Article | PubMed | ChemPort |

Jones SJ, Ledgerwood EC, Prins JB, Galbraith J, Johnson DR, Pober JS, Bradley JR (1999) TNF recruits TRADD to the plasma membrane but not the trans-Golgi network, the principal subcellular location of TNF-R1. J Immunol 162: 1042–1048 | PubMed | ISI | ChemPort |

Kuwahara I, Lillehoj EP, Lu W, Singh IS, Isohama Y, Miyata T, Kim KC (2006) Neutrophil elastase induces IL-8 gene transcription and protein release through p38/NF-{kappa}B activation via EGFR tr. Am J Physiol Lung Cell Mol Physiol 291: L407–L416 | PubMed | ChemPort |

Lowy FD (1998) Staphylococcus aureus infections. N Engl J Med 339: 520–532 | Article | PubMed | ISI | ChemPort |

Mezyk R, Bzowska M, Bereta J (2003) Structure and functions of tumor necrosis factor-alpha converting enzyme. Acta Biochim Pol 50: 625–645 | PubMed | ISI | ChemPort |

Miller WE, Mosialos G, Kieff E, Raab-Traub N (1997) Epstein-Barr virus LMP1 induction of the epidermal growth factor receptor is mediated through a TRAF signaling pathway distinct from NF-kappaB activation. J Virol 71: 586–594 | PubMed | ISI | ChemPort |

Moks T, Abrahmsen L, Nilsson B, Hellman U, Sjoquist J, Uhlen M (1986) Staphylococcal protein A consists of five IgG-binding domains. Eur J Biochem 156: 637–643 | Article | PubMed | ISI | ChemPort |

Pear WS, Nolan GP, Scott ML, Baltimore D (1993) Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci USA 90: 8392–8396 | Article | PubMed | ChemPort |

Peschon JJ, Slack JL, Reddy P, Stocking KL, Sunnarborg SW, Lee DC, Russell WE, Castner BJ, Johnson RS, Fitzner JN, Boyce RW, Nelson N, Kozlosky CJ, Wolfson MF, Rauch CT, Cerretti DP, Paxton RJ, March CJ, Black RA (1998) An essential role for ectodomain shedding in mammalian development. Science 282: 1281–1284 | Article | PubMed | ISI | ChemPort |

Rahman MM, McFadden G (2006) Modulation of tumor necrosis factor by microbial pathogens. PLoS Pathog 2: e4 | Article | PubMed | ChemPort |

Rajan S, Cacalano G, Bryan R, Ratner AJ, Sontich CU, van Heerckeren A, Davis P, Prince A (2000) Pseudomonas aeruginosa induction of apoptosis in respiratory epithelial cells: analysis of the effects of cystic fibrosis transmembrane conductance regulator dysfunction and bacterial virulence factors. Am J Respir Cell Mol Biol 23: 304–312 | PubMed | ISI | ChemPort |

Ratner AJ, Bryan R, Weber A, Nguyen S, Barnes D, Pitt A, Gelber S, Cheung A, Prince A (2001) Cystic fibrosis pathogens activate Ca²⁺-dependent mitogen-activated protein kinase signaling pathways in airway epithelial cells. J Biol Chem 276: 19267–19275 | Article | PubMed | ISI | ChemPort |

Reddy P, Slack JL, Davis R, Cerretti DP, Kozlosky CJ, Blanton RA, Shows D, Peschon JJ, Black RA (2000) Functional analysis of the domain structure of tumor necrosis factor-alpha converting enzyme. J Biol Chem 275: 14608–14614 | Article | PubMed | ISI | ChemPort |

Roben PW, Salem AN, Silverman GJ (1995) VH3 family antibodies bind domain D of staphylococcal protein A. J Immunol 154: 6437–6445 | PubMed | ChemPort |

Sasso EH, Silverman GJ, Mannik M (1989) Human IgM molecules that bind staphylococcal protein A contain VHIII H chains. J Immunol 142: 2778–2783 | PubMed | ISI | ChemPort |

Saunders BM, Tran S, Ruuls S, Sedgwick JD, Briscoe H, Britton WJ (2005) Transmembrane TNF is sufficient to initiate cell migration and granuloma formation and provide acute, but not long-term, control of Mycobacterium tuberculosis infection. J Immunol 174: 4852–4859 | PubMed | ChemPort |

Schlessinger J (2000) Cell signaling by receptor tyrosine kinases. Cell 103: 211–225 | Article | PubMed | ISI | ChemPort |

Shao MX, Nadel JA (2005) Neutrophil elastase induces MUC5AC mucin production in human airway epithelial cells via a cascade involving protein kinase C, reactive oxygen species, and TNF-alpha-converting enzyme. J Immunol 175: 4009–4016 | PubMed | ChemPort |

Shao MX, Nakanaga T, Nadel JA (2004) Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor-alpha-converting enzyme in human airway epithelial (NCI-H292) cells. Am J Physiol Lung Cell Mol Physiol 287: L420–L427 | PubMed | ChemPort |

Tsakadze NL, Sithu SD, Sen U, English WR, Murphy G, D'Souza SE (2006) Tumor necrosis factor-alpha-converting enzyme (TACE/ADAM-17) mediates the ectodomain cleavage of intercellular adhesion molecule-1 (ICAM-1). J Biol Chem 281: 3157–3164 | Article | PubMed | ChemPort |

Uhlen M, Guss B, Nilsson B, Gatenbeck S, Philipson L, Lindberg M (1984) Complete sequence of the staphylococcal gene encoding protein A. A gene evolved through multiple duplications. J Biol Chem 259: 1695–1702 | PubMed | ISI | ChemPort |

Wang J, Al-Lamki RS, Zhang H, Kirkiles-Smith N, Gaeta ML, Thiru S, Pober JS, Bradley JR (2003a) Histamine antagonizes tumor necrosis factor (TNF) signaling by stimulating TNF receptor shedding from the cell surface and Golgi storage pool. J Biol Chem 278: 21751–21760 | Article | ChemPort |

Wang X, Huong SM, Chiu ML, Raab-Traub N, Huang ES (2003b) Epidermal growth factor receptor is a cellular receptor for human cytomegalovirus. Nature 424: 456–461 | Article | PubMed | ISI | ChemPort |

Xanthoulea S, Pasparakis M, Kousteni S, Brakebusch C, Wallach D, Bauer J, Lassmann H, Kollias G (2004) Tumor necrosis factor (TNF) receptor shedding controls thresholds of innate immune activation that balance opposing TNF functions in infectious and inflammatory diseases. J Exp Med 200: 367–376 | Article | PubMed | ISI | ChemPort |

Zhang Z, Oliver P, Lancaster JJ, Schwarzenberger PO, Joshi MS, Cork J, Kolls JK (2001) Reactive oxygen species mediate tumor necrosis factor alpha-converting, enzyme-dependent ectodomain shedding induced by phorbol myristate acetate. Faseb J 15: 303–305 | PubMed | ISI |