A Competitive Mechanism for Staphylococcal Toxin SSL7 Inhibiting the Leukocyte IgA Receptor, FcαRI, Is Revealed by SSL7 Binding at the Cα2/Cα3 Interface of IgA*

Leukocyte recruitment and effector functions like phagocytosis and respiratory burst are key elements of immunity to infection. Pathogen survival is dependent upon the ability to overwhelm, evade or inhibit the immune system. Pathogenic group A and group B streptococci are well known to produce virulence factors that block the binding of IgA to the leukocyte IgA receptor, FcαRI, thereby inhibiting IgA-mediated immunity. Recently we found Staphylococcus aureus also interferes with IgA-mediated effector functions as the putative virulence factor SSL7 also binds IgA and blocks binding to FcαRI. Herein we report that SSL7 and FcαRI bind many of the same key residues in the Fc region of human IgA. Residues Leu-257 and Leu-258 in domain Cα2 and residues 440–443 PLAF in Cα3 of IgA lie at the Cα2/Cα3 interface and make major contributions to the binding of both the leukocyte receptor FcαRI and SSL7. It is remarkable this S. aureus IgA binding factor and unrelated factors from streptococci are functionally convergent, all targeting a number of the same residues in the IgA Fc, which comprise the binding site for the leukocyte IgA receptor, FcαRI.

Staphylococcus aureus, a commensal organism of the human skin and nose (1), is also a significant human pathogen responsible for conditions such as Scarlet fever, toxin shock, septicemia, and endocarditis. The S. aureus genome contains three clusters of superantigen and superantigen-like genes, designated SaPIn1, -2, and -3 (2). SaPIn2 contains the Staphylococcus superantigen-like (ssl) genes, previously designated as staphylococcal enterotoxin-like (SET) genes (3). These genes are highly represented in clinical isolates of S. aureus and are inferred to contribute to pathogenicity of these strains (4). Crystallographic studies of SSL5 (SET3) (5) and SSL7 (SET1) (6,7) proteins have indicated structural similarity to classical superantigens with the SSLs also comprising an OB-fold and ␤-grasp domain. We have recently defined two binding activities of the SSL7 protein demonstrating that SSL7 binds simultaneously to human complement factor C5 and IgA (8). The binding of IgA by SSL7 inhibited IgA binding to the leukocyte IgA receptor Fc␣RI (CD89) thus providing a potential mechanism for evasion of IgA-mediated cellular immunity, via the blockade of IgA-mediated leukocyte effector functions such as phagocytosis and respiratory burst (9 -11). General similarities with staphylococcal protein A are clear which, although an unrelated protein, is also capable of multiple interactions targeting the host immune response, including binding TNFR1 (12), C1qR (13), and IgG via V H 3 Fabs (14) and the Fc at the C␥2/C␥3 interface region (15). This study investigated the site of human IgA binding to SSL7. SSL7 was found to bind to the C␣2/C␣3 interface of IgA Fc and require residues essential for the interaction of Fc␣RI with IgA. Thus the SSL7 and Fc␣RI binding sites are likely to be the same or at least closely overlapping. Furthermore the interaction of SSL7 was independent of the IgA Asn-263 linked carbohydrate, which extends close to the C␣2/C␣3 interface (16).

MATERIALS AND METHODS
Biotinylated SSL7-Recombinant SSL7 was prepared as described previously (8). Purified SSL7 150 g/ml (7.5 M) was reacted in phosphate-buffered saline with 1 mg/ml of EZ-Link sulfo-NHS-LC-biotin (Pierce) at 25°C, 1 h, and then the remaining biotinylation reagent was reacted by the addition of 100 l of 0.5 M ethanolamine pH 8.5. Prior to use biotinylated SSL7 was dialyzed extensively against phosphate-buffered saline.
Transferrin Receptor (TfR)-IgA Fc Construct-The expression of the transferrin receptor-IgA Fc fusion protein was based on the approach of Stabila et al. (17). In brief, DNA encoding the N terminus and transmembrane region of the human TfR 2 was amplified from cDNA prepared from K562 cells (ATCC, Manassas, VA) using a cDNA synthesis kit (Pharmacia, Melbourne, Australia) and RNAzol (Invitrogen). PCR used the primers oBW207 CCCGAATTCGCCACCATGATGGAT-CAAGCTAGATCAGC and oBW208 CCCGGGCCCCTCAGTTTT-TGGTTCTACCCC and the thermostable proofreading polymerase Pwo (Roche Diagnostics), and this PCR product was digested with EcoRI and ApaI (New England Biolabs, Beverly, MA). A fragment encoding the IgA1 Fc was obtained by NotI and ApaI digest of pBAR233. The baculovirus expression vector pBAR233 was a derivative of pFastBac (Invitrogen) containing a Fc␥RIIa leader sequence, a hexahistidine tag sequence, and a human IgA1-Fc sequence derived by PCR amplification from cDNA prepared from the IgAϩ cell line Dakiki (18)  TfR fragment and the ApaI/NotI IgA Fc fragment were simultaneously ligated into the EcoRI and NotI restriction sites of the gateway vector pENTR1A (Invitrogen) yielding pBAR355. The LR clonase reaction was used with gateway reading frame-A cassette adapted pCR3 (Invitrogen) to create the construct pBAR357 expressing the N-terminal transmembrane region of the transferrin receptor fused, at the hinge region, to the IgA Fc. The IgA1-Fc mutants were constructed by PCR of pBAR355 using Turbo Pfu (Stratagene) followed by phosphorylation and ligation of the linear PCR product as previously described (19). The LL257,258MI mutation reaction used the primer pair oBW308 GGT-TCAGAAGCGAACCTCACG and oBW269 GATCATCAGGTC-CTCGAGGGCCG, the A442R mutation used the primer pair oBW266 ACACAGAAGACCATCGACCG and oBW202 GAAGCGCAGCG-GCAGGGCCTCG, the PLAF(440 -443)HNHY mutation used oBW-270 GCCCTGCACAACCACTACACACAGAAGACCATCGACCG and oBW271 CTCGTGGCCCACCATGCAG, and the N263T mutation used the primer pair oBW268 GGTTCAGAAGCGACCCTCAC and oBW309 TAAGAGCAGGTCCTCGAGTGCCG.
Fc␣RI-Fc␥2b Construct-The Fc region of mouse IgG2b was amplified from cDNA prepared from Balb/c splenocytes using the primers oBW137 GGGATCCGAGCCCAGCGGGCCCATTTC and oBW172 GCCCGGGCTATTTACCCGGAGACCGGGA as described above. Splice overlap PCR was used to add the sequence GGGCCCCCTGCA-GAACTGGTTCCGCGTGGATCC onto the 3Ј-end of the cDNA encoding the Fc␣RI ectodomains (pBAR152) (20) immediately following the codon for Ile-208 (i.e. corresponding amino acid sequence I208-GPPAELVPRGS, thrombin cleavage site in italics). These two DNAs were ligated at their BamHI sites, and the chimeric DNA was subcloned into the StuI site of pFastBac (Life Tech) making pBAR213. The chimeric DNA was then further subcloned into the EcoRI/XbaI sites of pcDNA3 (Invitrogen). CHO-K1 cells were transfected with this construct, pBAR225, using Lipofectamine 2000 (Invitrogen) and resistant colonies selected with 1 mg/ml G418. The cell line 225CHO expressing Fc␣RI-Fc␥2b was cloned by two rounds of single cell limiting dilution.
Biosensor Analysis of rSSL7 Binding to Chimeric IgA2-A BIAcore CM5 chip was derivatized with 3-nitro-4-hydroxyphenylacetate (NP-OSu, Genosys, Cambridgeshire, UK) as described in Ref. 21, and an equilibrium binding analysis was performed as described previously (19) using a BIAcore2000 TM (BIAcore, Melbourne, Australia). Briefly, 1 g/ml recombinant chimeric IgA2 anti-NIP (gift of Drs. Margaret Goodall and Roy Jefferies) was reacted (30 l, flow rate of 10 l/min) with the derivatized chip. The immobilized IgA2 layer was then reacted with rSSL7 in the concentration range 0.6 -80 nM (100 l, flow rate of 2 l/min), and the response end point of each injection, above that of the injection of buffer alone, was taken as approaching the equilibrium binding response for the SSL7:IgA2 interaction. The data were fitted to the single binding site model, where A is the free analyte concentration, and B is the binding capacity.
Expression and Binding to TfR-IgA Fc Fusion Proteins-Transient expression in CHOP cells (22) in 10 cm 2 wells was performed by transfection with 5 g of plasmid DNA and Lipofectamine 2000 reagent according to the manufacturer's instructions. After 48 or 72 h the expression of TfR-IgA Fc was measured by incubating cells (50 l, 10 5 cells) 1 h on ice with FITC-conjugated sheep FabЈ 2 anti-human IgA (Silenus/Chemicon, Melbourne, Australia) diluted 1/200 in phosphatebuffered saline containing 0.1% bovine serum albumin. After incubation the cells were resuspended in 3 ml of phosphate-buffered saline containing 0.1% bovine serum albumin and centrifuged (1000 rpm, 5 min), and the collected cells were analyzed using a FACSCalibur (Becton Dickinson). SSL7 binding activity of the mutants of IgA Fc was meas-ured by incubating cells (10 5 ) 1 h on ice with 0.2 g (10 pmol) of biotinylated SSL7. Unbound SSL7 was removed, and the cells were incubated in 1/400 dilution of 0.5 mg/ml phycoerythrin-conjugated streptavidin (Pharmingen) for 1 h on ice. Fc␣RI binding activity of the mutants of IgA Fc was measured by incubating cells (10 5 ) 1 h on ice with 0.5 ml of 225CHO cell supernatant containing Fc␣RI-Fc␥2b fusion protein (ϳ0.5 g, 5 pmol). Unbound receptor was removed, and the cells were incubated in a 1/200 dilution of FITC-conjugated sheep FabЈ 2 anti-mouse Ig (Silenus/Chemicon) for 1 h on ice.

RESULTS
Recombinant SSL7 Binding to IgA-S. aureus colonizes mucosal sites and this prompted us to characterize the interaction of SSL7 with IgA2, the major subclass of IgA at the mucosa. Here we report rSSL7 binding to a human chimeric IgA2 anti-NP antibody. The antibody was reacted with a NP-derivatized biosensor surface with ϳ700 response units of antibody binding and a variation between cycles of Յ10 response units (Fig. 1A). The binding to this immobilized IgA2 of SSL7 at different concentrations was recorded (Fig. 1B) after 3000 s when equilibrium was being approached. Plotting the equilibrium binding responses fitted best to a single binding site with an apparent affinity of 5.0 Ϯ 1.5 nM (n ϭ 5, Fig. 1C, one representative experiment K D ϭ 6.8 Ϯ 0.8 nM).  by staining with anti-IgA FITC ( Fig. 2A) and the rSSL7 (Fig. 2B), and Fc␣RI-Ig (Fig. 2C) binding to the same transfectants was also determined. In agreement with our previous data the binding of Fc␣RI-Ig to WT IgA Fc fusion protein expressing cells (mean fluorescent intensity (MFI) ϭ 38.6 Ϯ 2) was inhibited 90% in the presence of 2 M (40 g/ml) rSSL7 (MFI ϭ 8 Ϯ 2, background staining MFI ϭ 5 Ϯ 1, Fig. 2D). Hence in this experimental system Fc␣RI-Ig binding to the TfR-IgA Fc fusion protein is inhibited by rSSL7 just as rSSL7 inhibits serum IgA binding to isolated leukocytes (8).

Expression of Fusion Proteins of Transferrin Receptor with Mutant IgA Fc
Regions-Because SSL7 binds to both IgA1 and IgA2 with nanomolar affinity and SSL7 inhibits Fc␣RI binding to IgA, SSL7 may bind directly to the Fc␣RI binding site, which is conserved in IgA1 and IgA2.
Molecular analysis of the SSL7 binding site was performed by mutating residues at the C␣2/C␣3 interface of IgA Fc comprising the Fc␣RI binding site. The MFIs from FACS profiles showed the level of expression of the WT IgA fusion protein varied between experiments from a MFI ϭ ϳ150 ( Figs. 2A and 3) in one set of transfections to ϳ60 in another (Fig. 4). In each experiment the A442R mutant IgA Fc was always apparently expressed ϳ2-3-fold lower than the WT. This may indicate that a proportion of this mutant IgA Fc may not fold correctly and not progress through the secretory pathway. Another possibility is that the mutation could cause a significant local alteration of the protein such that a major epitope(s) among those recognized by the anti-IgA polyclonal antiserum is disrupted leading to loss of polyclonal reactivity, rather than a lower surface expression. To go some way to address this, WT IgA Fc fusion protein-expressing cells were incubated with 5 M (100 g/ml) SSL7 prior to staining with the anti-IgA polyclonal antiserum (Fig. 3). Although the WT cells stained with a MFI ϭ 146 (Ϯ5, n ϭ 4) the staining of the cells preincubated with SSL7 (MFI ϭ 53 Ϯ 2) was reduced to that of the A442R mutant-expressing cells (MFI ϭ 57 Ϯ 2). Thus SSL7 binding to IgA Fc inhibits binding of the anti-human IgA antiserum indicating some of the recognized epitopes lie close to the SSL7 binding site, and these epitopes may be affected by mutation of this site. Thus it is possible that the A442R mutation may likewise affect the reactivity with the antiserum. Although the A442R mutation has been used to elucidate the IgA binding site of Fc␣RI (23,24), to avoid any ambiguity another mutation of this region, PLAF440-443HNHY was made where the FG loop of C␣3, PLAF, was altered to the equivalent sequence of human IgG1. The PLAF440-443HNHY mutant (MFI ϭ 140 Ϯ 7) was expressed at levels equivalent to the WT protein (MFI ϭ 146 Ϯ 5) and hence was unlikely to be misfolded (Fig. 3). Other residues of IgA, Leu-257 and Leu-258 important in Fc␣RI binding (16,(23)(24)(25) were also mutated in the TfR-IgA Fc fusion protein to the equivalent residues in IgG1. This mutant LL257,258MI was also expressed at levels equivalent to that of the WT fusion protein (MFI ϭ 54 Ϯ 2, c.f. 61 Ϯ 10; Fig. 4A). Finally a mutation of the IgA Fc fusion protein N263T abolished the IgA C␣2 N-glycosylation site. The expression of this protein (MFI ϭ 54 Ϯ 6, n ϭ 4) was also not significantly different from that of the WT protein (MFI ϭ 61 Ϯ 10, n ϭ 4) (Fig. 4A).

DISCUSSION
SSL7, a putative staphylococcal virulence factor, binds serum and secretory IgA from multiple species and prevents human IgA interaction with the leukocyte IgA receptor, Fc␣RI (8). S. aureus infection of the bloodstream can lead to septicemia, and asymptomatic carriage of S. aureus can occur in the nose while life-threatening infections of the respiratory tract also occur (12). We previously had characterized SSL7 binding to serum IgA, of which the major form is IgA1 (8). The importance of S. aureus adaptation to mucosal environments prompted us to characterize the interaction of SSL7 with IgA2, the major mucosal subclass of IgA. Here we report that IgA2 also bound SSL7 with nanomolar affinity. This high affinity interaction marks SSL7 as a potential factor in the survival of S. aureus not only in the blood, but also at mucosal sites. This study has further shown that the SSL7 binding site and that of Fc␣RI utilize common residues in the Fc region of human IgA. Residues Leu-257 and Leu-258 in the AB loop/helix of domain C␣2 of IgA and residues 440 -443 PLAF in the FG loop of domain C␣3 of IgA have been found in domain swap, mutagenesis (23)(24)(25), and x-ray crystallographic studies (16) to make major contributions to the binding of the leukocyte receptor Fc␣RI (Fig. 5). Mutation of these residues resulted in an expected decrease in soluble Fc␣RI-Ig binding consistent with the structural data (16) and, in addition, completely abrogated SSL7 binding indicating the binding sites of SSL7 and Fc␣RI comprise many of the same residues. In the crystal structure of IgA bound to Fc␣RI, the N-linked carbohydrate of IgA extends down the external face of the C␣2 domain to the C␣2/C␣3 interface (16) (Fig. 5) and thus may potentially be involved in interaction with molecules binding to this region. The role of the carbohydrate in SSL7 binding was examined using the N-linked glycosylation site point mutant N263T. The N263T IgA Fc mutant was expressed at levels equivalent to WT and bound similar levels of SSL7 indicating that the carbohydrate did not contribute to SSL7 binding. Thus despite the fact that other bacterial proteins containing an OB-fold domain have been shown to bind oligosaccharides (26), we conclude that this is not the case for the SSL7/IgA interaction. Interestingly the IgA Asn-263-linked glycan is close to (8 Å), but not contacting, Fc␣RI in the crystal complex (16), and in this study only a modest effect on the binding of Fc␣RI-Ig to the glycan deficient N263T mutant was observed.
Leukocyte recruitment, phagocytosis, and respiratory burst are critical aspects of protective immunity to Gram-positive bacteria. Interfering with Fc␣RI-mediated recognition of IgA opsonized bacteria by blocking the receptor binding site on IgA is a strategy utilized by pathogenic group A (27,28) and group B streptococci (29,30) to frustrate IgA-mediated protective immunity. Our report that SSL7 binds IgA with high affinity and inhibits interaction with Fc␣RI (8) clearly demonstrates that this evasion strategy is utilized by staphylococci as well as streptococci. It is notable that these two organisms also share superantigens, which are structurally similar to the SSL structure. It is striking these unrelated IgA binding factors from group A and group B streptococci (29) and from S. aureus are functionally convergent, all targeting the same residues at the C␣2/C␣3 interface of IgA (Fig. 5), the binding site for Fc␣RI. A second noteworthy parallel is that the different streptococcal proteins in addition to IgA binding also bind complement proteins, with the M proteins (Sir22/Arp4) also binding the classical complement regulator C4bp (and IgG) (31) and the ␤-protein also binding the complement regulator factor H (32). An unrelated S. aureus protein, extracellular fibrinogen binding protein (Efb) also inhibits the complement pathway, in this case by binding C3 (33). Taken together these observations, including the binding of C5 and the inhibition of complement subsequent to C5 by SSL7 (8), suggest strong selection of pathogenic group A and group B streptococci (27)(28)(29)(30) and pathogenic S. aureus to evade both host complement and IgA/Fc receptor effector systems.