View all hidden authors and organizations

Edited by Richard P. Novick, New York University School of Medicine, New York City, New York, approved on December 26, 2020 (review received on August 10, 2020)

Antibodies are essential for the immune response against bacteria. In order to drive bacterial killing, antibodies should bind to bacterial cells and induce a complement response. This requires the IgG bound to the target to form a hexameric IgG platform, which is held together by non-covalent Fc-Fc interactions. Interestingly, pathogenic bacteria produce IgG binding molecules that specifically bind to the Fc region required for hexamerization. Here, we demonstrate that Staphylococcal Protein A (SpA) from Staphylococcus aureus specifically prevents the formation of IgG hexamers and the activation of downstream complement. In addition, we show that IgG3 antibodies (not recognized by SpA) have excellent ability to activate complement and induce human phagocytes to kill Staphylococcus aureus. These insights provide an important basis for optimizing antibody therapy against Staphylococcus aureus.

The immunoglobulin (Ig) G molecule is an important participant in the human immune response to bacterial infections. An important effector of IgG-dependent immunity is the induction of complement activation, which triggers various responses that help kill bacteria. Antibody-dependent complement activation is promoted by organizing target-bound IgG into hexamers, which are held together through non-covalent Fc-Fc interactions. Here, we show that Staphylococcal Protein A (SpA), an important Staphylococcus aureus virulence factor and candidate vaccine, effectively prevents IgG hexamerization and subsequent complement activation. Using native mass spectrometry and high-speed atomic force microscopy, we demonstrated that SpA prevents IgG hexamerization through competitive binding with the Fc-Fc interaction interface on the IgG monomer. Consistently, we show that SpA interferes with the formation of the (IgG)6:C1q complex and prevents the activation of downstream complement on the surface of Staphylococcus aureus. Finally, we demonstrated that IgG3 antibodies against Staphylococcus aureus can effectively induce complement activation and opsonize phagocytosis even in the presence of SpA. In summary, our results identify SpA as an immune evasion protein that can specifically block IgG hexamerization.

Antibodies play a key role in the body's immune response to bacterial infections. Although antibodies can bind and neutralize bacterial virulence factors, they can also signal components of the innate immune system and induce bacterial killing. To this end, the antibody binds to the bacterial cell through the variable (Fab) region of the bacterial cell, and then triggers the Fc-mediated effector function (1). The complement system is a huge network of plasma proteins, which is an important effector for antibody-dependent immune protection against invading bacteria. The activated complement cascade results in bacteria being effectively modified by C3-derived molecules, which are essential for triggering efficient phagocyte uptake via complement receptors on phagocytes. In addition, complement produces chemical attractants and induces direct killing of Gram-negative bacteria. Because effective complement activation is an important effect mechanism of cancer therapeutic antibodies (2), the ability of complement to kill bacteria can also be used for antibacterial therapy against (antibiotic resistant) pathogens (3⇓ –5).

When the circulating C1 complex is recruited to the surface of the antibody-labeled target, the antibody-driven "classical" complement pathway begins (6). The most abundant antibody isotype in serum is immunoglobulin (Ig) G, which is subdivided into IgG1, IgG2, IgG3, and IgG4 subclasses in order of decreasing abundance. IgG antibodies can bind to surface antigens through their Fab region and subsequently recruit C1 through their Fc region (SI Appendix, Figure S1A). The C1 complex consists of three large units: C1q, C1r and C1s. C1q includes the antibody recognition unit of the C1 complex and consists of six globular heads connected by collagen-like stems. When binding to C1q, its related proteases C1r and C1s are activated to cleave other complement proteins. Together, these complement proteins form enzymes on the surface to catalyze the covalent deposition of C3b molecules on the bacterial surface (SI Appendix, Figure S1A). The C3b molecule is recognized by complement receptors on phagocytes (neutrophils, macrophages), which engulf and digest bacteria within the cells. The deposition of C3b can also lead to the amplification of the complement cascade and the activation of downstream complement effector functions.

In recent years, it has become obvious that the effective binding of C1 to target-bound IgG molecules requires IgG to form an ordered hexameric ring structure (7, 8). Cryo-electron tomography and atomic force microscopy studies have shown that the six spherical heads of C1q can simultaneously bind each of the six IgG molecules to form a hexamer binding platform (7) (SI appendix, Figure S1B). The formation of these hexamers is induced by the binding of antibodies to surface-bound antigens and is driven by non-covalent interactions between the Fc regions of adjacent IgG molecules (9) (Figure 1A).

The Ig binding domain of SpA binds to the residues of the IgG-Fc region involved in IgG hexamerization. (A) IgG1-b12 IgG hexamer crystal packaging (Protein Database [PDB] ID code 1HZH). A single IgG is shown in gray, and the IgG-Fc domain is enclosed in a dashed box. (B) Schematic diagram of SpA organization. SpA is composed of a signal sequence, five Ig binding domains (E, D, A, B, and C), an Xr region (the number of octapeptide repeats is variable), and a cell wall attachment and sorting region containing a constant Xc region, LPETG based Order, hydrophobic anchor and positively charged residues. (C) Sequence alignment of the five highly homologous Ig binding domains of SpA. Amino acid residues that are conserved in all five domains are highlighted in green. The residues involved in the interaction with the IgG Fc region are shown in pink. (D) Space filling display, depicting the Fc domain of IgG1-b12 and its interaction with SpA-B (PDB ID code 1FC2; the complementary Fc docking domain of SpA-B is hidden). The residues involved in the Fc-Fc interaction required to form the IgG hexamer ring are shown in green, and the crystal structure of SpA-B is shown in red.

Interestingly, some bacteria produce IgG binding molecules that recognize the Fc domain of IgG (10). The most famous of these is Staphylococcal Protein A (SpA), which is a 42 kDa protein with high affinity for the Fc region of IgG, so it is often used as a tool for affinity chromatography to purify monoclonal antibodies. SpA is produced by Staphylococcus aureus, which is an important human pathogen and the main cause of serious hospital-acquired infections such as bacteremia, sepsis, and endocarditis (11). Due to the dramatic increase in antibiotic resistance and the lack of suitable vaccines, doctors often do not have useful or suboptimal alternatives when treating these infections.

SpA is considered an important virulence factor (12, 13) and vaccine candidate (14, 15). The protein is abundantly present on the bacterial cell wall (16⇓ –18), but it is also released in the extracellular environment (19, 20). SpA consists of a signal sequence, five consecutive Ig binding domains (denoted as E, D, A, B, and C), an Xr region, and a cell wall attachment and sorting region (21) (Figure 1B). Each of the five repeated Ig binding domains adopts a triple helix structure, which can bind to the Fc region of IgG through helices I and II (22), and bind to the Fab region of the VH3 antibody family through helices II and III (23) ⇓ –25). The binding of SpA to the Ig-Fc region is thought to protect Staphylococcus aureus from phagocytic killing (14), while the cross-linking of the Ig-Fab region triggers the proliferation and apoptotic collapse of B cells (26). The five Ig binding domains are highly homologous, sharing 74% to 91% of the amino acid sequence relative to the A domain (27) (Figure 1C). It has been demonstrated that the binding interface of SpA (SpA-B) and the B domain of IgG1-Fc involves 11 amino acid residues of SpA-B and 9 residues of IgG1-Fc (22). Interestingly, due to the substitution of one of the nine Fc contact residues in IgG3 (His435 in IgG1 becomes Arg435 in IgG3), SpA binds to all IgG subclasses except IgG3 (28). It has been suggested that residue Arg435 sterically hinders SpA when binding to IgG3-Fc (29). The reported crystal structure of SpA-B and its IgG-Fc interaction site are shown in Figure 1D. It is worth noting that SpA and IgG Fc-Fc interact at the same interface to form a hexameric IgG platform required for complement activation.

Here, we studied the influence of the IgG-Fc binding properties of SpA on the assembly of IgG molecules into hexamers in solution and on the antigen surface. We show that SpA prevents the formation of the hexameric C1q binding platform, thereby inhibiting Staphylococcus aureus IgG-dependent complement activation and opsonophagocytosis (OPK). Our data has made an important contribution to understanding the molecular mechanism of complement escape, which is essential for the intelligent design of new treatment strategies for infectious diseases.

To investigate whether the interaction between SpA and IgG-Fc affects IgG-dependent complement activation, we first checked the IgG binding properties of SpA. Most S. aureus strains express SpA with five highly homologous Ig binding domains (A to E; Figure 1C) that can bind to the Fc region of IgG. However, the single B domain of SpA has been widely used in structural and biochemical studies (22, 30), so here we tested a soluble SpA construct containing all five domains (SpA) and a SpA construct consisting of B only Body-domain (SpA-B).

In order to rule out the potential interaction between SpA protein and IgG Fab domain, we used human monoclonal IgG that does not bind SpA through the Fab domain. This was verified by comparing the binding of these antibodies to beads coated with different forms of SpA-B (SI appendix, Figure S2A). Although the SpA-B domain wild-type (SpA-B) or the SpA-B domain (SpA-BAA; D36A and D37A mutations) with Fab-binding elimination but complete Fc-binding properties (SpA-BAA; D36A and D37A mutations) binds to IgG1 antibodies, it has SpA that eliminates Fc -B domain binding but complete Fab binding properties (SpA-BKK; Q9K and Q10K mutations) do not interact with IgG1 (SI appendix, Figure S2A and Table S1). As expected, none of the SpA-B forms bind to IgG3 antibodies (SI appendix, Figure S2A). As a reference binding experiment for all antibodies (including IgG3), we used protein G beads (protein G binds to all IgG subclasses, including IgG3), and also measured the binding of VH3 family antibodies (anti-Hla IgG1) as a control for Fab binding. SpA-BKK. It is worth noting that we further confirmed by natural mass spectrometry (natural MS) that the KK mutation of SpA-BKK effectively weakened the binding to the Fc domain (SI appendix, Figure S2 B and C). Native MS analyzes a large number of intact protein complexes in their natural state, allowing non-covalent interactions to remain intact (31, 32).

We used enzyme-linked immunosorbent assay and natural MS to study the interaction between SpA constructs and human IgG. Consistent with previous studies, our results indicate that both SpA constructs bind strongly to the Fc region of all human IgG subclasses except IgG3 (Figure 2 and SI appendix, Figure S3 and Table S2). Interestingly, the binding stoichiometry between SpA and SpA-B is different. As expected by the presence of two identical binding sites on IgG-Fc, the single domain SpA-B can—and indeed does—bind to IgG molecules at a 2:1 stoichiometry (Figure 2A). However, although SpA was found to contain five IgG binding domains, it was found to mainly bind to a single IgG molecule with a stoichiometric ratio of 1:1 (Figure 2B). This indicates that full-length SpA can simultaneously bind to two IgG-Fc binding sites.

The single domain SpA-B binds IgG1 at a 2:1 stoichiometry, while the five domain SpA binds IgG1 at a 1:1 stoichiometry. (A and B) The deconvoluted natural mass spectrum shows that the mass of IgG1 (black) changes when incubated with SpA-B (orange) (A) or SpA (blue) (B). This transition corresponds to the binding of one or two copies of SpA-B to an IgG1 molecule, and the binding of only one SpA molecule to a single IgG1 molecule. The cartoon showing the binding of (IgG1)1:SpA1 is speculative because it is still unknown which SpA domain interacts with which binding site on the IgG molecule.

Next, we evaluated whether SpA binding can reduce IgG hexamerization in solution. Although the formation of IgG oligomers is a superficial phenomenon and requires the binding of IgG to the antigen surface, the process can be simulated in solution by introducing three mutations in the Fc region of IgG that enhance the Fc-Fc interaction (7, 32) . The combined mutations of residues E345R (Glu345→Arg), E430G (Glu430→Gly) and S440Y (Ser440→Tyr) lead to IgG-RGY, which easily forms hexamers in a dynamic equilibrium solution (7, 32). Here, we use native MS to study how SpA affects the monomer-hexamer balance of IgG-RGY. As previously reported (7, 32), the natural mass spectrum of IgG1-RGY showed the presence of monomers [denoted as (IgG1)1] and hexamers [(IgG1)6], and intermediate states were observed at lower abundances ( Figure 3A and SI appendix, Table S3). For SpA-bound IgG subclasses, when incubated with SpA-B or SpA, the relative abundance of IgG oligomers was significantly reduced (Figure 3A and B and SI Appendix, Table S3). Under these conditions, we observed strong binding of SpA-B/SpA to IgG monomers, but not to hexameric IgG species (SI Appendix, Table S3).

The binding of SpA-B or SpA prevents the assembly of IgG-RGY monomers into higher order oligomers. (A) The native mass spectrum of IgG1-RGY in the absence (green) and the presence of SpA-B (orange) or SpA (blue). (B) The relative mass abundance of IgG-RGY subclass hexamers in the absence and presence of SpA-B or SpA, as assessed by natural MS. Error bars indicate the SD of three replicate samples. Although the RGY mutation effectively induced the formation of hexamers of IgG1, IgG2, and IgG4, IgG3-RGY has a lower tendency to form hexamers in solution. (C) HS-AFM image of IgG1-RGY on DNP-SLB in the absence and presence of SpA-B or SpA after pre-incubation in solution. The white arrow represents (IgG1)6; the red arrow, (IgG1)1. (Scale bar: 100 nm.) (D) Distribution of IgG oligomers on DNP-SLB alone (n = 372) and in the presence of SpA-B (n = 697) or SpA (n = 386) after pre-incubation In the solution. The histogram shows the fraction of IgG that makes up each oligomer class. The oligomer distribution is quantified by force-induced dissociation. n refers to the number of individually characterized IgG. The two-tailed Mann-Whitney U test was used to evaluate the statistical significance between the three experiments. Control and SpA-B, P <0.00001; Control and SpA, P <0.00001; SpA-B and SpA, P <0.001.

High-speed atomic force microscopy (HS-AFM) experiments on 2,4-dinitrophenol (DNP)-labeled lipid-containing supporting lipid bilayers (DNP-SLB) confirmed the native MS measurements. The pre-incubation of DNP-SLB with the anti-DNP antibody IgG1-RGY alone or in combination with SpA-B or SpA resulted in a unique distribution of IgG1-RGY oligomers of different sizes (Figure 3C). The force-induced oligomer dissociation experiment (8) checks the oligomer size and overall IgG1 surface density, allowing us to compile a quantitative oligomer distribution (Figure 3D). Pre-incubation of anti-DNP IgG1-RGY with multi-domain SpA resulted in a sharp reduction of high-order IgG1-RGY oligomers on the surface of DNP-SLB (Figure 3D), consistent with our findings in the natural MS experiment (Figure 3B)) . However, although the two SpA constructs had similar effects on the IgG1-RGY hexamer population in solution (Figure 3B), when IgG1-RGY was pre-incubated with SpA-B, we observed that the IgG1-RGY hexamer was only reduced About 50% of the domains on DNP-SLB (Figure 3D). In summary, these data indicate that SpA competitively binds to the Fc-Fc interaction site on the IgG monomer, which effectively prevents IgG from forming higher-order IgG oligomers.

After demonstrating that SpA prevents the hexamerization of IgG in solution, we next explored whether SpA affects the formation of (IgG)6:C1q complexes. We use native MS to study the formation and behavior of these complexes in the presence and absence of SpA. Consistent with the earlier data (32), the natural mass spectrum of IgG1-RGY incubated with C1q showed clearly distinguishable species, and its masses corresponded to (IgG1)1, (IgG1)6 and (IgG1)6:C1q (Figure 4A) And SI) appendix, table S4). Experiments to study the effects of SpA were conducted in two ways. When SpA or SpA-B was added to IgG1-RGY pre-incubated with C1q, the abundance of (IgG1)6:C1q complex was significantly reduced (Figure 4A and SI appendix, Table S4). Similarly, after pre-incubation of IgG1-RGY with SpA constructs, the addition of C1q does not result in detectable levels of (IgG1)6:C1q complexes. Therefore, regardless of the mixing order of IgG1-RGY, C1q, and SpA, we mainly detect a complex of monomeric IgG bound to the SpA construct and free unbound C1q. Similar to IgG1-RGY, the two SpA constructs also prevented the assembly of the (IgG)6:Clq complex of IgG2-RGY and IgG4-RGY (Figure 4B). Overall, these results indicate that SpA prevents the binding of C1q to IgG by preventing the formation of hexameric IgG platforms in solution.

SpA-B and SpA prevent the assembly (IgG) 6:C1q complex in solution. (A) The native mass spectrum of IgG1-RGY:C1q in the absence (green) and the presence of SpA-B (orange) or SpA (blue). (B) The relative mass abundance of (IgG)6:C1q complex in the absence and presence of SpA-B or SpA, as assessed by native MS. Error bars indicate the SD of three replicate samples. We observed lower amounts of (IgG)6:C1q complexes using RGY mutants of IgG3 and IgG4. This is because, compared with other subclasses, IgG3 mutants have a lower tendency to hexamerize, while IgG4 has a lower affinity for C1q.

Next, we evaluated how these observations of stable hexamers of mutant IgG-RGY in solution compared to the behavior of wild-type IgG bound to the antigen surface. Since SpA and SpA-B prevent the formation of IgG hexamers through the same mechanism, we will focus on SpA-B in the following experiments. Using our previously used quartz crystal microbalance (QCM) to quantitatively study the binding and oligomerization of IgG1 to antigenic SLB (9), we evaluated the association of wild-type IgG1 with similar DNP-SLBs, such as for HS-AFM Experiment and subsequent binding of C1q in the presence or absence of SpA-B (Figure 5A and B). Monitor the change in the resonance frequency of the SiO2 coated quartz crystal covered by DNP-SLB, which is proportional to the change in binding quality, and produce a characteristic binding curve of the interaction between anti-DNP IgG1-WT and DNP-SLB (Figure 5A-C, green time interval) . It is obvious from the stability of the sensorgram in the subsequent time interval (Figure 5A-C; gray time interval) that the removal of IgG1-WT from the running buffer does not cause a large amount of IgG1-WT to dissociate, reflecting stable binding. After establishing equal amounts of surface-bound IgG in all three experiments, we added C1q in the absence (Figure 5A, green time interval) and the presence of SpA-B (Figure 5B, orange time interval), which resulted in obvious The binding curve. Although C1q is closely related to the IgG1-WT on our antigen membrane (Figure 5A), incubating C1q and SpA-B at the same time strongly reduces the binding signal (Figure 5B). The addition of SpA-B alone (without C1q; Figure 5C) resulted in a strong association between SpA-B and IgG1-WT, and there was no detectable dissociation when SpA-B was removed from the running buffer, indicating a high-affinity interaction . It is worth noting that the remaining frequency shift (and related mass) at the end of the dissociation phase is equal to the corresponding frequency shift of the C1q SpA-B mixture, indicating that the presence of SpA-B effectively excludes stable C1q binding (Figure 2). 5B).

SpA-B inhibits the binding of C1q to the target surface antigen-bound IgG. (AC) C1q alone (A), C1q and SpA-B (B) or SpA-B alone (C) combined with anti-DNP wild-type IgG1 antibody (IgG1-WT) QCM sensorgram, combined with DNP-load balancer . In the absence or presence of SpA-B, the binding of C1q (14 nM) is tracked in real time. SpA-B binding alone was monitored in a similar experiment without C1q. The bars represent the respective equilibrium level () and the level at the end of the dissociation phase (*). (D and E) After incubating C1q (D) or C1 complex (E) in the absence (solid line) or presence (dashed line) of SpA, C1q on IgG1-WT and IgG3-WT binds to DNP coating The bead binding-B is detected by flow cytometry with FITC-conjugated rabbit F(ab')2 anti-human C1q. The bars represent only the same data at 20 nM IgG concentration, and the black dotted line shows the background fluorescence from beads that have not been incubated with IgG. Data are expressed as geometric mean fluorescence intensity (GeoMFI) ± SD of three or four independent experiments. Statistical analysis was performed using an unpaired two-tailed t test to compare buffer and SpA-B conditions. ** P <0.01; *** P <0.001; ****P <0.0001.

QCM observations were confirmed by flow cytometry analysis, in which DNP-coated beads (33) were first labeled with anti-DNP IgG1-WT or IgG3-WT as a control. As expected, the presence of SpA-B inhibited the binding of C1q to IgG1-WT-labeled beads, but the binding to IgG3-WT did not change (Figure 5D). As a control, we confirmed that IgG1-WT and IgG3-WT have similar binding to DNP-coated beads (SI Appendix, Figure S4A), while SpA-B can only bind to the target antigen loaded with IgG1-WT (SI Appendix, Figure S4B ). Interestingly, SpA-B also blocked the binding of C1q when incubated after the IgG1-WT:Clq complex was formed (SI appendix, Figure S4C).

Because the classical complement pathway is initiated by C1q complexed with C1r and C1s proteases, we repeated the same experiment using the C1qr2s2 complex instead of C1q alone. In addition, for this fully assembled C1 complex, we observed that SpA-B strongly reduced the binding of C1q to DNP-bound IgG1-WT, and its presence did not affect the binding of C1q to IgG3-WT (Figure 5E and SI Appendix, Figure 5). S4D). Finally, we confirmed that, similar to SpA-B, SpA also greatly reduced the binding of C1q to the bead surface (SI appendix, Figure S5A) and the IgG bound on the upper surface of the lipid membrane (SI appendix, Figure S5B). In summary, these data indicate that both SpA constructs can effectively block the binding of C1q to IgG1 subclass surface-bound antibodies.

We next determined whether SpA affects antibody-dependent complement activation on the surface of Staphylococcus aureus. In order to prevent the cell surface SpA (and Sbi, another Ig binding protein of Staphylococcus aureus) (34) from binding to the Fc region of the antibody, we used a strain without SpA and Sbi (NewmanΔspa/sbi) here. NewmanΔspa/sbi is labeled with a human monoclonal antibody directed against wall teichoic acid (WTA) (35), a highly abundant anionic glycopolymer that is covalently anchored to the peptidoglycan layer (36). Since the anti-WTA antibody (clone 4497) belongs to the VH3 type family (37), it is expected to bind to SpA-B or SpA through the Fab region. Nevertheless, we found that the anti-WTA IgG1 antibody did not bind to the beads coated with SpA-BKK (SpA-B mutant that only binds to the IgG-Fab region) (SI appendix, Figure S6A), and further confirmed SpA-BKK by natural MS. B and SpA only bind to the Fc region of the antibody (SI appendix, Figure S6 BE).

To measure C1q binding and downstream complement activation on the bacterial surface, we incubated IgG-labeled bacteria with human serum binding buffer, SpA-B or SpA. Because serum contains not only complement proteins, but also many different antibodies, including IgG that does not bind SpA (IgG3) and antibodies that bind SpA through its Fab region (VH3 type family Igs), we use serum that does not contain natural antibodies (ΔIgG/ IgM serum). In this way, we can specifically determine the effect of SpA on complement activation with monoclonal anti-WTA antibodies. Consistent with the experimental results using DNP beads, we observed that both SpA constructs strongly reduced the binding of C1q to Staphylococcus aureus labeled with anti-WTA IgG1 antibody (Figure 6A). When the bacteria were labeled with anti-WTA IgG3, Clq binding was not inhibited by SpA-B or SpA (Figure 6B).

SpA reduces IgG-mediated C1q binding and downstream complement of Staphylococcus aureus. (A and B) C1q does not bind to anti-WTA wild-type IgG1 (IgG1-WT) (A) or IgG3 antibody (IgG3-WT) (B) The binding of bacteria to the surface of NewmanΔspa/sbi after incubation with 1% ΔIgG/ IgM human serum with the presence (green) or the presence of SpA-B (orange) or SpA (blue) is detected by flow cytometry with chicken anti-human C1qA antibody. (C and D) After the bacteria are incubated with IgG1-WT (C) or IgG3-WT (D), 1% ΔIgG/IgM human serum and buffer (green), and SpA-B, C3b is deposited on the surface of NewmanΔspa/sbi (Orange) or SpA (blue), detected by flow cytometry with monoclonal mouse anti-human C3d antibody. The data is expressed as the mean ± SD fold change of the 40 nM IgG control concentration of at least three independent experiments. The bars only represent the same data at 40 nM IgG concentration, and the black dotted line shows the background fluorescence from bacteria that have not been incubated with IgG. Use one-way analysis of variance for statistical analysis to compare buffer conditions with SpA-B and SpA conditions. *** P <0.001; ****P <0.0001.

To determine whether inhibition of C1q binding leads to downstream inhibition of the complement cascade, we also measured the deposition of C4b and C3b. The binding of C1q to the target antibody activates its attached C1r/C1s proteases, which cleave C4 and C2 and generate C3 convertase (C4b2b) (38). Subsequently, the covalently linked C3 convertase catalyzes the deposition of C3b on the target surface (38). After incubating IgG1-labeled Staphylococcus aureus with human (Ig-removed) serum and SpA-B or SpA, the surface deposits of C4b and C3b completely disappeared (SI appendix, Figure S7A and Figure 6C). As expected, the deposition of C4b and C3b on NewmanΔspa/sbi labeled with anti-WTA IgG3 antibody remained unchanged in the presence of SpA-B or SpA (SI appendix, Figure S7B and Figure 6D). It is worth noting that when the C3b deposition assay was repeated to include an antibody that recognizes the hapten DNP as an isotype control, we did not detect the C3 product on the surface of Staphylococcus aureus. Only incubation with an anti-WTA antibody that recognizes Staphylococcus aureus can produce detectable C3b levels, confirming that this signal reflects the presence of covalently bound C3 products deposited during complement activation (SI appendix, Figure S8). Taken together, these findings indicate that SpA effectively prevents C1q recruitment and downstream activation of the S. aureus complement cascade.

Finally, we assessed whether the inhibitory effect of SpA on complement activation can reduce the killing of Staphylococcus aureus by neutrophils. Neutrophils are the first cells recruited from the blood to the site of infection, where they engulf and internalize bacteria through phagocytosis, and then kill them by contact with antimicrobial agents (such as antimicrobial peptides, reactive oxygen species, and enzymes) (39 ). The previous work of our group showed that the deposition of C3-derived opsonins greatly enhanced the phagocytosis of Staphylococcus aureus (40), because C3b molecules can be recognized by complement receptors on phagocytes. To compare the OPK activity of anti-WTA IgG1 and IgG3 antibodies, we used a wild-type Staphylococcus aureus strain (Newman WT) expressing cell wall anchor SpA and cell-associated Sbi. Newman WT is incubated with IgG1 or IgG3 and 1% ΔIgG/IgM serum as a source of complement, and then mixed with freshly isolated human neutrophils to achieve phagocytosis and killing. We found that IgG3 antibodies were more effective in inducing Newman WT killing than IgG1 antibodies (Figure 7A).

SpA reduced the OPK killing of Staphylococcus aureus by IgG1 but not IgG3. (A) In the presence of 1% ΔIgG/IgM human serum, after incubating with IgG1-WT (green), IgG3-WT (blue) or no IgG (grey), the CFU of Newman WT is counted, and then it is compared with human Sex granulocytes are incubated. (B) The CFU count of NewmanΔspa/sbi after incubation with 2.5 nM anti-WTA IgG1-WT (green), IgG3-WT (blue) or no IgG (gray) in the presence of 1% ΔIgG/IgM human serum and buffer, SpA-B or SpA is then incubated with human neutrophils. Data are expressed as log10 CFU/mL ± SD of at least three independent experiments. Use unpaired two-tailed t-test to compare IgG1-WT and IgG3-WT conditions (A) or use one-way analysis of variance to compare buffer conditions with SpA-B and SpA conditions (B) for statistical analysis. ** P <0.01; *** P <0.001; ****P <0.0001.

To determine whether this was indeed caused by the interaction of SpA and IgG1 antibodies, we also killed NewmanΔspa/sbi in the absence or presence of exogenous SpA-B or SpA. Although anti-WTA IgG1 antibody induced OPK of Staphylococcus aureus (Figure 7B), we observed that the addition of soluble SpA-B or SpA blocked OPK (Figure 7B). As expected, killing Staphylococcus aureus in the presence of IgG3 was not affected in the presence of SpA (Figure 7B). Collectively, these data indicate that both soluble and cell-exposed SpA reduce the OPK of Staphylococcus aureus through IgG1 but not through IgG3.

Antibody-dependent complement activation is an important immune mechanism that accelerates bacterial killing (1). To effectively trigger complement, antibodies should bind to bacterial cells and subsequently form oligomeric IgG clusters to recruit C1 (7, 8). In this article, we use SpA in Staphylococcus aureus as an example of a bacterial immune evasion molecule that specifically blocks antibody aggregation by inhibiting IgG Fc-Fc contact. These findings are related to the basic pathophysiology of Staphylococcus infection and the development of immunotherapy against Staphylococcus aureus. In addition, SpA can be used as a tool to better understand the role of IgG clustering in various disease processes involving antibodies and complement.

Our research shows that by binding to the Fc region of IgG, soluble SpA can block IgG hexamerization, thereby inhibiting C1q recruitment, downstream complement activation on the surface of Staphylococcus aureus, and the killing of bacteria by human phagocytes. Although the interference of SpA on the classical complement pathway has been noticed before, the exact molecular mechanism is still elusive, and its role as an activator or inhibitor of the complement system (41⇓ ⇓ ⇓ –45) is controversial. Thanks to recent in-depth understanding of IgG oligomerization (7) and more advanced methods for directly visualizing the process, we have been able to unravel the mechanism of SpA-dependent complement inhibition under highly purified conditions. The binding of SpA to the Fc region of the monomeric rather than hexameric IgG-RGY class indicates that SpA only binds to the free, unoccupied Fc region. Therefore, by binding to monomeric IgG, SpA may prevent the transition to a hexameric state.

Since Staphylococcus aureus strains produce SpA with four or five Ig-binding domains (46), we compared the complement inhibitory activity of SpA with five Ig-binding domains to a single SpA-B domain. AFM experiments show that, compared with SpA-B, SpA prevents the hexamerization of IgG molecules on the antigen membrane more effectively. Since multi-domain SpA may bind IgG in a bivalent manner, its dissociation rate is lower than that of monovalently bound SpA-B. The resulting binding affinity of SpA relative to SpA-B enables it to more effectively compete with IgG Fc-Fc. In fact, multi-domain SpA is more effective than SpA-B in inhibiting the binding of C1q to target-bound IgG, which is consistent with the view that multivalent SpA molecules have complement inhibitory advantages over single SpA domains. However, when measuring downstream complement activation, the inhibitory advantage of SpA on a single SpA domain is not so obvious. We speculate that for cell-anchored SpA complement inhibition, the requirement for multiple domains may be more important. Since most of the SpA produced by Staphylococcus aureus is anchored to the bacterial cell wall, multiple SpA domains may be required to provide the molecule with sufficient length and flexibility to bind to the Fc domain of bacteria-bound IgG. Although most SpA is cell-anchored, 6.5% to 15% are secreted before sorting (20) or released from the cell wall after LytM enzymatic lysis (19). We have observed that the concentration of recombinant multidomain SpA required to reduce the deposition of C1q on the antigen surface is lower than the amount reported to be secreted by Newman or USA300 strains in vitro (47).

In addition to SpA, there are other bacterial proteins that bind to Ig (10). The most well-known are G protein (from group C and G streptococci), M and M-like proteins (group A streptococci), L protein (Peptococcus major), Sbi and SSL10 (Staphylococcus aureus). Protein G, protein M, M-like protein, and Sbi all bind to the Fc domain of IgG near the SpA binding site (48⇓ –50), indicating that these IgG-Fc binding proteins may be able to block IgG hexamerization as well.

Overall, our data provides crucial insights for the development of effective immunotherapy against Staphylococcus aureus. It is well known that both neutrophils and complement play an important role in killing Staphylococcus aureus. Although neutrophils can directly engulf Staphylococcus aureus, the previous work of our group showed that modification of bacteria with C3-derived opsonins can strongly enhance effective phagocytosis (40). Therefore, the identification of antibacterial antibodies with strong complement activation potential provides an interesting method for enhancing the host immune system and preventing or treating these infections. From this research, it is now clear that this method will be more successful if we take SpA-dependent antibody modulation into account. Although most of the active and passive immunization methods used to develop or induce antibodies against the surface components of Staphylococcus aureus (such as capsular polysaccharides, lipoteichoic acid, various surface adhesin) have failed in clinical trials (5 ), but we suggest that these strategies may be more effective when also blocking the effects of SpA. In this case, although our comparison of the potency of IgG1 and IgG3 in activating Staphylococcus aureus OPK cannot distinguish between SpA's FcγR-mediated inhibition and complement-mediated OPK, it shows that it targets golden grapes Monoclonal antibodies against surface components of cocci. Staphylococcus aureus should be developed as IgG3 (or its variants) not targeted by SpA. Alternatively, any amino acid modification in the IgG1 or IgG2 subclass that prevents SpA from binding to IgG but does not prevent IgG hexamerization is valuable. We also proposed that it may be necessary to use SpA as a vaccine antigen or a monoclonal antibody against the SpA Fc binding domain to prevent the anti-complement effect of SpA, thereby increasing the chance of bacterial clearance.

In addition, SpA can also be used as a research tool to specifically examine the role of IgG hexamerization in various disease processes. For example, SpA or a single domain of SpA (mutated to only bind to the IgG-Fc region) can be used to study whether antibodies induced during infection require Fc-Fc contact to induce complement activation on invading pathogens. In fact, our data on IgG1 antibodies against WTA indicate that naturally induced antibodies against Staphylococcus aureus do require Fc-Fc contact to induce complement activation on the bacterial surface. Although we use monoclonal antibodies, it is well known that IgG1 antibodies against WTA are produced during Staphylococcus aureus infection in the body (51⇓ ⇓ –54). In addition, given that the excessive activation of complement is related to the clinical manifestations of several autoimmune diseases, SpA can be used to study whether autoreactive antibodies can induce IgG aggregation on altered host cells. Finally, SpA or molecules that target the SpA binding site in IgG may prevent unwanted antibody responses. In fact, the therapeutic potential of SpA has been tested for the treatment of autoimmune diseases (55⇓ –57), although the basic principle of using SpA is based on its effectiveness as a B cell superantigen.

In conclusion, the identification of SpA as a biological inhibitor of IgG hexamerization will increase our understanding of antibody-dependent immune mechanisms and may help accelerate the development of immune interventions for infection and inflammation.

For more detailed information on the materials and methods used in this study, please refer to the SI appendix Materials and Methods.

The original MS analysis was performed on a modified LCT time-of-flight instrument (Waters) or a standard Exactive Plus EMR Orbitrap instrument (Thermo Fisher Scientific). Before analysis, using Amicon Ultra centrifugal filters (Merck) with 3-kDa or 10-kDa molecular weight cut-off, the buffer was replaced with 150 mM ammonium acetate (pH 7.5). The protein complex is assembled by mixing the subcomponents in the desired molar ratio and then incubating at room temperature (RT) for at least 30 minutes. For experiments to study the influence of SpA constructs, due to the relatively slow decomposition rate of IgG-RGY hexamer, the incubation step with SpA was carried out at 37°C for at least 3 hours. The IgG-RGY hexamer is measured at a total IgG concentration of 2 µM in the presence or absence of 10 µM SpA-B or SpA (ProSpec). For the measurement of the (IgG)6:C1q complex, 0.5 µM C1q (complement technology) was used. The sample is loaded into a gold-plated borosilicate capillary (prepared in-house) for direct injection from a static nanoelectrospray ionization source. The relative abundance of protein complexes is determined using an internal script that summarizes and compares the ionic intensities of different species, similar to the previously described method (58). Use UniDec (59) to generate deconvoluted mass spectra through Bayesian deconvolution.

HS-AFM (Institute of Biomolecular Metrology) is performed in tapping mode in RT buffer with a free amplitude of 1.5 to 2.5 nm and an amplitude set point >90%. Silicon nitride cantilever with electron beam deposition tip (USC-F1.2-k0.15; Nanoworld AG), nominal spring constant is 0.15 N m-1, resonance frequency is about 500 kHz, quality factor in liquid About 2 used. Imaging was performed in buffer 1 (10 mM Hepes, 150 mM NaCl, and 2 mM CaCl2, pH 7.4). All IgG are diluted and incubated in the same buffer.

The DNP-labeled SLB used for HS-AFM was prepared on muscovite. The liposomes were incubated on the freshly cut surface (500 µg mL-1 in buffer 1), placed in a humidity chamber to prevent evaporation, and heated to 60 °C for 30 minutes. Then gradually cool the temperature to RT within 30 minutes, and then exchange the solution with buffer 1. After 10 minutes of equilibration at RT and another 15 buffer exchanges, the SLB is ready for imaging. To inactivate any exposed mica, incubate SLB with 333 nM IgG1-b12 (an unrelated human IgG1 control antibody against HIV-1 gp120) (60) for 10 minutes before adding the molecule of interest.

The QCM experiment is done using a dual-channel QCM-I system (MicroVacuum). Use AT-cut SiO2 coated quartz crystal (Quartz Pro AB) with a diameter of 14.0 mm and a resonance frequency of 5 MHz. All sensorgrams are recorded at the first, third and fifth harmonic frequencies. The displayed data is related to the third harmonic. Before each set of experiments, SiO2 coated crystals were immersed in 2% sodium dodecyl sulfate (SDS) for 30 minutes, and then rinsed thoroughly with Milli-Q H2O. The chip is dried in a gentle stream of N2 and oxidized using air plasma (4 minutes at 80 W), and then installed in the measurement chamber. Before the measurement, the sensor surface was cleaned directly with 2% SDS at a flow rate of 250 µL min-1 for 5 minutes, and then with Milli-Q H2O at a flow rate of 250 µL min-1 for 5 minutes. Finally, inject buffer without IgG to reach an equilibrium of 50 µL min-1. All subsequent injections were performed at 50 µL min-1. In order to generate DNP-SLB on the QCM chip, the DPPC:DPPE:DNP-cap-DPPE liposome stock solution was heated to 60 °C for 30 minutes, and then slowly cooled to room temperature within 30 minutes. The solution can be used for injection after being diluted to 200 µg/mL with buffer 1. The formation of DNP-SLB is usually completed after 30 minutes at 50 µL min-1, after which the flow medium is changed to buffer 1 for equilibration.

Wash streptavidin beads (Dynabeads M-270; Invitrogen) in PBS-TH (phosphate buffered saline [PBS], 0.05% [vol/vol] Tween-20 and 0.5% human serum albumin [HSA]) And incubate (diluted 100×) with 1 µg/mL biotinylated DNP (DNP-PEG2-GSGSGSGK (biotin)-NH2; Pepscan Therapeutics) in PBS-TH at 4 °C and shake for 30 minutes. For each condition, use 0.5 µL magnetic beads (~3 × 105 beads/condition). After washing twice with PBS-TH, incubate the DNP-coated beads with 20 nM anti-DNP IgG or a two-fold serial dilution of anti-DNP IgG (starting with 20 nM IgG) at 4°C with shaking (±700 rpm) for 30 minute). Unless otherwise noted, the following incubation steps are performed at 4°C under shaking conditions (±700 rpm) for 30 minutes. The IgG-bound DNP coated beads were washed twice with VBS-TH (Veronal buffered saline [pH 7.4], 0.5 mM CaCl2, 0.25 mM MgCl2, 0.05% [vol/vol] Tween-20 and 0.5% HSA), and combined with 1.3 nM purified C1q or C1 alone (complement technology), 200 nM or 200 nM of recombinant SpA-B or SpA (ProSpec) or a four-fold dilution (starting from 1 µM) were incubated in VBS-TH at 37°C. Finally, the beads were washed twice with PBS-TH and incubated with rabbit F(ab')2 anti-human Clq conjugated with 4 µg/mL fluorescein isothiocyanate (FITC). Use flow cytometry (BD FACSVerse) to detect the binding of C1q to beads, and use FlowJo software to analyze the data based on a single bead population.

Staphylococcus aureus NewmanΔspa/sbi strain was transformed with pCM29 plasmid for fluorescent labeling, and constitutively expressed mAmetrine under the control of the sarA promoter, as described above (61, 62). The bacteria were grown overnight in Todd Hewitt broth (THB) plus 10 µg/mL chloramphenicol, diluted to an OD600 of 0.05 in fresh THB plus chloramphenicol, and cultured to mid-logarithmic phase (OD600 = 0.5). The cells were collected, washed, resuspended in RPMI-H medium (RPMI 0.05% HSA), and aliquoted at -20 °C.

Similar to Dynabeads analysis, all incubation steps are performed at 4°C (unless otherwise noted) under shaking conditions (±700 rpm) for 30 minutes, and then a single wash with RPMI-H by centrifugation. Bacteria (7.5 × 105 CFU) were incubated in RPMI-H with double titration (starting from 40 nM) IgG, and then incubated with 1% ΔIgG/IgM serum with buffer and 200 nM SpA-B or SpA in RPMI-H. Keep at 37°C for 30 minutes. For C1q detection, incubate the bacteria with 0.5 µg/mL chicken anti-human C1qA (Sigma-Aldrich), then incubate with phycoerythrin-conjugated donkey F(ab')2 anti-chicken (Jackson ImmunoResearch), diluted 1:500 RPMI -H. For C4b and C3b detection, 1 µg/mL mouse anti-C4d (Quidel) or anti-C3d (Quidel) antibody was incubated with bacteria, respectively. Subsequently, the bacteria were incubated with FITC-conjugated goat F(ab')2 anti-mouse (Dako) and diluted 1:100 in RPMI-H. After labeling, the sample was fixed with 1% paraformaldehyde in RPMI-H, and the binding of C1q, C4b, and C3b to the bacteria was detected by flow cytometry (BD FACSVerse). Use FlowJo software to analyze the data.

Purification of human neutrophils from the blood of healthy donors by the Ficoll/Histopaque density gradient method (63). The constitutive expression of Staphylococcus aureus Newman WT and NewmanΔspa/sbi mAmetrine was freshly grown to mid-log phase, washed and conditioned as described below. Newman WT was incubated with anti-WTA IgG1 or IgG3 and 1% ΔIgG/IgM serum in a fourfold titration (starting at 10 nM) in Hank's Balanced Salt Solution (HBSS) 0.1% HSA (HBSS-H). In HBSS-H, in the presence or absence of exogenous SpA-B or SpA (200 nM), NewmanΔspa/sbi is incubated with 2.5 nM anti-WTA IgG1 or IgG3 and 1% ΔIgG/IgM serum. After culturing for 30 minutes at 37 °C, bacteria (8.5 × 105 CFU) and freshly isolated neutrophils were cultured at 37 °C with a 1:1 ratio of bacteria to cells under 5% CO2 for 90 minutes. Subsequently, neutrophils were solubilized with cold 0.3% (wt/vol) saponin on ice for up to 15 minutes. The samples were serially diluted in PBS and seeded on TSA plates in duplicate. Incubate the plate overnight at 37°C, and quantify live bacteria by CFU counting.

According to the Declaration of Helsinki, after obtaining the informed consent of all subjects, human serum and blood were obtained from healthy donors. Approved by the Medical Ethics Committee of Utrecht University Medical Center (METC Agreement 07-125/C, approved on March 1, 2010).

All research data are included in the main text and SI appendix.

We thank Annette Stemerding for the fruitful discussions. This research was supported by the Dutch Science and Technology Foundation STW, which is part of the Netherlands Organization for Scientific Research (NWO) and partly funded by the Ministry of Economic Affairs (TTW-NACTAR Grant #16442 [to AJRH and SHMR]). This work was supported by the European Union Horizon 2020 research program H2020-MSCA-ITN (675106, for JAGvS and FB) and the European Research Council (ERC) start-up grant (639209, for SHMR). MAdB and AJRH through the X-omics roadmap plan (project 184.034.019) and the EU Horizon 2020 plan Epic-XS (project 823839) further recognized the funding of the Dutch Proteomics Center, a large-scale proteomics facility. AJRH and SHMR recognize Utrecht University's Center for Molecular Immunology. JP is supported by the European Regional Development Fund (EFRE, IWB2020), the Federal State of Upper Austria and the Austrian Science Foundation (FWF, P33958 and P34164).

↵1A. RC and MAdB contribute the same to this work.

↵2A.JRH and SHMR have made equal contributions to this work.

Author contributions: ARC, MAdB, J. Strasser, SAZ, FJB, GW, RNdJ, FB, JAGvS, KPMvK, J. Schuurman, JP, AJRH and SHMR design research; ARC, MAdB, J. Strasser and KPMvK conducted research ; SAZ, CJdH and PCA contributed new reagents/analysis tools; ARC, MAdB, J. Strasser, CJdH, PCA and KPMvK analyzed data; ARC, MAdB, J. Strasser, KPMvK, JP, AJRH and SHMR wrote this paper.

Competitive interest statement: ARC participated in the GlaxoSmithKline (GSK) graduate scholarship program. FJB, JAGvS, KPMvK, J. Schuurman and SHMR are listed as co-inventors of a patent describing antibody therapy against Staphylococcus aureus. FJB, RNdJ and J. Schuurman are Genmab employees. FB is an employee of the GSK group of companies and is the co-inventor of the patent for Staphylococcus aureus vaccine candidate.

This article is directly contributed by PNAS.

This article contains online support information https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2016772118/-/DCSupplemental.

This open access article is distributed under the Creative Commons Attribution-Non-Commercial-No Derivative License 4.0 (CC BY-NC-ND).

Thank you for your interest in advertising on PNAS.

Note: We only ask you to provide your email address so that the people you recommend the page to know that you want them to see it, and that it is not spam. We do not capture any email addresses.

Feedback privacy/legal

Copyright © 2021 National Academy of Sciences. Online ISSN 1091-6490. PNAS is a partner of CHORUS, COPE, CrossRef, ORCID and Research4Life.