Interaction of Human Complement with Sbi, a Staphylococcal Immunoglobulin-binding Protein

Staphylococcal immunoglobulin-binding protein, Sbi, is a 436-residue protein produced by many strains of Staphylococcus aureus. It was previously characterized as being cell surface-associated and having binding capacity for human IgG and β2-glycoprotein I. Here we show using small angle x-ray scattering that the proposed extracellular region of Sbi (Sbi-E) is an elongated molecule consisting of four globular domains, two immunoglobulin-binding domains (I and II) and two novel domains (III and IV). We further show that together domains III and IV (Sbi-III-IV), as well as domain IV on its own (Sbi-IV), bind complement component C3 via contacts involving both the C3dg fragment and the C3a anaphylatoxin domain. Preincubation of human serum with either Sbi-E or Sbi-III-IV is inhibitory to all complement pathways, whereas domain IV specifically inhibits the alternative pathway. Monitoring C3 activation in serum incubated with Sbi fragments reveals that Sbi-E and Sbi-III-IV both activate the alternative pathway, leading to consumption of C3. By contrast, inhibition of this pathway by Sbi-IV does not involve C3 consumption. The observation that Sbi-E activates the alternative pathway is counterintuitive to intact Sbi being cell wall-associated, as recruiting complement to the surface of S. aureus would be deleterious to the bacterium. Upon re-examination of this issue, we found that Sbi was not associated with the cell wall fraction, but rather was found in the growth medium, consistent with it being an excreted protein. As such, our data suggest that Sbi helps mediate bacterial evasion of complement via a novel mechanism, namely futile fluid-phase consumption. Staphylococcal immunoglobulin-binding protein, Sbi, is a 436-residue protein produced by many strains of Staphylococcus aureus. It was previously characterized as being cell surface-associated and having binding capacity for human IgG and β2-glycoprotein I. Here we show using small angle x-ray scattering that the proposed extracellular region of Sbi (Sbi-E) is an elongated molecule consisting of four globular domains, two immunoglobulin-binding domains (I and II) and two novel domains (III and IV). We further show that together domains III and IV (Sbi-III-IV), as well as domain IV on its own (Sbi-IV), bind complement component C3 via contacts involving both the C3dg fragment and the C3a anaphylatoxin domain. Preincubation of human serum with either Sbi-E or Sbi-III-IV is inhibitory to all complement pathways, whereas domain IV specifically inhibits the alternative pathway. Monitoring C3 activation in serum incubated with Sbi fragments reveals that Sbi-E and Sbi-III-IV both activate the alternative pathway, leading to consumption of C3. By contrast, inhibition of this pathway by Sbi-IV does not involve C3 consumption. The observation that Sbi-E activates the alternative pathway is counterintuitive to intact Sbi being cell wall-associated, as recruiting complement to the surface of S. aureus would be deleterious to the bacterium. Upon re-examination of this issue, we found that Sbi was not associated with the cell wall fraction, but rather was found in the growth medium, consistent with it being an excreted protein. As such, our data suggest that Sbi helps mediate bacterial evasion of complement via a novel mechanism, namely futile fluid-phase consumption. The human pathogen Staphylococcus aureus produces an arsenal of virulence factors that aid the organism in effectively evading the immune system of the host. The ability of S. aureus to evade the adaptive immune response of the host has long been recognized (1Foster T.J. Nat. Rev. Microbiol. 2005; 3: 948-958Crossref PubMed Scopus (887) Google Scholar). Staphylococcal cell wall-associated protein A (SpA), 7The abbreviations used are: SpA, Staphylococcus protein A; Sbi, Staphylococcus aureus binder of immunoglobulin; Efb, extracellular fibrinogen-binding molecule; SAXS, small angle x-ray scattering; CP, classical pathway; MBLP, mannose-binding lectin pathway; AP, alternative pathway; RU, response units; CR, complement receptor; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; SPR, surface plasmon resonance; NHS, N-hydroxysuccinimide; β2-GPI, β2-glycoprotein I. for instance, binds immunoglobulin G Fc fragment, and it interacts with certain Fab fragments, thus characterizing SpA as a B-cell superantigen (2Sasso E.H. Silverman G.J. Mannik M. J. Immunol. 1991; 147: 1877-1883PubMed Google Scholar, 3Silverman G.J. Goodyear C.S. Siegel D.L. Transfusion (Bethesda). 2005; 45: 274-280Crossref PubMed Scopus (43) Google Scholar). The Fc binding capacity of SpA has also been found to counteract the innate immune defenses of the host by interfering with the activation of the classical pathway of the complement system (4Goward C.R. Scawen M.D. Murphy J.P. Atkinson T. Trends Biochem. Sci. 1993; 18: 136-140Abstract Full Text PDF PubMed Scopus (52) Google Scholar). More recently a group of small excreted proteins has been discovered that also aid S. aureus in evading complement-mediated bacterial clearance (5Lee L.Y. Liang X. Hook M. Brown E.L. J. Biol. Chem. 2004; 279: 50710-50716Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 6de Haas C.J. Veldkamp K.E. Peschel A. Weerkamp F. Van Wamel W.J. Heezius E.C. Poppelier M.J. Van Kessel K.P. van Strijp J. 2004; PubMed Scopus Google Scholar, M. A. van Wamel W.J. van Kessel K.P. van Strijp Nat. Immunol. 2005; PubMed Scopus Google Scholar, I. T. J. Immunol. 2005; PubMed Scopus Google Scholar). The of evasion and the of the of of not to a of in the of bacterial but is also the in as to SpA, a immunoglobulin-binding protein, Sbi, has been J. M. PubMed Scopus Google that in many S. aureus strains and Sbi is a protein that immunoglobulin-binding domain and a immunoglobulin-binding both with to the immunoglobulin-binding and of SpA but to it was that the immunoglobulin-binding of Sbi is a domain and in to SpA, Sbi interacts with the IgG Fc fragment van Immunol. Scopus Google Scholar). SpA, Sbi the cell wall but it does a cell J. M. PubMed Scopus Google Scholar). It has further been that Sbi is associated with the bacterial surface J. M. PubMed Scopus Google Scholar). Sbi has been to bind protein β2-glycoprotein a protein that has been in M. PubMed Scopus Google Scholar, van A. M.J. J. J. 18: PubMed Scopus Google Scholar). 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Immunol. 2005; PubMed Scopus Google binds to the classical and alternative pathway C3 a bacterial surface and inhibits the of the bacterial in would of the to on the extracellular fibrinogen-binding protein and its recently discovered the region not but also in C3 (5Lee L.Y. Liang X. Hook M. Brown E.L. J. Biol. Chem. 2004; 279: 50710-50716Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, M. S. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Nat. Immunol. PubMed Scopus Google Scholar). It has recently been that to both and a that in the activation of complement M. S. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Nat. Immunol. 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