Abstract
Among the recently discovered Staphylococcus aureus immune evasion proteins, Sbi is unique in its ability to interact with components of both the adaptive and innate immune systems of the host. Sbi domains I and II (Sbi-I and Sbi-II) bind IgG. Sbi domain IV (residues 198–266) binds the central complement protein C3. When linked to Sbi-III, Sbi-IV induces a futile consumption of complement via alternative pathway activation, whereas isolated Sbi-IV specifically inhibits the alternative pathway without complement consumption. Here we have determined the three-dimensional structure of Sbi-IV by NMR spectroscopy, showing that Sbi-IV adopts a three-helix bundle fold similar to those of the S. aureus complement inhibitors Efb-C, Ehp, and SCIN. The 1H-15N HSQC spectrum of Sbi-III indicates that this domain, essential for futile complement consumption, is natively unfolded, at least when isolated from the rest of Sbi. Sbi-IV and Sbi-III-IV both bind C3dg with 1:1 stoichiometry and submicromolar affinity. Despite low overall sequence identity, Sbi possesses the same residues as Efb at two positions essential for Efb-C binding to C3d. Mutation to alanine of either of these residues, Arg-231 and Asn-238, abolishes both Sbi-IV binding to C3dg and Sbi-IV alternative pathway inhibition. The almost complete conservation of Sbi-III and Sbi-IV amino acid sequences across more than 30 strains isolated from human and animal hosts indicates that the unique mechanism of Sbi in complement system subversion is a feature of infections of both humans and economically important animals.
Highlights
Complement comprises a finely regulated cascade of proteins that links the innate and adaptive immune systems [5,6,7]. The complement system both eliminates bacterial cells directly via the membrane attack complex and recruits effector molecules that label bacterial cells and target them for destruction by immune effector cells, for example neutrophils. This process of complement fixation occurs via two specific recognition pathways: the lectin pathway, which is a component of innate immunity and the classical pathway, which requires specific interaction with antibodies bound to antigens on target cells
The lectin and classical pathways are amplified by the alternative pathway, the alternative pathway may initiate on a target independent of the classical and lectin pathways
We have recently demonstrated that Sbi is a multifunctional protein that interferes directly with the adaptive immune system through its two N-terminal IgG binding domains (Sbi-I and Sbi-II) [24], and modulates the innate immune system through its third and fourth domains (Sbi-III and Sbi-IV) [23]
Summary
Expression, and Purification of Recombinant Sbi Fragments—DNA sequences encoding several Sbi fragments were amplified by PCR using S. aureus strain Mu50 genomic DNA as template and cloned into a pET-based vector with a TEV-cleavable N-terminal hexahistidine tag [27], as described previously [23, 24]. The column was washed with binding buffer, and the bound proteins were eluted using a 0.05–1.0 M imidazole gradient. For NMR data acquisition, the N-terminal hexahistidine tag was removed by incubation with TEV protease (Invitrogen), and the hexahistidine tag-free Sbi fragment was separated from TEV protease (itself His-tagged) by a second passage through a HisTrap column. Following TEV removal of the hexahistidine tag, the Sbi proteins contained N-terminal residues GAM from the expression vector. Ensembles of NMR structures were analyzed for violated restraints using the VMD-Xplor visualization package [35]. Columns were washed with PBS, and bound proteins were eluted with a 0 –1 M NaCl gradient over 10 ml. Wild-type or mutant Sbi-IV analytes were injected at a flow rate of 20 l/min for 60 s at the concentrations indicated, followed by a 60-s buffer flow before initiating the washing phase.
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