Abstract
Streptococcus pyogenes AP1, a strain of the highly virulent M1 serotype, uses exclusively protein H to bind the complement inhibitor C4b-binding protein (C4BP). We found a strong correlation between the ability of AP1 and its isogenic mutants lacking protein H to inhibit opsonization with complement C3b and binding of C4BP. C4BP bound to immobilized protein H or AP1 bacteria retained its cofactor activity for degradation of (125)I-C4b. Furthermore, C4b deposited from serum onto AP1 bacterial surfaces was processed into C4c/C4d fragments, which did not occur on strains unable to bind C4BP. Recombinant C4BP mutants, which (i) lack certain CCP domains or (ii) have mutations in single aa as well as (iii) mutants with additional aa between different CCP domains were used to determine that the binding is mainly mediated by a patch of positively charged amino acid residues at the interface of domains CCP1 and CCP2. Using recombinant protein H fragments, we narrowed down the binding site to the N-terminal domain A. With a peptide microarray, we identified one single 18-amino acid-long peptide comprising residues 92-109, which specifically bound C4BP. Biacore was used to determine KD = 6 × 10(-7) M between protein H and a single subunit of C4BP. C4BP binding also correlated with elevated levels of adhesion and invasion to endothelial cells. Taken together, we identified the molecular basis of C4BP-protein H interaction and found that it is not only important for decreased opsonization but also for invasion of endothelial cells by S. pyogenes.
Highlights
Pathogens such as Streptococcus pyogenes acquire protection by binding complement inhibitors to their surface
Protein H Protects S. pyogenes from Opsonization and Promotes Endothelial Adherence and Invasion—First, we confirmed that protein H but not M1 protein is important for S. pyogenes ability to bind C4b-binding protein (C4BP)
We addressed those questions by using C4BP mutants, which lacked individual complement control protein (CCP) domains or had two additional alanines inserted within the interface between the first CCP domains
Summary
Pathogens such as Streptococcus pyogenes acquire protection by binding complement inhibitors to their surface. Streptococcus pyogenes AP1, a strain of the highly virulent M1 serotype, uses exclusively protein H to bind the complement inhibitor C4b-binding protein (C4BP). Recombinant C4BP mutants, which (i) lack certain CCP domains or (ii) have mutations in single aa as well as (iii) mutants with additional aa between different CCP domains were used to determine that the binding is mainly mediated by a patch of positively charged amino acid residues at the interface of domains CCP1 and CCP2. We identified one single 18-amino acid-long peptide comprising residues 92–109, which bound C4BP. We identified the molecular basis of C4BP-protein H interaction and found that it is important for decreased opsonization and for invasion of endothelial cells by S. pyogenes
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