Binding of complement inhibitor C4b-binding protein to a highly virulent Streptococcus pyogenes M1 strain is mediated by protein H and enhances adhesion to and invasion of endothelial cells.
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.
From the Department of Laboratory Medicine, Medical Protein Chemistry, Lund University, SE-205 02 Malmö, Sweden.
David
D
Ermert
From the Department of Laboratory Medicine, Medical Protein Chemistry, Lund University, SE-205 02 Malmö, Sweden.
Antonin
A
Weckel
From the Department of Laboratory Medicine, Medical Protein Chemistry, Lund University, SE-205 02 Malmö, Sweden.
Vaibhav
V
Agarwal
Department of Clinical Sciences, Division of Infection Medicine, Lund University, SE-221 00 Lund, Sweden.
Inga-Maria
IM
Frick
Department of Clinical Sciences, Division of Infection Medicine, Lund University, SE-221 00 Lund, Sweden.
Lars
L
Björck
From the Department of Laboratory Medicine, Medical Protein Chemistry, Lund University, SE-205 02 Malmö, Sweden. Electronic address: anna.blom@med.lu.se.
Anna M
AM
Blom
D001422:Bacterial Adhesion / Q000502:physiology*; D001665:Binding Sites; D002478:Cells, Cultured; D015935:Complement C4b / Q000235:genetics / Q000378:metabolism*; D050716:Complement C4b-Binding Protein; D005260:Female; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism* / Q000382:microbiology / Q000473:pathology; D006801:Humans; D008297:Male; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013297:Streptococcus pyogenes / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D020349:Surface Plasmon Resonance; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
C055609:C4BPA protein, human; D050716:Complement C4b-Binding Protein; D011994:Recombinant Proteins; D037521:Virulence Factors; D015935:Complement C4b
Complement; Host-pathogen Interactions; Peptide Arrays; Protein-protein Interactions; Streptococcus pyogenes; Virulence Factors