Interactions of human C4BP with Bordetella pertussis and Streptococcus pyogenes

Abstract: Many microorganisms have developed mechanisms to protect themselves against attack from the complement system of the host. One possible mechanism for a microorganism to evade complement attack is to bind a human complement regulator, which may allow the microorganism to down-regulate complement activation. This thesis describes studies of such interactions between human C4b-binding protein (C4BP), a complement regulator present in plasma, and two bacteria pathogenic for humans: Bordetella pertussis and Streptococcus pyogenes. All clinical isolates of B. pertussis, the etiologic agent of whooping cough, were shown to bind C4BP. The binding was found to be dependent on at least two different surface components, one of which is the virulence factor filamentous hemagglutinin (FHA). The region in C4BP that binds B. pertussis is very similar, but not identical, to the region used by the natural ligand C4b. Many, but not all, strains of S. pyogenes bind C4BP. The binding is due to M proteins, a group of surface proteins important for virulence. The binding site in C4BP for M proteins was studied and found to overlap with the binding site for C4b. Thus, two very different pathogens, B. pertussis and S. pyogenes, bind to the same region in C4BP as the natural ligand C4b. The M proteins of S. pyogenes are characterized by the presence of a hypervariable region (HVR), which allows the bacteria to evade host immunity due to antigenic variation. The HVR is required for the ability of S. pyogenes to resist phagocytosis, but the mechanism of action of the region has remained unknown. Previously, it has been demonstrated that C4BP binds to the HVR, but evidence has been lacking that bacteria-bound C4BP plays a role in phagocytosis resistance. A functional study presented in this thesis provided several lines of evidence that C4BP indeed contributes to phagocytosis resistance. Our data provide the first molecular explanation for the ability of an HVR to confer resistance to phagocytosis. We found that isolated HVRs retain the ability to bind C4BP, allowing direct characterization of the C4BP-binding HVRs. Synthetic peptides/HVRs with very little residue identity were found to bind C4BP with high specificity and computational modeling suggested that they have similar folds. However, the C4BP-binding HVRs were immunologically completely unrelated. These data show that a bacterial protein domain can exhibit extreme variability with regard to sequence and immunological properties, while retaining the ability to bind a ligand with high specificity.