Background: Group A Streptococcus (GAS) M proteins have an exceptional ability to induce phagocytosis resistance and aid in establishing infections. Binding of the potent inhibitor of complement, C4BP, to M proteins has been characterized to a structural level. Binding of C4BP to whole GAS has been demonstrated for 89/100 emm-types. However, studies using purified proteins found no interaction with M proteins from 13/22 emm-types positive for whole cell binding, suggesting other C4BP binding partners exist.
Methods & Results: C4BP-binding motifs were defined based on sequence patterns in C4BP-binding M proteins. We discovered predicted binding sites in several GAS Enn proteins. The gene for Enn is present in 90% of over 1400 GAS genomes and up to two thirds contain predicted C4BP-binding motifs. 9 C4BP-binding GAS strains with M proteins negative for C4BP-binding were selected. The ability of the whole bacteria to bind C4BP was confirmed using surface plasmon resonance. Enn proteins from these C4BP-binding GAS strains with or without the predicted C4BP-binding motif were produced (n=7 & 2 respectively). Binding of C4BP was observed by pull-down assays for 2 proteins containing the motif, but was negative for another without a motif. We have mapped binding to the N-terminus of Enn for the two C4BP binding proteins and determined essential residues required for C4BP-binding using targeted mutagenesis.
Conclusions: This work suggests that Enn proteins may play a significant role in binding of C4BP at the GAS surface. The impact of this interaction on virulence and vaccination requires further investigation.