Poster Presentation 20th Lancefield International Symposium on Streptococci and Streptococcal Diseases 2017

A novel interaction between C4BP and the Enn protein of Group A streptococcus (#191)

Hannah RC Frost 1 2 , Martina Sanderson-Smith 3 , David De Oliveira 3 , Ailish Cleary 3 , Mark Davies 4 , Mark J Walker 5 , Andrew C Steer 6 7 , Anne Botteaux 1 , Pierre R Smeesters 1 8
  1. Bacterology moleculaire, Universite Libre de Bruxelles, Brussels, Belgium
  2. Universite Libre de Bruxelles, Anderlecht, BRUSSELS, Belgium
  3. Illawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia
  4. Microbiology and Immunology, Peter Doherty Institute, University of Melbourne, Melbourne, Victoria, Australia
  5. Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD, Australia
  6. Group A Streptococcus Research Group, Murdoch Childrens Research Institute, Melbourne, Victoria, Australia
  7. Centre for International Child Health, University of Melbourne, Melbourne, Victoria, Australia
  8. Department of Pediatrics, Academic Children Hospital Queen Fabiola, Brussels, Belgium

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.