Abstract
Streptococcus pyogenes is one of the most significant bacterial pathogens in the human population mostly causing superficial and uncomplicated infections (pharyngitis and impetigo) but also invasive and life-threatening disease. We have previously identified a virulence determinant, protein sHIP, which is secreted at higher levels by an invasive compared to a non-invasive strain of S. pyogenes. The present work presents a further characterization of the structural and functional properties of this bacterial protein. Biophysical and structural studies have shown that protein sHIP forms stable tetramers both in the crystal and in solution. The tetramers are composed of four helix-loop-helix motifs with the loop regions connecting the helices displaying a high degree of flexibility. Owing to interactions at the tetramer interface, the observed tetramer can be described as a dimer of dimers. We identified three residues at the tetramer interface (Leu84, Leu88, Tyr95), which due to largely non-polar side-chains, could be important determinants for protein oligomerization. Based on these observations, we produced a sHIP variant in which these residues were mutated to alanines. Biophysical experiments clearly indicated that the sHIP mutant appear only as dimers in solution confirming the importance of the interfacial residues for protein oligomerisation. Furthermore, we could show that the sHIP mutant interacts with intact histidine-rich glycoprotein (HRG) and the histidine-rich repeats in HRG, and inhibits their antibacterial activity to the same or even higher extent as compared to the wild type protein sHIP. We determined the crystal structure of the sHIP mutant, which, as a result of the high quality of the data, allowed us to improve the existing structural model of the protein. Finally, by employing NMR spectroscopy in solution, we generated a model for the complex between the sHIP mutant and an HRG-derived heparin-binding peptide, providing further molecular details into the interactions involving protein sHIP.
Highlights
Streptococcus pyogenes, known as group A streptococci (GAS) is a significant human pathogen that infects and colonizes the skin and the upper respiratory tract where it causes relatively mild clinical conditions such as impetigo and pharyngitis
Through structure-based protein engineering of a selected set of residues in the dimer-dimer interface of sHIP, we were able to generate a dimeric form of the protein with retained functional properties
We have through a combination of protein engineering, biophysics, and structural biology methods, provided the first molecular details describing the interaction between the antimicrobial protein histidine-rich glycoprotein (HRG) and the novel virulence factor sHIP
Summary
Streptococcus pyogenes, known as group A streptococci (GAS) is a significant human pathogen that infects and colonizes the skin and the upper respiratory tract where it causes relatively mild clinical conditions such as impetigo and pharyngitis. S. pyogenes produces a number of proteins that enable the bacterium to attach to host tissues, evade the immune response, and spread by penetrating host tissue layers These virulence factors are predominantly secreted or surface associated proteins, and they include the family of M proteins (Lancefield, 1962; Swanson et al, 1969; Phillips et al, 1981), fibronectin-binding proteins (Talay et al, 1994; Kreikemeyer et al, 1995; Jaffe et al, 1996; Courtney et al, 1999; Rocha and Fischetti, 1999; Terao et al, 2001), superantigenic exotoxins (Stevens et al, 1989; Abe et al, 1991; Tomai et al, 1992; Mollick et al, 1993; Norrby-Teglund et al, 1994), and the secreted streptococcal inhibitor of complement referred to as protein SIC (Åkesson et al, 1996; Fernie-King et al, 2002; Frick et al, 2003). The generation of stable dimers enabled further characterization of the interaction between sHIP and a peptide from HRG through NMR experiments, providing the first molecular details of an interaction involving this novel virulence determinant
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