Many bacterial pathogens present adhesins at the tips of long macromolecular filaments known as pili that are often important virulence determinants. Very little is known about how pili presented by Gram-positive pathogens mediate host cell binding. The crystal structure of a pilus adhesin from the important human pathogen Streptococcus pyogenes reveals an internal thioester bond formed between the side chains of a cysteine and a glutamine residue. The presence of the thioester was verified using UV-visible spectroscopy and mass spectrometry. This unusual bond has only previously been observed in thioester domains of complement and complement-like proteins where it is used to form covalent attachment to target molecules. The structure also reveals two intramolecular isopeptide bonds, one of these formed through a Lys/Asp residue pair, which are strategically positioned to confer protein stability. Removal of the internal thioester by allele-replacement mutagenesis in S. pyogenes severely compromises bacterial adhesion to model host cells. Although current paradigms of bacterial/host cell interaction envisage strong non-covalent interactions, the present study suggests cell adhesion could also involve covalent bonds.Adhesion to a host cell surface is often a key step in establishing a successful infection. Many specific adhesins are projected outwards from bacterial surfaces by extended polymeric protein filaments known as pili. These structures have received considerable attention due to their roles in enabling host colonization and potential as vaccine candidates (1-6). In Grampositive bacteria, pili are assembled through intermolecular covalent linkages, formed by the action of sortases, which recognize specific sequence motifs on substrate proteins (7-10). An emerging paradigm for the structure of Gram-positive pili includes a minor pilus subunit displayed at the tip followed by a polymer of repeating subunits (composed of a protein frequently referred to as the "shaft" or "major" pilus subunit); this structure is then attached to the bacterial surface, frequently through a second minor pilus subunit that acts as a cell wall linker. In addition to intermolecular links between subunits, major pilus subunits also contain intramolecular isopeptide bonds (11-14) that confer proteolytic, thermodynamic and mechanical stability on these proteins (12,13,15,16). Together, these bonds are thought to provide structural stability to these thin (1 molecule), extended (100s of molecules) structures and maintain pilus integrity during the early stages of interaction with host cells.Streptococcus pyogenes (Group A Streptococcus) is an important human host-specific pathogen that can cause a wide variety of infections including potentially life-threatening diseases such as acute rheumatic fever, streptococcal toxic shock syndrome, and necrotizing fasciitis (17). Pili presented on the surface of the serotype M1 Group A Streptococcus strain SF370 (GAS) 3 are required for efficient adhesion to model host cells and clinically rele...

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ϳ1/3 and C-terminal ϳ2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.

Adhesion of the serotype M1 Streptococcus pyogenes strain SF370 to human tonsil explants and cultured keratinocytes requires extended polymeric surface structures called pili. In this important human pathogen, pili are assembled from three protein subunits: Spy0125, Spy0128 and Spy0130 through the action of sortase enzymes. For this study, the structural properties of these pili proteins have been investigated in solution. Spy0125 and Spy0128 display characteristics of globular, folded proteins. Circular dichroism suggests a largely beta-sheet composition for Spy0128 and Spy0125; Spy0130 appears to contain little secondary structure. Each of the proteins adopts a monodisperse, monomeric state in solution as assessed by analytical ultracentrifugation. Further, small-angle X-ray scattering curves for Spy0125, Spy0128 and Spy0130 suggest each protein adopts an elongated shape, likely comprised of two domains, with similar maximal dimensions. Based on the scattering data, dummy atom models of each of the pili subunits have been reconstructed ab initio. This study provides the first insights into the structure of Streptococcus pyogenes minor pili subunits, and possible implications for protein function are discussed.

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