Structural characterisation of adhesins from the human pathogen Streptococcus pyogenes

The Gram-positive bacterium Streptococcus pyogenes is a common human pathogen that causes a range of diseases such as tonsillitis, skin infections, toxic shock and rheumatic heart fever. Proteins called adhesins mediate the first step of infection by enabling S. pyogenes to adhere to and colonise human epithelia. This study characterises the atomic structure of two S. pyogenes adhesins, Lbp and FctB, by X-ray crystallography. Lbp is known to bind the human extracellular matrix protein laminin in vitro and appears to be important for streptococcal cell adhesion. Sequence alignments, however, show that Lbp is related to the family of metal-binding receptors (MBRs). To address the question whether Lbp is an adhesin or an MBR, or both, Lbp was characterised structurally and biochemically. The crystal structure of Lbp was solved to a resolution of 2.45 Å and consists of two domains that are connected by a seemingly rigid α-helical backbone, enclosing a zinc ion in a typical zinc-binding site. The presence of zinc was confirmed by inductively coupled plasma-mass spectrometry and an absorption scan. The fold of Lbp is similar to that of MBRs involved in the transport of zinc, iron and manganese, but distinct in the architecture of its metal-binding site. Whereas the interaction between Lbp and laminin could be confirmed and quantified in an enzymelinked immunosorbent assay in vitro, it was also demonstrated that Lbp is localised to the bacterial cell membrane. Thus, Lbp may be completely covered by the thick bacterial cell wall surrounding the bacterium and hence be prevented from interacting with human laminin in vivo. Therefore, Lbp may function primarily as a zinc receptor, a notion supported by recently published biological data showing that Lbp has an essential role in zinc transport and homeostasis. FctB is part of the FCT islet, which has recently been shown to encode subunits of pili in S. pyogenes. Pili are long, hair-like appendages that are crucial for streptococcal adhesion to human tonsils. The shaft of the pilus is assembled by the covalent polymerisation of a backbone pilin (BP) by sortases, a mechanism unique to Grampositive bacteria. At the tip of the pilus, an ancillary pilin (AP1) is the adhesin per se, and at the pilus base, a second ancillary pilin (AP2) called FctB anchors the pilus to the bacterial cell wall. In order to shed light on how FctB is incorporated into the pilus and how it anchors the pilus to the cell wall, the structure of FctB was solved to a resolution of 1.9 Å. FctB has an immunoglobulin (Ig)-like domain followed by a Cterminal, proline-rich extension. The latter forms a polyproline-II-like helix that appears to be a common motif in the cell wall-anchoring of AP2s from Gram-positive bacteria. Unexpectedly, the Ig-like domain displayed close structural homology to the N-terminal domain of the BP, leading to the identification of a conserved lysine residue involved in the covalent linkage of FctB to the pilus shaft. This linkage was subsequently confirmed by mass spectral analysis of native pili from S. pyogenes. In addition, the C-terminal