Staphylococcus aureus and Streptococcus pyogenes, two important human pathogens, target host fibronectin (Fn) in their adhesion to and invasion of host cells. Fibronectin-binding proteins (FnBPs), anchored in the bacterial cell wall, have multiple Fn-binding repeats in an unfolded region of the protein. The bacterium-binding site in the amino-terminal domain (1-5F1) of Fn contains five sequential Fn type 1 (F1) modules. Here we show the structure of a streptococcal (S. dysgalactiae) FnBP peptide (B3) in complex with the module pair 1F12F1. This identifies 1F1- and 2F1-binding motifs in B3 that form additional antiparallel beta-strands on sequential F1 modules-the first example of a tandem beta-zipper. Sequence analyses of larger regions of FnBPs from S. pyogenes and S. aureus reveal a repeating pattern of F1-binding motifs that match the pattern of F1 modules in 1-5F1 of Fn. In the process of Fn-mediated invasion of host cells, therefore, the bacterial proteins seem to exploit the modular structure of Fn by forming extended tandem beta-zippers. This work is a vital step forward in explaining the full mechanism of the integrin-dependent FnBP-mediated invasion of host cells.

The SasG surface protein of Staphylococcus aureus has been shown to promote the formation of biofilm. SasG comprises an N-terminal A domain and repeated B domains. Here we demonstrate that SasG is involved in the accumulation phase of biofilm, a process that requires a physiological concentration of Zn 2؉ . The B domains, but not the A domain, are required. Purified recombinant B domain protein can form dimers in vitro in a Zn 2؉ -dependent fashion. Furthermore, the protein can bind to cells that have B domains anchored to their surface and block biofilm formation. The full-length SasG protein exposed on the cell surface is processed within the B domains to a limited degree, resulting in cleaved proteins of various lengths being released into the supernatant. Some of the released molecules associate with the surface-exposed B domains that remain attached to the cell. Studies using inhibitors and mutants failed to identify any protease that could cause the observed cleavage within the B domains. Extensively purified recombinant B domain protein is very labile, and we propose that cleavage occurs spontaneously at labile peptide bonds and that this is necessary for biofilm formation.

SummaryMany pathogenic Gram-positive bacteria produce cell wall-anchored proteins that bind to components of the extracellular matrix (ECM) of the host. These bacterial MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) are thought to play a critical role in infection. One group of MSCRAMMs, produced by staphylococci and streptococci, targets fibronectin (Fn, a glycoprotein found in the ECM and body fluids of vertebrates) using repeats in the C-terminal region of the bacterial protein. These bacterial Fn-binding proteins (FnBPs) mediate adhesion to host tissue and bacterial uptake into nonphagocytic host cells. Recent studies on interactions between the host and bacterial proteins at the residue-specific level and on the mechanism of host cell invasion are providing a much clearer picture of these processes.

Staphylococcus aureus can adhere to and invade endothelial cells by binding to the human protein fibronectin (Fn). FnBPA and FnBPB, cell wall-attached proteins from S. aureus, have multiple, intrinsically disordered, high-affinity binding repeats (FnBRs) for Fn. Here, 30 years after the first report of S. aureus/Fn interactions, we present four crystal structures that together comprise the structures of two complete FnBRs, each in complex with four of the N-terminal modules of Fn. Each Ϸ40-residue FnBR forms antiparallel strands along the triple-stranded ␤-sheets of four sequential F1 modules ( 2-5 F1) with each FnBR/ 2-5 F1 interface burying a total surface area of Ϸ4,300 Å 2 . The structures reveal the roles of residues conserved between S. aureus and Streptococcus pyogenes FnBRs and show that there are few linker residues between FnBRs. The ability to form large intermolecular interfaces with relatively few residues has been proposed to be a feature of disordered proteins, and S. aureus/Fn interactions provide an unusual illustration of this efficiency.intrinsic disorder ͉ tandem ␤-zipper ͉ host-pathogen interaction S taphylococcus aureus is a dangerous human pathogen that causes a wide range of debilitating and life-threatening infections (1). Incidence of S. aureus resistance to antibiotics (2) makes the understanding of its mechanisms of pathogenesis imperative. S. aureus/Fn interactions were first reported 30 years ago, and an S. aureus Fn-binding protein was isolated and characterized Ϸ20 years ago (3). Our recent work has dissected the 363-residue C-terminal region of FnBPA into 11 FnBRs (4) (FnBPA1-11; Fig. 1 A and B), six of which bind the NTD (N-terminal domain) of Fn (comprising modules 1-5 F1) with dissociation constants in the nanomolar range (5). The Cterminal region of FnBPB, a second S. aureus Fn-binding protein, is very similar to FnBPA but lacks one of the shorter FnBRs (5). In FnBPA, which also binds fibrinogen, the fibrinogen-and Fn-binding regions (Fig. 1 A) appear to cooperate in disease progression, with the FnBR region being particularly associated with persistence of infection (6). FnBPA/Fn interactions both mediate S. aureus invasion of (7) and activate endothelial cells, evoking both the proinflammatory and procoagulant responses typical of infective endocarditis (8). FnBPAs ability to mediate platelet activation, a key step in thrombus formation, is also likely to play a role in cardiovascular disease (9) and FnBPA has been implicated in cardiac device infections through its ability to mediate S. aureus attachment to implanted prosthetic materials (10). We previously predicted that in Fn-BPA each FnBR binds a string of three or four F1 modules in the NTD of Fn through a longer version of the tandem ␤-zipper mechanism that we discovered in Streptococcus dysgalactiae interactions with 1 F1 2 F1 (4). Results and DiscussionCrystal Structure of FnBPA-1/ 2-5 F1. Fig. 1C shows two F1 module pair/peptide structures that together comprise the structure of the most N-terminal S. aureus FnBR (...

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Entry of Staphylococcus aureus into the bloodstream can lead to metastatic abscess formation and infective endocarditis. Crucial to the development of both these conditions is the interaction of S. aureus with endothelial cells. In vivo and in vitro studies have shown that the staphylococcal invasin FnBPA triggers bacterial invasion of endothelial cells via a process that involves fibronectin (Fn) bridging to α5β1 integrins. The Fn-binding region of FnBPA usually contains 11 non-identical repeats (FnBRs) with differing affinities for Fn, which facilitate the binding of multiple Fn molecules and may promote integrin clustering. We thus hypothesized that multiple repeats are necessary to trigger the invasion of endothelial cells by S. aureus. To test this we constructed variants of fnbA containing various combinations of FnBRs. In vitro assays revealed that endothelial cell invasion can be facilitated by a single high-affinity, but not low-affinity FnBR. Studies using a nisin-inducible system that controlled surface expression of FnBPA revealed that variants encoding fewer FnBRs required higher levels of surface expression to mediate invasion. High expression levels of FnBPA bearing a single low affinity FnBR bound Fn but did not invade, suggesting that FnBPA affinity for Fn is crucial for triggering internalization. In addition, multiple FnBRs increased the speed of internalization, as did higher expression levels of FnBPA, without altering the uptake mechanism. The relevance of these findings to pathogenesis was demonstrated using a murine sepsis model, which showed that multiple FnBRs were required for virulence. In conclusion, multiple FnBRs within FnBPA facilitate efficient Fn adhesion, trigger rapid bacterial uptake and are required for pathogenesis.

Binding of the fibronectin-binding protein FnBPA fromStaphylococcus aureus to the human protein fibronectin has previously been implicated in the development of infective endocarditis, specifically in the processes of platelet activation and invasion of the endothelium. We recently proposed a model for binding of fibronectin to FnBPA in which the bacterial protein contains 11 potential binding sites (FnBPA-1 to FnBPA-11), each composed of motifs that bind to consecutive fibronectin type 1 modules in the N-terminal domain of fibronectin. Here we show that six of the 11 sites bind with dissociation constants in the nanomolar range; other sites bind more weakly. The high affinity binding sites include FnBPA-1, the sequence of which had previously been thought to be encompassed by the fibrinogen-binding A domain of FnBPA. Both the number and sequence conservation of the type-1 module binding motifs appears to be important for high affinity binding. The in vivo relevance of the in vitro binding studies is confirmed by the presence of antibodies in patients with S. aureus infections that specifically recognize complexes of these six high affinity repeats with fibronectin.Staphylococcus aureus is one of the most important bacterial pathogens to affect humans. Clinical manifestations of infection range from superficial skin infections (1) to life-threatening conditions, such as endocarditis (2, 3) and difficult to treat infections of the bones and joints (4, 5). The increasing virulence and antibiotic resistance exhibited by this major source of both community and hospital-acquired infection presents an urgent challenge. Furthering our understanding of the mechanism by which staphylococcal pathogenesis occurs is thus imperative for the development of novel therapeutic and preventative strategies.Since attachment to host tissue is a critical early step in infection, one particular group of targets for intervention is the microbial surface components recognizing adhesive matrix molecules (MSCRAMM) 4 family of surface-expressed adhesins (6, 7). These proteins exploit extracellular matrix proteins, such as fibronectin (Fn), using them as a bridge between the bacterial cell surface and host cell receptors that effect downstream signaling (8, 9). Although classically regarded as an exclusively extracellular pathogen, S. aureus has been shown to adhere to and invade several host cell types (10 -14), and the Fn-binding subfamily of MSCRAMMs (FnBPs) appears to be involved in this process (15). There is an emerging view that S. aureus can exist intracellularly, hijacking and invading host cells to establish persistence (16). Conceivably, this mechanism could facilitate rapid and effective bloodstream dissemination while allowing the bacterium to evade antibiotics and host immune surveillance, an apposite theory given the prevalence of bacterial metastasis (3) and infection relapse in staphylococcal disease (17).Fn is a large glycoprotein present in a soluble form in human plasma and other body fluids and in an insoluble form in...

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