Staphylococcus aureus is an important opportunistic pathogen and persistently colonizes about 20% of the human population. Its surface is ‘decorated’ with proteins that are covalently anchored to the cell wall peptidoglycan. Structural and functional analysis has identified four distinct classes of surface proteins, of which microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) are the largest class. These surface proteins have numerous functions, including adhesion to and invasion of host cells and tissues, evasion of immune responses and biofilm formation. Thus, cell wall-anchored proteins are essential virulence factors for the survival of S. aureus in the commensal state and during invasive infections, and targeting them with vaccines could combat S. aureus infections.

The fibrinogen (Fg) binding MSCRAMM Clumping factor A (ClfA) from Staphylococcus aureus interacts with the C-terminal region of the fibrinogen (Fg) γ-chain. ClfA is the major virulence factor responsible for the observed clumping of S. aureus in blood plasma and has been implicated as a virulence factor in a mouse model of septic arthritis and in rabbit and rat models of infective endocarditis. We report here a high-resolution crystal structure of the ClfA ligand binding segment in complex with a synthetic peptide mimicking the binding site in Fg. The residues in Fg required for binding to ClfA are identified from this structure and from complementing biochemical studies. Furthermore, the platelet integrin αIIbβ3 and ClfA bind to the same segment in the Fg γ-chain but the two cellular binding proteins recognize different residues in the common targeted Fg segment. Based on these differences, we have identified peptides that selectively antagonize the ClfA-Fg interaction. The ClfA-Fg binding mechanism is a variant of the “Dock, Lock and Latch” mechanism previously described for the Staphylococcus epidermidis SdrG–Fg interaction. The structural insights gained from analyzing the ClfANFg peptide complex and identifications of peptides that selectively recognize ClfA but not αIIbβ3 may allow the design of novel anti-staphylococcal agents. Our results also suggest that different MSCRAMMs with similar structural organization may have originated from a common ancestor but have evolved to accommodate specific ligand structures.

Dengue virus type 2 (DEN2), a member of the Flaviviridae family, is a re-emerging human pathogen of global significance. DEN2 nonstructural protein 3 (NS3) has a serine protease domain (NS3-pro) and requires the hydrophilic domain of NS2B (NS2BH) for activation. NS3 is also an RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase and a 5-RNA triphosphatase (RTPase). In this study the first biochemical and kinetic properties of full-length NS3 (NS3 FL )-associated NTPase, RTPase, and RNA helicase are presented. The NS3 FL showed an enhanced RNA helicase activity compared with the NS3-pro-minus NS3, which was further enhanced by the presence of the NS2BH (NS2BH-NS3 FL ). An active protease catalytic triad is not required for the stimulatory effect, suggesting that the overall folding of the N-terminal protease domain contributes to this enhancement. In DEN2-infected mammalian cells, NS3 and NS5, the viral 5-RNA methyltransferase/ polymerase, exist as a complex. Therefore, the effect of NS5 on the NS3 NTPase activity was examined. The results show that NS5 stimulated the NS3 NTPase and RTPase activities. The NS5 stimulation of NS3 NTPase was dose-dependent until an equimolar ratio was reached. Moreover, the conserved motif, 184 RKRK, of NS3 played a crucial role in binding to RNA substrate and modulating the NTPase/RNA helicase and RTPase activities of NS3.The mosquito-borne Flavivirus genus, in the Flaviviridae family, includes human pathogens of global distribution and prevalence (for reviews, see Refs. 1-3), and Dengue viruses (DEN) 1 types 1-4 cause the most common infection encountered in humans (4). The diseases caused by DEN infections include from dengue fever, usually a self-limiting disease, to more severe forms, dengue hemorrhagic fever and dengue shock syndrome. These diseases pose a significant threat to humans living in DEN-infected Aedes aegypti mosquitoes endemic in areas that inhabit two-thirds of world population (5) DEN genome is a single-stranded RNA (10,723 nt in length for DEN2 New Guinea C strain used in this study (6)) of positive polarity. The viral RNA contains a long open reading frame coding for a polyprotein precursor. The polyprotein is processed into mature structural proteins, capsid (C), precursor membrane (prM), and envelope (E) and at least seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, by cellular signal peptidase and viral serine protease in the endoplasmic reticulum (for review, see Ref. 7). The 5Ј-end of the viral RNA is modified by a type I cap structure (m 7 GpppN; 2Ј-OH moiety of N is methylated). DEN2 NS3 is a multifunctional protein of about 69 kDa. It includes a serine catalytic triad within the N-terminal 185 amino acid residues. The protease domain is activated by the hydrophobic protein NS2B which serves as a cofactor for the protease and forms a complex in the infected cells (8 -12). NS2B has three hydrophobic regions flanking a conserved hydrophilic domain. The hydrophilic domain of NS2B (NS2BH) alone is sufficient for protease act...

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Clumping factor B (ClfB) from Staphylococcus aureus is a bifunctional protein that binds to human cytokeratin 10 (K10) and fibrinogen (Fg). ClfB has been implicated in S. aureus colonization of nasal epithelium and is therefore a key virulence factor. People colonized with S. aureus are at an increased risk for invasive staphylococcal disease. In this study, we have determined the crystal structures of the ligand-binding region of ClfB in an apo-form and in complex with human K10 and Fg ␣-chainderived peptides, respectively. We have determined the structures of MSCRAMM binding to two ligands with different sequences in the same site showing the versatile nature of the ligand recognition mode of microbial surface components recognizing adhesive matrix molecules. Both ligands bind ClfB by parallel ␤-sheet complementation as observed for the clumping factor A⅐␥-chain peptide complex. The ␤-sheet complementation is shorter in the ClfB⅐Fg ␣-chain peptide complex. The structures show that several residues in ClfB are important for binding to both ligands, whereas others only make contact with one of the ligands. A common motif GSSGXG found in both ligands is part of the ClfB-binding site. This motif is found in many human proteins thus raising the possibility that ClfB recognizes additional ligands.

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We have determined the crystal structure of the ligand binding segment of the Enterococcus faecalis collagen binding MSCRAMM ACE (microbial surface components recognizing adhesive matrix molecules adhesin of collagen from enterococci). This segment is composed of two subdomains, N 1 and N 2 , each adopting an IgG-like fold and forming a putative collagen binding surface at the interface between the two subdomains. This structure is very similar to that recently reported for CNA, the collagen binding MSCRAMM of Staphylococcus aureus, for which a unique ligand binding mechanism called the Collagen Hug was proposed. We suggest that ACE binds collagen by a similar mechanism and present the first biochemical evidence for this binding model. Replacing residues in the putative collagen binding trench of ACE N 2 with Ala residues affected collagen binding. A closed conformation of ACE stabilized by an engineered disulfide bond is unable to bind collagen. Finally, the importance of the residues in the N 2 extension in stabilizing the MSCRAMM-ligand complex is demonstrated by selected point and truncation mutations.During the last decade, Enterococcus faecalis has emerged as a common cause of nosocomial infections, and the organism has been associated with infections such as septicemia, endocarditis, and urinary tract infections (1).The ability of E. faecalis to readily exchange DNA by conjugation allows for the rapid spread of genetic elements and is probably the reason for the observed increase in multidrug resistance among clinical enterococcal isolates (2-4).The molecular pathogenesis of different enterococcal infections has not been elucidated but presumably involves multiple sets of virulence factors (5). As with most other bacterial infections, the adherence of E. faecalis to host tissues likely represents an early critical step in the infection process (6, 7). Extracellular pathogens, such as enterococci, staphylococci, and streptococci, often target extracellular matrix components for attachment and colonization. The adhesins on the surface of microbes mediating these interactions collectively have been named MSCRAMMs 3 (8). Most MSCRAMMs on Gram-positive pathogens belong to a family of structurally related cell wall-anchored proteins. These MSCRAMMs contain an N-terminal signal peptide followed by a non-repetitive region called the A region, which in most cases is responsible for ligand binding. The A regions are composed of two or more subdomains each adopting an immunoglobulin G-like (IgG-like) fold. Following the A region is often a segment composed of repeated sequences or motifs that is referred to as the B region. The C-terminal segment has features required for cell wall attachment including an LPXTG-like motif and a hydrophobic transmembrane region followed by a short positively charged cytoplasmic tail at the end (8).ACE, a collagen binding MSCRAMM, was the first MSCRAMM identified on E. faecalis (9 -11). ACE has a structural organization similar to that of the Staphylococcus aureus collagen adhesin CNA...

Coagulase (Coa) and Efb, secreted Staphylococcus aureus proteins, are important virulence factors in staphylococcal infections. Coa interacts with fibrinogen (Fg) and induces the formation of fibrin(ogen) clots through activation of prothrombin. Efb attracts Fg to the bacterial surface and forms a shield to protect the bacteria from phagocytic clearance. This communication describes the use of an array of synthetic peptides to identify variants of a linear Fg binding motif present in Coa and Efb which are responsible for the Fg binding activities of these proteins. This motif represents the first Fg binding motif identified for any microbial protein. We initially located the Fg binding sites to Coa’s C-terminal disordered segment containing tandem repeats by using recombinant fragments of Coa in enzyme-linked immunosorbent assay-type binding experiments. Sequence analyses revealed that this Coa region contained shorter segments with sequences similar to the Fg binding segments in Efb. An alanine scanning approach allowed us to identify the residues in Coa and Efb that are critical for Fg binding and to define the Fg binding motifs in the two proteins. In these motifs, the residues required for Fg binding are largely conserved, and they therefore constitute variants of a common Fg binding motif which binds to Fg with high affinity. Defining a specific motif also allowed us to identify a functional Fg binding register for the Coa repeats that is different from the repeat unit previously proposed.