Streptococcus pyogenes is a gram-positive human pathogen that causes a wide spectrum of disease, placing a significant burden on public health. Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. The identification of these proteins for vaccine development is an important goal of bacterial proteomics. Here we describe a method of proteolytic digestion of surface-exposed proteins to identify surface antigens of S. pyogenes. Peptides generated by trypsin digestion were analyzed by multidimensional tandem mass spectrometry. This approach allowed the identification of 79 proteins on the bacterial surface, including 14 proteins containing cell wall-anchoring motifs, 12 lipoproteins, 9 secreted proteins, 22 membrane-associated proteins, 1 bacteriophage-associated protein, and 21 proteins commonly identified as cytoplasmic. Thirty-three of these proteins have not been previously identified as cell surface associated in S. pyogenes. Several proteins were expressed in Escherichia coli, and the purified proteins were used to generate specific mouse antisera for use in a whole-cell enzymelinked immunosorbent assay. The immunoreactivity of specific antisera to some of these antigens confirmed their surface localization. The data reported here will provide guidance in the development of a novel vaccine to prevent infections caused by S. pyogenes.Streptococcus pyogenes, also known as group A Streptococcus, is a gram-positive bacterium that causes a wide spectrum of diseases ranging from mild localized infections, such as pharyngitis and impetigo, to severe invasive diseases, such as necrotizing fasciitis and streptococcal toxic shock-like syndrome. Invasive streptococcal disease is associated with high morbidity and mortality rates (37). S. pyogenes is also associated with a variety of autoimmune sequelae such as acute rheumatic fever, which after repeated episodes can result in rheumatic valvular heart disease, the most common cause of pediatric heart disease worldwide (11). In spite of the high mortality and substantial economic losses caused by these diseases, there is currently no licensed vaccine to prevent human S. pyogenes infections.For many years, efforts to develop a vaccine to protect against S. pyogenes infections were focused on the surfaceassociated M protein (19, 38), a major virulence factor of S. pyogenes. However, there are at least two significant limitations for using M protein as a vaccine antigen. First, the M protein contains a highly variable amino-terminal region that determines the S. pyogenes serotype. With over 150 different M serotypes identified, it is difficult to envision using the M protein as a broadly efficacious vaccine. Second, M protein elicits antibodies that are cross-reactive with human cardiac myosin and are associated with the development of acute rheumatic fever (10). To circumvent these issues, we have initiated an alternative strategy to identify other proteins localized to the sur...

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.

Direct measurements of the structure and function of the COOH-terminal portion of the A alpha chain (residues 220-610) of human fibrinogen have been hampered by the difficulty of isolating intact fragments derived from this protease-sensitive region. Here, we overcame this problem by expressing two fragments, alpha C45K (A alpha 221-610) and a truncated version of it, alpha C30K (A alpha 368-610), in Escherichia coli. Both proteins were purified to homogeneous state, and their integrity was confirmed at protein level by sequencing. Upon treatment with factor XIIIa, the alpha C45K fragment but not the alpha C30K fragment formed polymers similar to those derived from fibrin clots. Sequence analysis of cross-linked alpha C45K polymers revealed involvement in the cross-linking reaction of at least three Gln residues (221, 237, 328) in the NH2-terminal region of the fragment and four Lys residues (539, 556, 580, 601) located in the COOH-terminal part of the molecule. In addition, a fraction of alpha C45K fragment was found in an intramolecular cross-linked form, suggesting a high level of flexibility of its polypeptide chain and consistent with the location of its donor and acceptor residues in clusters near the ends of the molecule. The alpha C30K fragment, lacking the NH2-terminal Gln residues, was not able to form polymers or internally cross-linked monomers. Thus, the C-terminal part of the A alpha chain comprises an autonomous, functionally active, and flexible region that plays a key role in alpha polymer formation and stabilization of fibrin clots by factor XIIIa.

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The Clostridium difficile toxins A and B are primarily responsible for symptoms of C. difficile associated disease and are prime targets for vaccine development. We describe a plasmid-based system for the production of genetically modified toxins in a non-sporulating strain of C. difficile that lacks the toxin genes tcdA and tcdB. TcdA and TcdB mutations targeting established glucosyltransferase cytotoxicity determinants were introduced into recombinant plasmids and episomally expressed toxin mutants purified from C. difficile transformants. TcdA and TcdB mutants lacking glucosyltransferase and autoproteolytic processing activities were ~10 000-fold less toxic to cultured human IMR-90 cells than corresponding recombinant or native toxins. However, both mutants retained residual cytotoxicity that could be prevented by preincubating the antigens with specific antibodies or by formalin treatment. Such non-toxic formalin-treated mutant antigens were immunogenic and protective in a hamster model of infection. The remaining toxicity of untreated TcdA and TcdB mutant antigens was associated with cellular swelling, a phenotype consistent with pore-induced membrane leakage. TcdB substitution mutations previously shown to block vesicular pore formation and toxin translocation substantially reduced residual toxicity. We discuss the implications of these results for the development of a C. difficile toxoid vaccine.

The structure of a cell surface enzyme from a Gram-positive pathogen has been determined to 2-Å resolution. Gram-positive pathogens have a thick cell wall to which proteins and carbohydrate are covalently attached. Streptococcal C5a peptidase (SCP), is a highly specific protease and adhesin͞invasin. Structural analysis of a 949-residue fragment of the [D130A,S512A] mutant of SCP from group B Streptococcus (S. agalactiae, SCPB) revealed SCPB is composed of five distinct domains. The N-terminal subtilisin-like protease domain has a 134-residue protease-associated domain inserted into a loop between two ␤-strands. This domain also contains one of two Arg-Gly-Asp (RGD) sequences found in SCPB. At the C terminus are three fibronectin type III (Fn) domains. The second RGD sequence is located between Fn1 and Fn2. Our analysis suggests that SCP binding to integrins by the RGD motifs may stabilize conformational changes required for substrate binding.bacterial adhesins ͉ endopeptidases ͉ molecular models ͉ Streptococcus agalactiae ͉ x-ray crystallography

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A recombinant streptococcal C5a peptidase was expressed in Escherichia coli and its catalytic properties and thermal stability were subjected to examination. It was shown that the NH 2 -terminal region of C5a peptidase (Asn32-Asp79/ Lys90) forms the pro-sequence segment. Upon maturation the propeptide is hydrolyzed either via an autocatalytic intramolecular cleavage or by exogenous protease streptopain. At pH 7.4 the enzyme exhibited maximum activity in the narrow range of temperatures between 40 and 43°C. The process of heat denaturation of C5a peptidase investigated by fluorescence and circular dichroism spectroscopy revealed that the protein undergoes biphasic unfolding transition with T m of 50 and 70°C suggesting melting of different parts of the molecule with different stability.Unfolding of the less stable structures was accompanied by the loss of proteolytic activity. Using synthetic peptides corresponding to the COOH-terminus of human complement C5a we demonstrated that in vitro peptidase catalyzes hydrolysis of two His67-Lys68 and Ala58-Ser59 peptide bonds. The high catalytic efficiency obtained for the SQLRANISHKDMQLGR extended peptide compared to the poor hydrolysis of its derivative Ac-SQLRANISH-pNA that lacks residues at P2¢-P7¢ positions, suggest the importance of C5a peptidase interactions with the P¢ side of the substrate.Keywords: maturation; propeptide; streptopain; denaturation; substrate binding.Group A streptococcus (Streptococcus pyogenes) is a common human pathogen causing a wide variety of diseases. These include relatively mild pathological conditions such as pharyngitis and impetigo, more serious nonsuppurative sequelae, acute rheumatic fever, glomerulonephritis, deadly toxic shock syndrome and necrotizing fasciitis. S. pyogenes has developed complex and sophisticated molecular mechanisms that allow it to avoid human defenses. One of the important virulence factors of streptococci involved in such activity is an extracellular C5a peptidase [2]. Streptococcal C5a peptidase is a surfaceassociated subtilisin-like serine protease with an unusually restricted substrate specificity. The only known protein substrate hydrolyzed by C5a peptidase is human complement fragment C5a [3,4]. C5a peptidase-generated cleavage within the COOH-terminal region of human C5a drastically reduces the ability of this anaphylatoxin to bind receptors on the surface of polymorphonuclear neutrophil leukocytes (PMNLs) and therefore abolishes its chemotactic activity [3]. It is believed that C5a peptidase plays an important role in bacterial colonization of the host by inhibiting the influx of PMNLs and impeding initial clearance of the streptococci.C5a peptidase from S. pyogenes is encoded by the chromosomal scpA gene and consists of 1167 amino residues (Fig. 1A). C5a peptidase is first produced as a precursor, with an NH 2 -terminal 31 amino acid residue signal peptide responsible for exporting the protein across the membrane [2]. The length of C5a peptidase prosequence segment and the mechanism of its cleavage rem...