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...
The multivalent pneumococcal conjugate vaccine is effective against both systemic disease and otitis media caused by serotypes contained in the vaccine. However, serotypes not covered by the present conjugate vaccine may still cause pneumococcal disease. To address these serotypes, and the remaining otitis media due to Streptococcus pneumoniae, efforts have been devoted to identifying protective protein antigens. Immunity to conserved surface proteins important for adhesion, nutrient acquisition, or other functions could result in a reduction of colonization and a lower disease potential. We have been searching for conserved surface-exposed proteins from S. pneumoniae that may be involved in pathogenesis to test as vaccine candidates. Here, an ϳ20-kDa protein that has significant homology to a nonheme iron-containing ferritin protein from Listeria innocua and other bactoferritins was identified as pneumococcal protective protein A (PppA). We expressed and purified recombinant PppA (rPppA) and evaluated its potential as a vaccine candidate. The antibodies elicited by purified rPppA were cross-reactive with PppA from multiple strains of S. pneumoniae and were directed against surface-exposed epitopes. Intranasal immunization of BALB/c mice with PppA protein and either a synthetic monophosphoryl lipid A analog, RC529AF, or a cholera toxin mutant, CT-E29H, used as an adjuvant reduced nasopharyngeal colonization in mice following intranasal challenge with a heterologous pneumococcal strain. PppA-specific systemic and local immunoglobulin G (IgG) and IgA antibody responses were induced. The antisera reacted with whole cells of a heterologous S. pneumoniae type 3 strain. These observations indicate that PppA may be a promising candidate for inclusion in a vaccine against pneumococcal otitis media.Infections with Streptococcus pneumoniae are a major cause of human diseases, such as otitis media, bacteremia, meningitis, and fatal pneumonia, worldwide (9). The rapid emergence of multidrug-resistant pneumococcal strains throughout the world has led to an increased emphasis on prevention of pneumococcal infections by vaccination (18). The presently available 23-valent pneumococcal capsular polysaccharide vaccine is not effective in children less than 2 years of age or immunocompromised patients, two of the major populations at risk for pneumococcal infection (14). A seven-valent pneumococcal polysaccharide-protein conjugate vaccine, recently licensed in the United States, was shown to be highly effective in infants and children against systemic pneumococcal disease caused by the vaccine serotypes and against cross-reactive capsular serotypes (36). However, parenteral immunization with the sevenvalent vaccine was only 60% effective against serotype-specific otitis media (17), demonstrating the need for additional immunization strategies (e.g., intranasal [i.n.] immunization), additional noncapsular antigens, or both. Therefore, there is an immediate need for a cost-effective vaccine to cover most or all of the disease-causin...
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