The PCR-based methodology applied to multiple-locus variable numbers of tandem repeat (VNTR) analysis was recently shown to be a useful technique for the molecular typing of clinical isolates of several bacterial species. We have adopted this method for the molecular typing of methicillin-resistant Staphylococcus aureus. Five staphylococcal VNTR loci (sdr, clfA, clfB, ssp, and spa) were subjected to analysis, and it was shown that the method allows typing of S. aureus strains with the discriminatory power and reproducibility of pulsed-field gel electrophoresis while at the same time being rapid and applicable to analysis of large numbers of isolates.During the past few years, the most remarkable advances in molecular typing were achieved by analysis of variable numbers of tandem repeat (VNTR) loci identified in the genomes of eukaryotic and prokaryotic species during genome sequencing projects. The number of repeat units at the same locus often varies from strain to strain and can be detected by PCR with flanking primers, a technique also commonly used for DNA fingerprinting of eukaryotic and prokaryotic species (1,5,7,9,11). However, to the best of our knowledge, this method has never been used to type Staphylococcus aureus strains, although several genes with repetitive sequences were analyzed for the purpose of S. aureus typing (16).The sequencing of the S. aureus genome indicated the presence of several VNTR loci, including sdr, clfA, clfB, ssp, coa, and spa. The sdr locus (10) comprises two or three closely linked and tandemly arrayed open reading frames containing sdrC, sdrD, and sdrE, which encode Sdr proteins. The Sdr proteins, together with clumping factor A (ClfA) (12) and clumping factor B (ClfB) (13), are members of a family of surface proteins which are characterized by the presence of an R region containing various numbers of the repeated Ser-Asp dipeptides encoded by an 18-nucleotide DNA repeat at the 3Ј region of the sdr genes. The ssp locus contains a gene (sspA) encoding a serine protease (V8 protease) (14), the C-terminal fragment of which is built of multiple, variable numbers of tripeptide repeats encoded by 9-nucleotide repeating units (2). Finally, coa and spa genes coding for collagen binding protein and protein A, respectively, have various numbers of degenerated repeats of 81 and 24 bp, respectively (15). This polymorphism has been commonly used for differentiating S. aureus isolates (6,8). In this paper, we describe a multiple-locus VNTR analysis (MLVA) system to discriminate among different S. aureus clinical isolates based on the analysis of five (sdr, clfA, clfB, ssp, and spa) tandem repeat loci composed of seven individual genes. This method of exploring the VNTR polymorphism enables the typing of clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates, determination of their diversity and evolutionary relationships with discriminatory power, and a reproducibility matching the commonly used pulsed-field gel electrophoresis (PFGE) technique.The strain collection encompa...
Protein ubiquitination requires the sequential activity of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitinligase (E3). The ubiquitin-transfer machinery is hierarchically organized; for every ubiquitin-activating enzyme, there are several ubiquitin-conjugating enzymes, and most ubiquitin-conjugating enzymes can in turn interact with multiple ubiquitin ligases. Despite the central role of ubiquitin-activating enzyme in this cascade, a crystal structure of a ubiquitin-activating enzyme is not available. The enzyme is thought to consist of an adenylation domain, a catalytic cysteine domain, a fourhelix bundle, and possibly, a ubiquitin-like domain. Its adenylation domain can be modeled because it is clearly homologous to the structurally known adenylation domains of the activating enzymes for the small ubiquitinlike modifier (SUMO) and for the protein encoded by the neuronal precursor cell-expressed, developmentally down-regulated gene 8 (NEDD8). Low sequence similarity and vastly different domain lengths make modeling difficult for the catalytic cysteine domain that results from the juxtaposition of two catalytic cysteine halfdomains. Here, we present a biochemical and crystallographic characterization of the two half-domains and the crystal structure of the larger, second catalytic cysteine half-domain of mouse ubiquitin-activating enzyme. We show that the domain is organized around a conserved folding motif that is also present in the NEDD8-and SUMO-activating enzymes, and we propose a tentative model for full-length ubiquitin-activating enzyme.
Staphostatins are the endogenous inhibitors of the major secreted cysteine proteases of Staphylococcus aureus, the staphopains. Our recent crystal structure of staphostatin B has shown that this inhibitor forms a mixed, eight-stranded -barrel with statistically significant similarity to lipocalins, but not to cystatins. We now present the 1.8-Å crystal structure of staphostatin B in complex with an inactive mutant of its target protease. The complex is held together through extensive interactions and buries a total surface area of 2300 Å 2 . Unexpectedly for a cysteine protease inhibitor, staphostatin B binds to staphopain B in an almost substratelike manner. The inhibitor polypeptide chain runs through the protease active site cleft in the forward direction, with residues IG-TS in P2 to P2 positions. Both in the free and complexed forms, the P1 glycine residue of the inhibitor is in a main chain conformation only accessible to glycines. Mutations in this residue lead to a loss of affinity of the inhibitor for protease and convert the inhibitor into a substrate.The emergence of multiple antibiotic resistance in Staphylococci, in particular Staphylococcus aureus, has become a major medical concern. With the prevalence of methicillin-resistant S. aureus in hospitals throughout the world (1, 2) and with the appearance of strains that are resistant to "last resort" glycopeptide antibiotics (3, 4), treatment of S. aureus infections in difficult cases could soon depend on a few new compounds that have either been recently introduced to the clinic or are still in clinical trials (5-9). A possible strategy for the development of new antibacterials is to target bacterial virulence factors. The S. aureus arsenal of virulence factors includes secreted toxins (10, 11), immune modulatory molecules (12), adhesion molecules (13), signaling factors (14, 15), and possibly secreted proteases (16).Staphopains A and B are the major secreted cysteine proteases of S. aureus. The literature contains evidence both for and against their role as virulence factors. Random mutagenesis has shown that transposon insertion into the V8-proteinase gene attenuates S. aureus virulence in three separate animal models (mouse abscess, bacteraemia, and wound infection models) (17). More recent work suggests that loss of virulence could be because of a polar effect of the insertion event on the downstream sspB gene encoding staphopain B (18). Consistent with this, inactivation of the sspB gene attenuates S. aureus virulence in the skin abscess model. In contrast, staphopain A, a close homologue of staphopain B, appears to be dispensable for virulence at least in this model. 1Staphopains A and B are remote members of the papain superfamily of enzymes and are encoded on the genome as preproenzymes in the scp and ssp loci, respectively. Recently, it was shown that a short open reading frame in the ssp operon termed sspC codes for an inhibitor of staphopain B, the cysteine protease in this operon (19). This initial finding could be extended to the scp op...
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