Biomedical subjects

Artur Sabat

Publications and source records attributed to Artur Sabat.

9 recordsLinked to original sources

PubMed2006

Comparison of PCR-based methods for typing Staphylococcus aureus isolates.

In this study, we compared the potentials of (i) a multiplex PCR-based multilocus variable-number tandem repeat (VNTR) assay; (ii) a triplex PCR coamplifying fragments of spa, coa, and the hypervariable region adjacent to the mecA gene; (iii) restriction profile analysis of the STAR repetitive element; (iv) randomly amplified polymorphic DNA analysis; (v) inter-IS256 PCR; and (vi) rep-MP3 PCR. Multilocus VNTR typing and triplex PCR (coa, spa, and hypervariable region) approaches showed excellent reproducibility and high discriminatory power; however, only multilocus VNTR typing could distinguish all pulsed-field gel electrophoresis and spa types. Multilocus VNTR typing appears to be the most useful PCR-based method for the rapid genotyping of Staphylococcus aureus strains.

Bacterial Typing Techniques↗

PubMed2006

Distribution of the serine-aspartate repeat protein-encoding sdr genes among nasal-carriage and invasive Staphylococcus aureus strains.

The sdr locus was found in all 497 investigated Staphylococcus aureus strains, although in 29 strains it contained only the sdrC gene (sdrD negative, sdrE negative). The sdrC-positive, sdrD-negative, sdrE-negative gene profile was exclusive to methicillin-sensitive S. aureus (MSSA) strains (Fisher's exact test; P = 0.0005) and was not found in the strains collected from bone infections (P = 0.0019). We also found a strong association between the presence of the sdrD gene and methicillin-resistant S. aureus strains (P < 0.0001). Our findings suggest that MSSA strains with the newly uncovered sdrC-positive, sdrD-negative, sdrE-negative gene profile have a substantially decreased potential to establish bone infection.

Bacterial Proteins↗

PubMed2005

Comparison of multiple-locus variable-number tandem-repeat analysis with pulsed-field gel electrophoresis, spa typing, and multilocus sequence typing for clonal characterization of Staphylococcus aureus isolates.

Multiple-locus variable-number tandem-repeat analysis (MLVA), a new PCR-based method of typing Staphylococcus aureus, was compared to pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) on a group of 59 S. aureus (mostly methicillin-resistant) clinical isolates. The aim of the study was to establish possible criteria of clustering MLVA patterns and to check concordance levels between the results produced by MLVA and the three other typing methods. As in our earlier study, MLVA turned out to have discriminatory power similar to that of PFGE. Comparison of data obtained by the two approaches allowed us to propose a 70% or ca. 80% cutoff value of the similarity between two MLVA patterns, depending on a cutoff level applied to interpret the PFGE results, 75% or ca. 90%, respectively. The cutoff values corresponded to the difference of up to six or four bands, respectively, among maximum 14 bands in total produced by two isolates in the analysis. The MLVA clusters matched well those obtained by PFGE, and they were also consistent in general with clusters generated by spa typing and MLST, these latter methods characterized lower resolution. Our results suggest that MLVA may be reliable in shorter-term S. aureus epidemiological studies, including analyses of outbreaks and hospital-to-hospital strain transmission events. Well-known advantages of typing methods based on PCR (low cost, short time, and easiness of performance) make MLVA a method that may be useful in a variety of laboratories, including those performing routine microbiological analyses within medical centers.

Anti-Bacterial Agents↗

PubMed2004

Prostaphopain B structure: a comparison of proregion-mediated and staphostatin-mediated protease inhibition.

Prostaphopain B is the precursor of staphopain B, a papain-type secreted cysteine protease from the pathogen Staphylococcus aureus. Here, we describe the 2.5 A crystal structure of the proenzyme. Its 21 kDa proregion is organized around a central half-barrel or barrel-sandwich hybrid and occludes primed, but not nonprimed, sites in the active site cleft of the protease. The structure of the mature part of the protease is similar to previously reported staphopain structures, and no distortion of the catalytic residues is apparent at 2.5 A resolution. A comparison of prostaphopain B with the staphopain B-staphostatin B complex shows that the proregion and the inhibitor interact with largely nonoverlapping parts of the protease surface. In a modeled complex of prostaphopain B with staphostatin B, clashes occur both inside and outside the active site cleft, but involve mostly poorly ordered regions of the protein that may be mobile.

Bacterial Proteins↗

PubMed2004

Genetic characterization of staphopain genes in Staphylococcus aureus.

Staphylococcus aureus , a leading cause of bacterial infections in humans, is endowed with a wealth of virulence factors that contribute to the disease process. Several extracellular proteolytic enzymes, including cysteine proteinases referred to as the staphopains (staphopain A, encoded by the scpA gene, and staphopain B, encoded by sspB ), have proposed roles for staphylococcal virulence. Here we present data regarding the distribution, copy number and genetic variability of the genes encoding the staphopains in a large number of S. aureus strains. The polymorphism of the scpA and sspB genes in three laboratory strains and 126 clinical isolates was analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Both genes were detected in all isolates by PCR amplification and, based on the PCR-RFLP patterns, classified as four types for scpA and six types for sspB . Those with the most divergent patterns were subjected to DNA sequencing and compared with genomic sequence data for the seven available strains of S. aureus . Southern blot analysis of the scpA and sspB sequences indicates that they are strongly conserved as single-copy genes in the genome of each S. aureus strain investigated. Taken together, these data suggest that the staphopains have important housekeeping and/or virulence functions, and therefore may constitute an interesting target for the development of therapeutic inhibitors for the treatment of staphylococcal diseases.

Amino Acid Sequence↗

PubMed2003

Staphostatins: an expanding new group of proteinase inhibitors with a unique specificity for the regulation of staphopains, Staphylococcus spp. cysteine proteinases.

A novel type of cysteine proteinase inhibitor (SspC) has been recently recognized in Staphylococcus aureus (Massimi, I., Park, E., Rice, K., Muller-Esterl, W., Sauder, D.N., and McGavin, M.J. (2002) J Biol Chem 277: 41770-41777). In this paper we have identified homologous proteins encoded in the genome of S. aureus and other coagulase-negative Staphylococci. Collectively we refer to these proteins as staphostatins as they specifically inhibit cysteine proteinases (staphopains) from Staphylococcus spp. The primary structure of staphostatins seems to be unique, although they resemble cystatins in size (105-108 residues). Recombinant staphostatin A, a product of the scpB gene and staphostatin B (SspC) from S. aureus have been characterized in details. Similar to the cystatins, the staphostatins interact specifically with their target proteinases forming tight and stable non-covalent complexes, staphostatin A with staphopain A and staphostatin B with staphopain B. However, in contrast to the cystatins, each of which inhibits broad range of cathepsins, complex formation between staphostatin and staphopain appears to be exclusive, with no cross interaction observed. In addition, the activities of several tested cysteine proteinases of prokaryotic- and eukaryotic-origin were not affected by staphostatins. Such narrow specificity limited to staphopains is presumed to be required to protect staphylococcal cytoplasmic proteins from being degraded by prematurely activated/folded prostaphopains. This function is guaranteed through the unique co-expression of the secreted proteinase and the intracellular inhibitor from the same operon, and represents a unique mechanism of regulation of proteolytic activity in Gram-positive bacteria.

Bacterial Proteins↗

PubMed2003

Staphostatins resemble lipocalins, not cystatins in fold.

Staphostatins are the endogenous inhibitors of the major secreted cysteine proteases of Staphylococcus aureus, the staphopains. Here, we present the 1.4 A crystal structure of staphostatin B and show that the fold can be described as a fully closed, highly sheared eight-stranded beta-barrel. Thus, staphostatin B is related to beta-barrel domains that are involved in the inhibition or regulation of proteases of various catalytic types and to the superfamily of lipocalins/cytosolic fatty acid binding proteins. Unexpectedly for a cysteine protease inhibitor, staphostatin B is not significantly similar to cystatins.

Carrier Proteins↗

PubMed2003

New method for typing Staphylococcus aureus strains: multiple-locus variable-number tandem repeat analysis of polymorphism and genetic relationships of clinical isolates.

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.

Bacterial Proteins↗

PubMed2002

The Clostridium ramosum IgA proteinase represents a novel type of metalloendopeptidase.

Clostridium ramosum is part of the normal flora in the human intestine. Some strains produce an IgA proteinase that specifically cleaves human IgA1 and the IgA2m(1) allotype. This prolylendopeptidase was purified from a broth culture supernatant, and N-terminal sequences of the native protein and tryptic fragments thereof were determined. A fragment of the iga gene encoding the IgA proteinase was isolated using degenerate primers in PCR, and the complete gene was obtained by inverse PCR. The identity of the iga gene was confirmed by heterologous expression in Escherichia coli. The deduced amino acid sequence indicated a signal peptide of 30 residues and a secreted proteinase of 133,828 Da. A typical Gram-positive cell wall anchor motif was identified in the C terminus. The presence of a putative zinc-binding motif His-Glu-Phe-Gly-His together with inhibition studies indicate that the proteinase belongs to the zinc-dependent metalloproteinases. However, the sequence of the C. ramosum IgA proteinase shows no overall similarity to other proteins except for significant identity around the zinc-binding motif with family M6 of metalloendopeptidases, and the unique sequence of the IgA proteinase in this area presumably establishes a new subfamily. The GC percentage of the iga gene is significantly higher than that for the entire genome of C. ramosum, suggesting that the gene was acquired recently in evolution.

Alanine↗