Proteolytic Cleavage of Staphylococcal Exoproteins Analyzed by Two‐Dimensional Gel Electrophoresis

Abstract

Extracellular proteases of Staphylococcus aureus are emerging as potential virulence factors that are relevant to the pathogenicity of staphylococcal infections. These proteases may also be involved in the proteolytic cleavage of other exoproteins released from this organism. To define the target exoproteins and their sites of cleavage by proteases, high-resolution two-dimensional polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing of exoprotein spots was performed. Two to three hundred exoprotein spots were detected at the early-stationary phase of cultures of S. aureus NCTC8325, and then at the late-stationary stage most of these high molecular protein spots became invisible due to further proteolytic degradation. As the result of N-terminal analysis, lipase, triacylglycerollipase, orf619 protein and orf388 protein were detected as multiple spots at the early-stationary phase. We found that these exoproteins were cleaved at 3, 7, 4 and 4 different sites, respectively, by proteases. According to the M.W. and pI of each peptide spot obtained from the gel and their matches with calculated values in addition to their Nterminal sequences, we showed that the positions of putative peptides resulted from proteolytic cleavage of these proteins. Key words: Staphylococcal exoproteins, Extracellular proteases, Two-dimensional polyacrylamide-gel electrophoresisStaphylococcus aureus is an important human pathogen implicated in a wide range of diseases including septicemia, meningitis, endocarditis, osteomyelitis, toxic-shock syndrome (TSS) and food poisoning (21,28,34). Incidences of both community-and hospitalacquired staphylococcal infections are increasing because of its rapid development of antibiotic resistance (29).S. aureus secretes a large number of extracellular proteins (exoproteins), which have been shown to contribute to the pathogenic activities or to enhance the virulence of this organism (15,21,25,33). Recently, renewed interest has focused on proteases secreted from S. aureus under the control of agr (accessory gene regulator) and sar (staphylococcal accessory regulator), the main regulators of S. aureus virulence determinant genes (6,7,12,24). The expression of a specific serine glutamyl endopeptidase, V8 protease, was found to be *Address correspondence to Dr. Michio Ohta, Department of Molecular Bacteriology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan. Fax: + 81-52-744-2107. E-mail: mohta @med.nagoya-u.ac.jp 285 important for virulence in animal models of staphylococcal infections (8). In addition to V8 protease, S. au reus secretes at least two other proteases, staphopain (papain-like thiol protease) (2) and aureolysin (typical metalloproteinase) (I). These proteases also encompass enzymes including Pseudomonas aeruginosa elastase (23), Legionella pneumophila (18) and Listeria monocytogenes metalloproteinases (9), Vibrio cholerae hemagglutinin protease (5), Staphylococcus epidermidis elastase (32), and the ...

Two‐Dimensional Analysis of Exoproteins of Methicillin‐Resistant Staphylococcus aureus (MRSA) for Possible Epidemiological Applications

“…The superantigenic exotoxins were likely rather refractory to the action of these proteases and were isolated as clear spots on a 2-DE gel after the culture. However, a few exoenzymes such as lipase and triacylglycerol lipase were detected as multiple spots, indicating probable proteolysis to generate active forms as described in the previous paper (26).…”

“…The N-terminal sequence of glutamic acid-specific endopeptidase (spot 4) found in strain MF330 was different from those of spots G and H of strain 2932. As our previous study showed, the multiple spots of these enzymes were generated from the proteolytic cleavage by various proteases after the protein synthesis (26). The multiple spots of toxins may be generated in a similar way by the action of own proteases, although we have had no evidence yet.…”

“…IPG gels were equilibrated with equilibration buffers A and B according to the manufacturer's instructions (26) and the second-dimensional separation was carried out using 9 16% linear gradient polyacrylamide gels (100 160 10 mm) (30). IPG gels were embedded on top of the gels with 0.5% w/v agarose, and electrophoresis was performed.…”

Type I Signal Peptidase and Protein Secretion in Staphylococcus aureus

“…There has been considerable effort directed toward defining the S. aureus secretome (9,14,17,36,37,54,55,71,79,99,(117)(118)(119). Some of the earliest efforts were computational approaches that relied on identifying conserved features of Sec-type N-terminal signal sequences.…”

An Outbreak of Neonatal Toxic Shock Syndrome‐Like Exanthematous Disease (NTED) Caused by Methicillin‐Resistant Staphylococcus aureus (MRSA) in a Neonatal Intensive Care Unit

“…Bacteria were incubated in 20 ml of LuriaBertani (LB) broth for 15 16 hr at 37 C with rotary shaking at 150 rpm, and the culture supernatant was obtained by centrifugation at 8,000 g for 15 min to precipitate bacterial cells. Total exoproteins were precipitated from the culture supernatant with 10% trichloroacetic acid (TCA) as previously described (8,18). Precipitated exoproteins were dissolved in 500 µl of immobilized pH gradient (IPG) rehydration solution (7 M urea, 2 M thiourea, 2% w/v 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 0.6% w/v dithiothreitol (DTT), 0.5% v/v IPG buffer, and orange G as a tracer, and the sample solution was then adjusted to a volume of 1 ml with double distilled water.…”