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

Abstract

Staphylococcus aureus releases a large number of exoproteins such as toxins and enzymes, as extracellular and surface-associated forms, many of which have been shown to contribute to pathogenic activity or to enhance virulence (17,35,48,57). These exoproteins include (i) factors involved in invasion/tissue penetration, (ii) factors involved in evasion of host defenses and (iii) factors involved in attachment. The first group of exoproteins are alpha-toxin (14), beta-hemolysin (47), gammahemolysin (46), delta-hemolysin (20), and phospholipase C (9). The second group are toxic shock syndrome toxin-1 (TSST-1) (29), enterotoxins (3,4,8,15,22,52), protein A (34), and others (6,31,32,50,53), and the third group contains clumping factor (37) and others (7,12,23,45). More than 30 of these exoproteins that are significantly linked to pathogenesis have been purified to homogeneity, and the genes encoding these exoproteins have been cloned and sequenced (5,48 Abstract: We applied two-dimensional gel electrophoresis (2-DE) to the total exoproteins secreted from pathogenic MRSA strains and identified major protein spots by N-terminal amino acid sequence analysis. In approximately 300 to 500 spots visualized on each gel, various exoproteins and cell-associated proteins were identified and their sites on the gels confirmed for construction of a reference map. Major exotoxins such as enterotoxins SEA, SEB, and SEC3, toxic shock syndrome toxin-1 (TSST-1), and hemolysins were distributed in the region of pI 6.8 to 8.1 and MW 21 to 35 kDa. Although the differences between calculated and observed values of pI and MW were relatively small in each exoprotein, those of several proteins including alpha-hemolysin and SEB were considerably deviated from the positions of the expected values. Some exoproteins were detected as multiple spots. These included beta-hemolysin, enterotoxins SEA, SEB, and SEC3, glutamic acid-specific endopeptidase, glycerophosphoryl diester phosphodiesterase and triacylglycerol lipase. The multiple spots of these exoproteins may be generated by the action of own proteases. Certain similarities of 2-DE patterns among strains belonging to the same coagulase types were observed. On the basis of 2-DE image analysis, coagulase type II strains secreted somewhat larger amounts of SEB and SEC3 as well as TSST-1 than the strains belonging to other coagulase types. Taken together, 2-DE analysis of exoproteins is applicable to epidemiological studies for MRSA, as compared with pulsed field gel electrophoresis of restricted chromosomal DNA.

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

“…Strains A20, O-16 and M51 produced TSST-1 as 3.15, 4.09, and 2.92%, respectively, and produced SEC 3 as 21.69, 21.24, and 17.63%, respectively, of the total exoproteins. These values are approximately two-fold higher than those of MRSA strains frequently isolated in Japanese hospitals (18).…”

“…3.00 software (Amersham Pharmacia Biotech AB). Spots of TSST-1 and SEC 3 found in strains A20, O-16, M51 and O-21 were analyzed quantitatively based on the spot intensity as reported previously (18). For accuracy of quantification, we performed the 2-DE procedure 3 times for these strains.…”

“…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.…”

Growth-dependent release of carbohydrate metabolism-related and antioxidant enzymes from Staphylococcus aureus strain 6 as determined by proteomic analysis

“…org] (14). A more detailed database of S. aureus is now available with both genomic and proteomic information (2).…”

Rot Repression of Enterotoxin B Expression in Staphylococcus aureus

“…A variety of virulence factors are produced by this organism, including multiple exoproteins and cell wall-associated components (5,22,23). Coordinated regulation of expression of the many virulence genes is a critical feature of the pathogenicity of S. aureus, and the regulatory networks might provide sites of possible therapeutic intervention for the treatment of staphylococcal infections.…”