Formation of Extracellular Protein A by Staphylococcus aureus

Movitz, Jan

doi:10.1111/j.1432-1033.1976.tb10788.x

Cited by 77 publications

(50 citation statements)

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“…The detergent SDS appears to be removing all other antigens rather than exposing hidden antigens (as seems to be the case in exposure of streptococcal M protein by acid extraction ;Beck & BergnerRabinowitz, 1977) since all rabbits immunized with unextracted whole bacteria had formed antibodies to antigens A and B. The fact that these wall-associated antigens can also be recovered from cell-free culture filtrates resembles the behaviour of wall proteins of a number of other bacteria, including protein A of Staphylococcus aureus (Movitz, 1976), M protein of group A streptococci (Pinney & Widdowson, 1977) and the murein-lipoprotein of Escherichia coli (Braun, 1975;Russell, 19763). The release of wall proteins greatly simplified their purification, but it is important to remember that there may be slight differences between the wall-bound and free forms of the protein, and also that there may exist other wall-bound antigens which are not readily released into the culture medium.…”

Section: Discussionmentioning

confidence: 99%

Wall-associated Protein Antigens of Streptococcus mutans

Russell

1979

Journal of General Microbiology

231

203

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109When heat-killed whole organisms of Streptococcus mutans strain Ingbritt (serotype c ) were injected into rabbits, antibodies to at least 12 antigens were detectable by crossed immunoelectrophoresis. In contrast, when rabbits were immunized with organisms which had been subjected to extraction with the detergent sodium dodecyl sulphate (SDS), antibodies to only two protein antigens were found. These two proteins (A and B), while existing in a form apparently closely associated with peptidoglycan, could also be recovered from homogenates of whole organisms after sonication and from culture filtrates. Antigenic material was excreted throughout growth. SDS-polyacrylamide gradient gel electrophoresis showed A to have a molecular weight of 29000, while B had a molecular weight of 190000. Antigen B was purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. All of six strains of serotype c examined produced antigen B. Strains of serotypes e and f also produce antigenically identical proteins and strains of serotypes d and g produce proteins which cross-reacted with antigen B. Antigen B was specifically precipitated by rabbit antiserum to human heart tissue. I N T R O D U C T I O NExperiments from a number of laboratories have clearly shown that immunization of animals with Streptococcus mutans or its subcellular fractions can confer protection against dental caries induced by this organism (Bowen et al., 1976), but the mechanism of this protection and the bacterial antigens involved remain unknown. For the development of an effective vaccine it is desirable to identify the antigen(s) involved in eliciting protective antibody, and the means by which protection is conferred. Identification and purification of such an antigen would aid in the understanding of the mechanism of protection, allow monitoring of vaccine preparations for efficacy, and facilitate study of its relationship with any toxic components of a crude vaccine. Of particular concern in the case of S. mutans is the demonstration of an antigenic relationship between S. mutans and human heart tissue (van de Rijn et al., 1976).An earlier report from this laboratory described successful protection of monkeys immunized with walls of S. mutans (Bowen et al., 1975). Walls treated with trypsin, on the other hand, fail to confer protection suggesting that the protective antigen might be protein in nature (Colman & Cohen, 1979). The present paper describes studies of proteins associated with S. mutans walls and the purification of one wall-associated antigen. The purified antigen is also demonstrated to be antigenically related to human heart tissue. M E T H O D R. R. B. R U S S E L Lyeast extract, 3 g; NaCI, 2 g; K2HP04, 2 g; NaHCO,, 1 g; glucose, 5 g; water, 1 1) and the chemically defined (CD) medium of Janda & Kuramitsu (1976) supplemented with 0.2% (w/v) Na,CO, (Terleckyj & Shockman, 1975). Antisera. New Zealand white rabbits (each 2 to 3 kg) were used for the preparation of antisera. Injection schedule...

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Section: Discussionmentioning

confidence: 99%

Wall-associated Protein Antigens of Streptococcus mutans

Russell

1979

Journal of General Microbiology

231

203

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“…It, like the cell wall-associated protein A, is mainly synthesized during exponential growth (3,7). In contrast, other extracellular proteins and toxins (e.g., serine protease; nuclease; lipase; fibrinolysin; oa-, P-, and 8-hemolysin; toxic shock syndrome toxin 1; and enterotoxin B) are produced after the end of exponential growth (1).…”

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confidence: 99%

Coagulase expression in Staphylococcus aureus is positively and negatively modulated by an agr-dependent mechanism

Lebeau¹,

Vandenesch²,

Greenland³

et al. 1994

J Bacteriol

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Expression of staphylocoagulase by agr+ Staphylococcus aureus depends on the growth phase, being maximal during exponential growth and decreasing sharply postexponentially, while an agr-deleted strain continuously expresses an intermediate level of coagulase. Therefore, coagulase expression appears to be both positively and negatively modulated by an agr-dependent mechanism.Staphylocoagulase is an extracellular protein produced by Staphylococcus aureus which promotes fibrin formation in human plasma by nonenzymatic activation of prothrombin (14). It, like the cell wall-associated protein A, is mainly synthesized during exponential growth (3, 7). In contrast, other extracellular proteins and toxins (e.g., serine protease; nuclease; lipase; fibrinolysin; oa-, P-, and 8-hemolysin; toxic shock syndrome toxin 1; and enterotoxin B) are produced after the end of exponential growth (1). The expression of these products is controlled by a complex regulatory locus, agr (6, 9, 10), whose unique feature is control of the expression of its target genes by means of an RNA molecule (RNAIII) (8). agr RNAIII acts as a positive regulator on genes which are preferentially expressed postexponentially, whereas protein A and coagulase are inhibited. The kinetics of protein A gene (spa) transcription has been investigated with agr+ and agr mutant strains (12) and shown to be inversely related to the level of RNAIII. The proposed mechanism was that RNAIII had a strict inhibitory effect on spa expression. To our knowledge, no similar study of agr regulation of the coagulase gene (coa) has been conducted. Our results indicate that the regulation of coa expression is different from that of spa, in that coagulase expression may be modulated both positively and negatively by the agr system according to the growth stage.Kinetics of coagulase activity. The coagulase activity of strain RN6390 (9), an agr+ derivative of S. aureus 8325-4, was compared with that of RN6911, which was constructed by allelic replacement of the agr locus in RN6390 (8). Brain heart infusion broth (50 ml) in 500-ml Erlenmeyer flasks was inoculated with 10 colonies of overnight cultures on blood agar plates and incubated at 37°C with agitation at 190 rpm. Growth was monitored spectrophotometrically. Titration of coagulase activity was performed hourly for supernatants as described by McDevitt et al. (5) The two strains had similar latency times and growth rates under the cultivation conditions used in this study (Fig. 1). The agr+ strain RN6390 displayed a maximum clotting activity of 18.6 after 2 to 3 h of growth (Fig. 1A), which decreased rapidly to become undetectable after 7 h. The agr mutant strain RN6911 had a relatively constant coagulase activity of 8 (8.57 ± 1.68) over the same period (Fig. 1B).Kinetics of coa mRNA. The kinetics of coa mRNA expression was studied in the two strains by Northern (RNA) blot hybridization with a coa-specific RNA probe synthesized by in vitro transcription of pLUG12 (13) described previously (12). Hybridization and detection of l...

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“…Of the total amount of protein A produced by this strain, approximately 8% is extracellular (20). In exponentially growing bacteria, protein A is incorporated into the cell wall or released into the culture medium directly after synthesis on the ribosomes (19,20).…”

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confidence: 99%

“…Most strains of Staphylococcus aureus produce cell-bound as well as extracellular protein A (6,20). Strain Cowan I is frequently used as a potent protein A producer because of its high content of the protein.…”

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confidence: 99%