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

Abstract

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...

VfrB Is a Key Activator of the Staphylococcus aureus SaeRS Two-Component System

“…S1 in the supplemental material). As described previously, coa transcriptional activity was maximal during the exponential growth phase (35). Similarly, hla promoter activity was maximal during the stationary growth phase, as documented previously (8,18,19), demonstrating that both reporter constructs accurately reflected previously published studies of these promoters.…”

Transcription of Staphylococcus aureus fibronectin binding protein genes is negatively regulated by agr and an agr-independent mechanism

“…Analysis of twofold serial dilutions of total RNA from both strains revealed that the agr mutant produced at least 16 times as much fnb mRNA as the wild-type strain (data not shown). Unlike that of other negatively regulated genes (i.e., spa and coa) which are constitutively expressed in agr mutants (23,40), transcription of fnb was restricted to the first hour of growth (lag phase and first cell division).…”

Staphylococcus aureus-induced clotting of plasma is an immune evasion mechanism for persistence within the fibrin network

“…The regulation of S. aureus-induced coagulation and fibrinolysis is a wellorchestrated series of events that should be beneficial for the bacterium. Timing is an essential aspect in these processes and it is worth noting that coagulase is produced during the early growth phase (Lebeau et al, 1994), whilst staphylokinase is produced in the late exponential phase of the S. aureus growth cycle (Bokarewa et al, 2006). It seems therefore plausible that the early release of coagulase will enable the bacteria to evade an innate immune response by promoting their uptake into the fibrin network, whilst staphylokinase released at a later time point will allow the bacteria to escape from the clot and cause systemic complications.…”