Inhibition of virulence factor expression in Staphylococcus aureus by the Staphylococcus epidermidis agr pheromone and derivatives

The agr quorum-sensing system in Staphylococci controls the production of surface proteins and exoproteins. In the pathogenic species Staphylococcus aureus, these proteins include many virulence factors. The extracellular signal of the quorum-sensing system is a thiolactone-containing peptide pheromone, whose sequence varies among the different staphylococcal strains. We demonstrate that a synthetic Staphylococcus epidermidis pheromone is a competent inhibitor of the Staphylococcus aureus agr system. Derivatives of the pheromone, in which the N-terminus or the cyclic bond structure was changed, were synthesized and their biological activity was determined. The presence of a correct N-terminus and a thiolactone were absolute prerequisites for an agr-activating effect in S. epidermidis, whereas inhibition of the S. aureus agr system was less dependent on the original structure. Our results show that effective quorum-sensing blockers that suppress the expression of virulence factors in S. aureus can be designed based on the S. epidermidis pheromone.     

Staphylococcus aureus and Staphylococcus epidermidis peptide pheromones produced by the accessory gene regulator agr system

The accessory gene regulator (agr) system of staphylococci regulates the expression of virulence factors in response to cell density. The extracellular signaling molecule encoded by this system is a thiolactone-containing pheromone peptide whose primary sequence varies among staphylococcal strains. A post-translational modification of the peptide is believed to be carried out by an enzyme with a novel function, AgrB. Staphylococcal pheromones show cross-inhibiting properties: Pheromones of self and pheromones of non-self induce and suppress the agr response, respectively, and have therefore been proposed as novel anti-staphylococcal drugs. As inhibition of agr leads to diminished expression of toxins, but to increased expression of colonization factors and biofilm formation, their therapeutic potential remains yet to be evaluated in depth.     

trans-Complementation of a Staphylococcus aureus agr Mutant by Staphylococcus lugdunensis agr RNAIII

RNAIII from Staphylococcus lugdunensis (RNAIII-sl) in a Staphylococcus aureus agr mutant partially restored the Agr phenotype. A chimeric construct consisting of the 5′ end of RNAIII-sl and the 3′ end of RNAIII from S. aureus restored the Agr phenotype to a greater extent, suggesting the presence of independent regulatory domains.     

Transposase-dependent formation of circular IS256 derivatives in Staphylococcus epidermidis and Staphylococcus aureus

IS256 is a highly active insertion sequence (IS) element of multiresistant staphylococci and enterococci. Here we show that, in a Staphylococcus epidermidis clinical isolate, as well as in recombinant Staphylococcus aureus and Escherichia coli carrying a single IS256 insertion on a plasmid, IS256 excises as an extrachromosomal circular DNA molecule. First, circles were identified that contained a complete copy of IS256. In this case, the sequence connecting the left and right ends of IS256 was derived from flanking DNA sequences of the parental genetic locus. Second, circle junctions were detected in which one end of IS256 was truncated. Nucleotide sequencing of circle junctions revealed that (i) either end of IS256 can attack the opposite terminus and (ii) the circle junctions vary significantly in size. Upon deletion of the IS256 open reading frame at the 3' end and site-directed mutageneses of the putative DDE motif, circular IS256 molecules were no longer detectable, which implicates the IS256-encoded transposase protein with the circularization of the element.     

In vitro serial passage of Staphylococcus aureus: changes in physiology, virulence factor production, and agr nucleotide sequence

Recently, we observed that Staphylococcus aureus strains newly isolated from patients had twofold-higher aconitase activity than a strain passaged extensively in vitro, leading us to hypothesize that aconitase specific activity decreases over time during in vitro passage. To test this hypothesis, a strain recovered from a patient with toxic shock syndrome was serially passaged for 6 weeks, and the aconitase activity was measured. Aconitase specific activity decreased 38% (P < 0.001) by the sixth week in culture. During serial passage, S. aureus existed as a heterogeneous population with two colony types that had pronounced (wild type) or negligible zones of beta-hemolytic activity. The cell density-sensing accessory gene regulatory (agr) system regulates beta-hemolytic activity. Surprisingly, the percentage of colonies with a wild-type beta-hemolytic phenotype correlated strongly with aconitase specific activity (rho = 0.96), suggesting a common cause of the decreased aconitase specific activity and the variation in percentage of beta-hemolytic colonies. The loss of the beta-hemolytic phenotype also coincided with the occurrence of mutations in the agrC coding region or the intergenic region between agrC and agrA in the derivative strains. Our results demonstrate that in vitro growth is sufficient to result in mutations within the agr operon. Additionally, our results demonstrate that S. aureus undergoes significant phenotypic and genotypic changes during serial passage and suggest that vigilance should be used when extrapolating data obtained from the study of high-passage strains.     

The Staphylococcus aureus ArlRS two-component system regulates virulence factor expression through MgrA

The Gram-positive bacterium, Staphylococcus aureus, is a versatile pathogen that can sense and adapt to a wide variety of environments within the human host, in part through its 16 two-component regulatory systems. The ArlRS two-component system has been shown to affect many cellular processes in S. aureus, including autolysis, biofilm formation, capsule synthesis and virulence. Yet the molecular details of this regulation remained largely unknown. We used RNA sequencing to identify the ArlRS regulon, and found 70% overlap with that of the global regulator MgrA. These genes included cell wall-anchored adhesins (ebh, sdrD), polysaccharide and capsule synthesis genes, cell wall remodeling genes (lytN, ddh), the urease operon, genes involved in metal transport (feoA, mntH, sirA), anaerobic metabolism genes (adhE, pflA, nrdDG) and a large number of virulence factors (lukSF, lukAB, nuc, gehB, norB, chs, scn and esxA). We show that ArlR directly activates expression of mgrA and identify a probable ArlR-binding site (TTTTCTCAT-N₄-TTTTAATAA). A highly similar sequence is also found in the spx P2 promoter, which was recently shown to be regulated by ArlRS. We also demonstrate that ArlS has kinase activity toward ArlR in vitro, although it has slower kinetics than other similar histidine kinases.     

Mathematical modelling of the agr operon in Staphylococcus aureus

Staphylococcus aureus is a pathogenic bacterium that utilises quorum sensing (QS), a cell-to-cell signalling mechanism, to enhance its ability to cause disease. QS allows the bacteria to monitor their surroundings and the size of their population, and S. aureus makes use of this to regulate the production of virulence factors. Here we describe a mathematical model of this QS system and perform a detailed time-dependent asymptotic analysis in order to clarify the roles of the distinct interactions that make up the QS process, demonstrating which reactions dominate the behaviour of the system at various timepoints. We couple this analysis with numerical simulations and are thus able to gain insight into how a large population of S. aureus shifts from a relatively harmless state to a highly virulent one, focussing on the need for the three distinct phases which form the feedback loop of this particular QS system.     

Metabolic control of virulence factor production in Staphylococcus aureus

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The influence of agr and sigmaB in growth phase dependent regulation of virulence factors in Staphylococcus aureus

The expression of many virulence determinants in Staphylococcus aureus is tightly coordinated generally by global regulatory elements such as accessory gene regulator (agr), staphylococcal accessory regulator and the alternative sigma factor sigmaB. We have compared the two-dimensional (2-D) protein pattern of extracellular protein extracts of wild-type cells with the 2-D patterns of the respective regulatory mutants in order to identify proteins whose amount is influenced by a mutation in agr or sigB. In order to quantify changes in the level of interesting proteins we used the Ettan-fluorescence difference gel electrophoresis technique (Amersham Biosciences). As in most bacteria, the amount of extracellular proteins was strongly regulated and increased mainly in the stationary phase of growth at high cell densities. By comparing the extracellular protein pattern of the RN6390 rsbU strain with that of an isogenic agr mutant RN6911 we show that the level of about 70 protein spots changed in the mutant. To analyze the role of sigmaB in virulence gene expression an RsbU+ (RN6390 RsbU+) derivative was included in this study. The protein pattern of the RsbU+ strain (RN6390 RsbU+) was very similar to that of the Deltaagr/DeltarsbU mutant strain (RN6911) indicating an opposing effect of agr and rsbU on the expression of the same genes.     

RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus

Cell-density-dependent gene regulation by quorum-sensing systems has a crucial function in bacterial physiology and pathogenesis. We demonstrate here that the Staphylococcus aureus agr quorum-sensing regulon is divided into (1) control of metabolism and PSM cytolysin genes, which occurs independently of the small regulatory RNA RNAIII, and (2) RNAIII-dependent control of additional virulence genes. Remarkably, PSM expression was regulated by direct binding of the AgrA response regulator. Our findings suggest that quorum-sensing regulation of PSMs was established before wide-ranging control of virulence was added to the agr regulon, which likely occurred by development of the RNAIII-encoding region around the gene encoding the PSM delta-toxin. Moreover, the agr regulon in the community-associated methicillin-resistant S. aureus MW2 considerably differed from that previously determined using laboratory strains. By establishing a two-level model of quorum-sensing target gene regulation in S. aureus, our study gives important insight into the evolution of virulence control in this leading human pathogen.     

Staphylococcus aureus CodY negatively regulates virulence gene expression

CodY is a global regulatory protein that was first discovered in Bacillus subtilis, where it couples gene expression to changes in the pools of critical metabolites through its activation by GTP and branched-chain amino acids. Homologs of CodY can be found encoded in the genomes of nearly all low-G+C gram-positive bacteria, including Staphylococcus aureus. The introduction of a codY-null mutation into two S. aureus clinical isolates, SA564 and UAMS-1, through allelic replacement, resulted in the overexpression of several virulence genes. The mutant strains had higher levels of hemolytic activity toward rabbit erythrocytes in their culture fluid, produced more polysaccharide intercellular adhesin (PIA), and formed more robust biofilms than did their isogenic parent strains. These phenotypes were associated with derepressed levels of RNA for the hemolytic alpha-toxin (hla), the accessory gene regulator (agr) (RNAII and RNAIII/hld), and the operon responsible for the production of PIA (icaADBC). These data suggest that CodY represses, either directly or indirectly, the synthesis of a number of virulence factors of S. aureus.     

Subinhibitory concentrations of perilla oil affect the expression of secreted virulence factor genes in Staphylococcus aureus

Background

The pathogenicity of staphylococcus aureus is dependent largely upon its ability to secrete a number of virulence factors, therefore, anti-virulence strategy to combat S. aureus-mediated infections is now gaining great interest. It is widely recognized that some plant essential oils could affect the production of staphylococcal exotoxins when used at subinhibitory concentrations. Perilla [Perilla frutescens (L.) Britton], a natural medicine found in eastern Asia, is primarily used as both a medicinal and culinary herb. Its essential oil (perilla oil) has been previously demonstrated to be active against S. aureus. However, there are no data on the influence of perilla oil on the production of S. aureus exotoxins.

Methodology/Principal Findings

A broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of perilla oil against S. aureus strains. Hemolysis, tumour necrosis factor (TNF) release, Western blot, and real-time RT-PCR assays were performed to evaluate the effects of subinhibitory concentrations of perilla oil on exotoxins production in S. aureus. The data presented here show that perilla oil dose-dependently decreased the production of α-toxin, enterotoxins A and B (the major staphylococcal enterotoxins), and toxic shock syndrome toxin 1 (TSST-1) in both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA).

Conclusions/Significance

The production of α-toxin, SEA, SEB, and TSST-1 in S. aureus was decreased by perilla oil. These data suggest that perilla oil may be useful for the treatment of S. aureus infections when used in combination with β-lactam antibiotics, which can increase exotoxins production by S. aureus at subinhibitory concentrations. Furthermore, perilla oil could be rationally applied in food systems as a novel food preservative both to inhibit the growth of S. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins.     

Inhibition of Staphylococcus aureus cysteine proteases by human serpin potentially limits staphylococcal virulence

Bacterial proteases are considered virulence factors and it is presumed that by abrogating their activity, host endogenous protease inhibitors play a role in host defense against invading pathogens. Here we present data showing that Staphylococcus aureus cysteine proteases (staphopains) are efficiently inhibited by Squamous Cell Carcinoma Antigen 1 (SCCA1), an epithelial-derived serpin. The high association rate constant (k(ass)) for inhibitory complex formation (1.9×10(4) m/s and 5.8×10(4) m/s for staphopain A and staphopain B interaction with SCCA1, respectively), strongly suggests that SCCA1 can regulate staphopain activity in vivo at epithelial surfaces infected/colonized by S. aureus. The mechanism of staphopain inhibition by SCCA1 is apparently the same for serpin interaction with target serine proteases whereby the formation of a covalent complex result in cleavage of the inhibitory reactive site peptide bond and associated release of the C-terminal serpin fragment. Interestingly, the SCCA1 reactive site closely resembles a motif in the reactive site loop of native S. aureus-derived inhibitors of the staphopains (staphostatins). Given that S. aureus is a major pathogen of epithelial surfaces, we suggest that SCCA1 functions to temper the virulence of this bacterium by inhibiting the staphopains.     

纤溶酶原在金黄色葡萄球菌感染中的作用

金黄色葡萄球菌菌体表面有多种纤溶酶原受体,包括次黄嘌呤单核苷酸脱氢酶、核糖核苷酸还原酶、α-烯醇化酶和3-磷酸甘油醛脱氢酶等,它们均可以与纤溶酶原结合。与细菌结合的纤溶酶原可被宿主的纤溶酶原激活剂(组织型纤溶酶原激活剂和尿激酶型纤溶酶原激活剂)或葡萄菌属的纤溶酶原激活剂(葡激酶)激活为纤溶酶。细菌表面的纤溶酶有利于其降解宿主胞外基质,穿越组织屏障,因此哺乳动物的纤溶酶原可能在金黄色葡萄球菌感染宿主过程中起重要作用。