Biosynthesis of Staphylococcus aureus autoinducing peptides by using the synechocystis DnaB mini-intein

The Agr quorum-sensing system of Staphylococcus aureus modulates the expression of virulence factors in response to autoinducing peptides (AIPs). The peptides are seven to nine residues in length and have the C-terminal five residues constrained in a thiolactone ring. We have developed a new method to generate AIP structures using an engineered DnaB mini-intein from Synechocystis sp. strain PCC6803. In the method, an oligonucleotide encoding the AIP is ligated to the intein and the fusion protein is expressed and purified by affinity chromatography. To produce the correct AIP structure, intein splicing is interrupted, allowing the cysteine side chain to catalyze thiolactone ring formation and release AIP from the resin. The technique is simple and robust, and we have successfully produced the three main classes of AIPs using the intein system. The intein-generated AIPs possessed the correct thiolactone ring modification based on biochemical analysis, and, importantly, all the samples were bioactive against S. aureus. The AIP activity was confirmed through Agr interference and activation profiling with developed S. aureus reporter strains. The simplicity of the method, benefits of DNA encoding, and scalable nature enable the production of S. aureus AIPs for many biological applications.     

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.     

Staphylococcus intermedius produces a functional agr autoinducing peptide containing a cyclic lactone

The agr system is a global regulator of accessory functions in staphylococci, including genes encoding exoproteins involved in virulence. The agr locus contains a two-component signal transduction module that is activated by an autoinducing peptide (AIP) encoded within the agr locus and is conserved throughout the genus. The AIP has an unusual partially cyclic structure that is essential for function and that, in all but one case, involves an internal thiolactone bond between a conserved cysteine and the C-terminal carboxyl group. The exceptional case is a strain of Staphylococcus intermedius that has a serine in place of the conserved cysteine. We demonstrate here that the S. intermedius AIP is processed by the S. intermedius AgrB protein to generate a cyclic lactone, that it is an autoinducer as well as a cross-inhibitor, and that all of five other S. intermedius strains examined also produce serine-containing AIPs.     

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.     

Random mutagenesis and topology analysis of the autoinducing peptide biosynthesis proteins in Staphylococcus aureus

The Staphylococcus aureus accessory gene regulator (agr) is a peptide signalling system that regulates the production of secreted virulence factors required to cause infections. The signal controlling agr function is a 7‐9 residue thiolactone‐containing peptide called an autoinducing peptide (AIP) that is biosynthesized from the AgrD precursor by the membrane peptidase AgrB. To gain insight into AgrB and AgrD function, the agrBD genes were mutagenized and screened for deficiencies in AIP production. In total, single‐site mutations at 14 different residues in AgrD were identified and another 20 within AgrB. In AgrD, novel mutations were characterized in the N‐terminal leader and throughout the central region encoding the AIP signal. In AgrB, most mutations blocked peptidase activity, but mutations in the K129–K131 residues were defective in a later step in AIP biosynthesis, separating the peptidase function from thiolactone ring formation and AIP transport. With the identification of residues in AgrB essential for AgrD processing, we reevaluated the membrane topology and the new model predicts four transmembrane helices and a potential re‐entrant loop on the cytoplasmic face. Finally, co‐immunoprecipitation studies indicate that AgrB forms oligomeric structures within the membrane. These studies provide further insight into the unique structural and functional properties of AgrB.     

Transmembrane topology and histidine protein kinase activity of AgrC, the agr signal receptor in Staphylococcus aureus

The agr P2 operon in Staphylococcus aureus codes for the elements of a density-sensing cassette made up of a typical two-component signalling system and its corresponding inducer. It is postulated that the autoinducer, a post-translationally modified octapeptide generated from the AgrD peptide, interacts with a receptor protein, coded by agrC , to transmit a signal via AgrA regulating expression of staphylococcal virulence genes through expression of agr RNA III. We show by analysis of PhoA fusions that AgrC is a transmembrane protein, and confirm using Western blotting that a 46 kDa protein corresponding to AgrC is present in the bacterial membrane. This protein is autophosphorylated on a histidine residue only in response to supernatants from an agr⁺ strain, and can also respond to the purified native octapeptide. A recombinant fusion protein where most of the N-terminal region of AgrC is replaced by the Escherichia coli maltose-binding protein is also autophosphorylated in response to stimulation by agr⁺ supernatants or purified octapeptide. We conclude that AgrC is the sensor molecule of a typical two-component signal system in S. aureus , and that the ligand-binding site of AgrC is probably located in the third extracellular loop of the protein.     

Requirement of the agr locus for colony spreading of Staphylococcus aureus

The important human pathogen Staphylococcus aureus is known to spread on soft agar plates. Here, we show that colony spreading of S. aureus involves the agr quorum-sensing system. This finding can be related to the agr-dependent expression of biosurfactants, such as phenol-soluble modulins, suggesting a connection between spreading motility and virulence.     

Impact of sar and agr on methicillin resistance in Staphylococcus aureus

Abstract The global regulators agr and sar control expression of cell wall and extracellular proteins. Inactivation of either sar and/or agr in a typical heterogeneously methicillin-resistant Staphylococcus aureus resulted in a small but reproducible decrease in the number of cells in the subpopulation expressing high methicillin resistance. The amount of low affinity penicillin-binding protein PBP2', the prerequisite for methicillin resistance, was apparently not affected, however, a reduction in PBP1 and PBP3 production was observed, suggesting that these resident PBPs of the cells might be involved somehow together with PBP2' in high level methicillin resistance.     

Exfoliatin-producing strains define a fourth agr specificity group in Staphylococcus aureus

The staphylococcal virulon is activated by the density-sensing agr system, which is autoinduced by a short peptide (autoinducing peptide [AIP]) processed from a propeptide encoded by agrD. A central segment of the agr locus, consisting of the C-terminal two-thirds of AgrB (the putative processing enzyme), AgrD, and the N-terminal half of AgrC (the receptor), shows striking interstrain variation. This finding has led to the division of Staphylococcus aureus isolates into three different agr specificity groups and to the division of non-aureus staphylococci into a number of others. The AIPs cross-inhibit the agr responses between groups. We have previously shown that most menstrual toxic shock strains belong to agr specificity group III but that no strong clinical identity has been associated with strains of the other two groups. In the present report, we demonstrate a fourth agr specificity group among S. aureus strains and show that most exfoliatin-producing strains belong to this group. A striking common feature of group IV strains is activation of the agr response early in exponential phase, at least 2 h earlier than in strains of the other groups. This finding raises the question of the biological significance of the agr autoinduction threshold.     

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.     

Staphylococcus aureus determinants for nasal colonization

Approximately 20% of the healthy human population is persistently colonized in the nasal cavity with Staphylococcus aureus, which constitutes a major risk for infection. S. aureus seems to predominantly colonize the anterior part of the nasal cavity by adhering to nasal surface structures and escaping the host innate and adaptive immune responses. Several bacterial and host factors that play a role in these processes have been identified in the past few years and were in part functionally evaluated in appropriate colonization models. However, the dynamics of host-pathogen crosstalk is only partially understood.     

Evolutionary genetics of the accessory gene regulator (agr) locus in Staphylococcus aureus

The accessory gene regulator (agr) locus influences the expression of many virulence genes in the human pathogen Staphylococcus aureus. Four allelic groups of agr, which generally inhibit the regulatory activity of each other, have been identified within the species. Interference in virulence gene expression caused by different agr groups has been suggested to be a mechanism for isolating bacterial populations and a fundamental basis for subdividing the species. To test the hypothesis that the species is phylogenetically structured according to agr groups, we mapped agr groups onto a clone phylogeny inferred from partial sequences of 14 genes from 27 genetically diverse strains. Shimodaira-Hasegawa and parametric bootstrap tests rejected the hypotheses that the species is subdivided into three or five monophyletic agr groups but failed to reject the hypothesis that the species is subdivided into two groups that each consist of multiple clonal complexes and multiple agr groups. Additional evidence for agr recombination is found from clustered polymorphisms in complete agr sequences. However, agr recombination has not occurred frequently or randomly through time, because the topology and branch lengths of the clone phylogeny are reflected within each agr group. To account for these observations, we propose a new evolutionary model that involves a genetically polymorphic ancestral population of S. aureus that horizontally transferred agr groups between two subspecies groups near the time that these subspecies groups diverged.     

Transposition of penicillinase determinants in methicillin-resistant Staphylococcus aureus

Abstract A methicillin-resistant Staphylococcus aureus (MRSA) typical of those being isolated in Australian hospitals has been studied. It contains two plasmids, one of 1.4 megadalton (MDa) and one of 18 MDa. When selection is made for resistance to nucleic acid-binding (NAB) compounds in mixed-culture transfer, two types of transcipients are obtained; those containing an 18-MDa plasmid and resistant to NAB compounds, trimethoprim and aminoglycosides such as gentamicin and kanamycin and those having a 22 MDa plasmid and the additional phenotype of penicillinase production. The penicillinase determinants on the 22-MDa plasmid have been found to transpose to the chromosome and from the chromosome to an 18-MDa plasmid similar to that found in the original isolate. Restriction enzyme analysis has shown that a 7.3-kilobase pair (kb) element is involved. This has been designated Tn 3852 .