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.     

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.     

Significance of Staphylococcus epidermidis in the Clinical Laboratory

Susceptibility to 11 antibiotics was determined for 63 cultures of Staphylococcus aureus and 63 cultures of Staphylococcus epidermidis obtained at random from the clinical laboratory. The incidence of resistance to nine of these antibiotics was greater for S. epidermidis than for S. aureus. Studies of the minimal inhibitory concentration of these cultures to clindamycin showed that 61 cultures of S. aureus were susceptible whereas only 46 cultures of S. epidermidis were susceptible to this antibiotic. Although cultures of S. aureus were more active in the production of seven virulence factors, some cultures of S. epidermidis produced virulence factors. By successive cultivation in increasing concentrations of clindamycin, resistant variants were obtained for 10 cultures of S. aureus and 3 cultures of S. epidermidis. The presence of subinhibitory concentrations of clindamycin inhibited the production of some virulence factors by the resistant variants. In view of the greater resistance of S. epidermidis to antibiotics and its ability to produce virulence factors, its isolation in the clinical laboratory should not be regarded lightly.     

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.     

Agr-mediated dispersal of Staphylococcus aureus biofilms

The agr quorum-sensing system of Staphylococcus aureus modulates the expression of virulence factors in response to autoinducing peptides (AIPs). Recent studies have suggested a role for the agr system in S. aureus biofilm development, as agr mutants exhibit a high propensity to form biofilms, and cells dispersing from a biofilm have been observed displaying an active agr system. Here, we report that repression of agr is necessary to form a biofilm and that reactivation of agr in established biofilms through AIP addition or glucose depletion triggers detachment. Inhibitory AIP molecules did not induce detachment and an agr mutant was non-responsive, indicating a dependence on a functional, active agr system for dispersal. Biofilm detachment occurred in multiple S. aureus strains possessing divergent agr systems, suggesting it is a general S. aureus phenomenon. Importantly, detachment also restored sensitivity of the dispersed cells to the antibiotic rifampicin. Proteinase K inhibited biofilm formation and dispersed established biofilms, suggesting agr-mediated detachment occurred in an ica-independent manner. Consistent with a protease-mediated mechanism, increased levels of serine proteases were detected in detaching biofilm effluents, and the serine protease inhibitor PMSF reduced the degree of agr-mediated detachment. Through genetic analysis, a double mutant in the agr-regulated Aur metalloprotease and the SplABCDEF serine proteases displayed minimal extracellular protease activity, improved biofilm formation, and a strongly attenuated detachment phenotype. These findings indicate that induction of the agr system in established S. aureus biofilms detaches cells and demonstrate that the dispersal mechanism requires extracellular protease activity.     

Genome sequence of Staphylococcus lugdunensis N920143 allows identification of putative colonization and virulence factors

Staphylococcus lugdunensis is an opportunistic pathogen related to Staphylococcus aureus and Staphylococcus epidermidis. The genome sequence of S. lugdunensis strain N920143 has been compared with other staphylococci, and genes were identified that could promote survival of S. lugdunensis on human skin and pathogenesis of infections. Staphylococcus lugdunensis lacks virulence factors that characterize S. aureus and harbours a smaller number of genes encoding surface proteins. It is the only staphylococcal species other than S. aureus that possesses a locus encoding iron-regulated surface determinant (Isd) proteins involved in iron acquisition from haemoglobin.     

Characterization of RAP,a quorum sensing activator of Staphylococcus aureus

Staphylococcus aureus are Gram-positive bacteria and cause diverse serious diseases in humans and animals through the production of toxins. The production of toxins is regulated by quorum sensing mechanisms, where proteins such as RNAIII activating protein (RAP) are secreted by the bacteria and induce virulence. Antibodies to RAP have been shown to protect mice from infection, but the molecular structure of RAP was not known and hindered vaccine development. To characterize RAP, recombinant protein was made and tested for its ability to induce genes important for pathogenesis (agr). In addition, monoclonal antibodies were produced to identify its cellular localization. Results shown here indicate that RAP is a 277-aa protein that is an ortholog of the ribosomal protein L2. Like the native molecule, recombinant RAP activates the production of RNAIII (encoded by agr). Using RAP specific monoclonal antibodies we demonstrate that RAP is continuously secreted and while RAP is expressed also in other bacteria (like Staphylococcus epidermidis, Staphylococcus xylosus and Escherichia coli), it is secreted to the culture medium only by S. aureus. Our results show that the ribosomal protein L2 has an extraribosomal function and that when secreted RAP acts as an autoinducer of virulence to regulate S. aureus pathogenesis.     

Regulated expression of pathogen-associated molecular pattern molecules in Staphylococcus epidermidis: quorum-sensing determines pro-inflammatory capacity and production of phenol-soluble modulins

Phenol-soluble modulin (PSM) is a peptide complex produced by the nosocomial pathogen Staphylococcus epidermidis that has a strong capacity to activate the human innate immune response. We developed a novel method based on liquid chromatography-mass spectrometry (LC-MS) to quantify the production of the individual PSM components. Each PSM peptide was abundant in most of the 76 S epidermidis strains tested. Importantly, none of the PSM components were secreted by an agr mutant strain, indicating that PSM synthesis is regulated strictly by the agr quorum-sensing system. Furthermore, the agr mutant strain failed to elicit production of TNFalpha by human myeloid cells and induced significantly less neutrophil chemotaxis compared with the wild-type strain. Thus, quorum-sensing in S. epidermidis dramatically influenced activation of human host defence. We propose that an agr quorum-sensing mechanism facilitates growth and survival in infected hosts by adapting production of the pro-inflammatory PSMs to the stage of infection.     

High Genetic Variability of the agr Locus in Staphylococcus Species

The agr quorum-sensing and signal transduction system was initially described in Staphylococcus aureus, where four distinct allelic variants have been sequenced. Western blotting suggests the presence of homologous loci in many other staphylococci, and this has been confirmed for S. epidermidis and S. lugdunensis. In this study we isolated agr-like loci from a range of staphylococci by using PCR amplification from primers common to the six published agr sequences and bracketing the most variable region, associated with quorum-sensing specificity. Positive amplifications were obtained from 14 of 34 staphylococcal species or subspecies tested. Sequences of the amplicons identified 24 distinct variants which exhibited extensive sequence divergence with only 10% of the nucleotides absolutely conserved on multiple alignment. This variability involved all three open reading frames involved in quorum sensing and signal transduction. However, these variants retained several protein signatures, including the conserved cysteine residue of the autoinducing peptide, with the exception of S. intermedius of pigeon origin, which contained a serine in place of cysteine at this position. We discuss hypotheses on the mode of action and the molecular evolution of the agr locus based on comparisons between the newly determined sequences.     

Role of ClpP in biofilm formation and virulence of Staphylococcus epidermidis

Infections caused by the leading nosocomial pathogen Staphylococcus epidermidis are characterized by biofilm formation on implanted medical devices. However, the molecular basis of biofilm formation and its regulation are not completely understood. Here, we describe an important role of the ClpP protease in biofilm development and virulence of S. epidermidis. We constructed an isogenic clpP mutant strain of a biofilm-forming clinical isolate of S. epidermidis. The mutant strain showed decreased biofilm formation in vitro and reduced virulence in a rat model of biofilm-associated infection. Biofilm forming ability of the mutant strain could be restored by expressing clpP on a plasmid, but not when a catalytically inactive allele of clpP gene was introduced. These observations indicate that the peptidase function of ClpP determines its role in biofilm formation. Experimental data in this work also suggested that clpP influenced initial attachment of bacteria on the plastic surface, the first step of biofilm formation. Furthermore, clpP was found to be regulated by the quorum-sensing agr, suggesting that part of the previously described influence of agr on the initial attachment to plastic surfaces may be mediated by clpP.     

Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain

Staphylococcus aureus is an opportunistic pathogen and the major causative agent of numerous hospital- and community-acquired infections. Staphylococcus epidermidis has emerged as a causative agent of infections often associated with implanted medical devices. We have sequenced the approximately 2.8-Mb genome of S. aureus COL, an early methicillin-resistant isolate, and the approximately 2.6-Mb genome of S. epidermidis RP62a, a methicillin-resistant biofilm isolate. Comparative analysis of these and other staphylococcal genomes was used to explore the evolution of virulence and resistance between these two species. The S. aureus and S. epidermidis genomes are syntenic throughout their lengths and share a core set of 1,681 open reading frames. Genome islands in nonsyntenic regions are the primary source of variations in pathogenicity and resistance. Gene transfer between staphylococci and low-GC-content gram-positive bacteria appears to have shaped their virulence and resistance profiles. Integrated plasmids in S. epidermidis carry genes encoding resistance to cadmium and species-specific LPXTG surface proteins. A novel genome island encodes multiple phenol-soluble modulins, a potential S. epidermidis virulence factor. S. epidermidis contains the cap operon, encoding the polyglutamate capsule, a major virulence factor in Bacillus anthracis. Additional phenotypic differences are likely the result of single nucleotide polymorphisms, which are most numerous in cell envelope proteins. Overall differences in pathogenicity can be attributed to genome islands in S. aureus which encode enterotoxins, exotoxins, leukocidins, and leukotoxins not found in S. epidermidis.     

Structure, activity and evolution of the group I thiolactone peptide quorum-sensing system of Staphylococcus aureus

In Staphylococcus aureus, the agr locus is responsible for controlling virulence gene expression via quorum sensing. As the blockade of quorum sensing offers a novel strategy for attenuating infection, we sought to gain novel insights into the structure, activity and turnover of the secreted staphylococcal autoinducing peptide (AIP) signal molecules. A series of analogues (including the L-alanine and D-amino acid scanned peptides) was synthesized to determine the functionally critical residues within the S. aureus group I AIP. As a consequence, we established that (i) the group I AIP is inactivated in culture supernatants by the formation of the corresponding methionyl sulphoxide; and (ii) the group I AIP lactam analogue retains the capacity to activate agr, suggesting that covalent modification of the AgrC receptor is not a necessary prerequisite for agr activation. Although each of the D-amino acid scanned AIP analogues retained activity, replacement of the endocyclic amino acid residue (aspartate) located C-terminally to the central cysteine with alanine converted the group I AIP from an activator to a potent inhibitor. The screening of clinical S. aureus isolates for novel AIP groups revealed a variant that differed from the group I AIP by a single amino acid residue (aspartate to tyrosine) in the same position defined as critical by alanine scanning. Although this AIP inhibits group I S. aureus strains, the producer strains possess a functional agr locus dependent on the endogenous peptide and, as such, constitute a fourth S. aureus AIP pheromone group (group IV). The addition of exogenous synthetic AIPs to S. aureus inhibited the production of toxic shock syndrome toxin (TSST-1) and enterotoxin C3, confirming the potential of quorum-sensing blockade as a therapeutic strategy.