A role for type I signal peptidase in Staphylococcus aureus quorum sensing

The Staphylococcus aureus Agr quorum-sensing system modulates the expression of extracellular virulence factors. The Agr system is controlled by an autoinducing peptide (AIP) molecule that is secreted during growth. In the AIP biosynthetic pathway, two proteolytic events are required to remove the leader and tail segments of AgrD, the peptide precursor of AIP. The only protein known to be involved in this pathway is AgrB, a membrane endopeptidase that removes the AgrD carboxy-tail. We designed a synthetic peptide substrate and developed an assay to detect peptidases that can remove the N-terminal leader of AIP. Several peptidase activities were detected in S. aureus extracts and these activities were present in both wild-type and agr mutant strains. Only one of these peptidases cleaved in the correct position and all properties of this enzyme were consistent with type I signal peptidase. Subsequent cloning and purification of the two known S. aureus signal peptidases, SpsA and SpsB, demonstrated that only SpsB catalysed this activity in vitro. To investigate the role of SpsB in AIP biosynthesis, SpsB peptide inhibitors were designed and characterized. The most effective inhibitor blocked SpsB activity in vitro and showed antibacterial activity against S. aureus. Importantly, the inhibitor reduced expression of an Agr-dependent reporter and inhibited AIP production in S. aureus, indicating a role for SpsB in quorum sensing.     

Differential recognition of Staphylococcus aureus quorum-sensing signals depends on both extracellular loops 1 and 2 of the transmembrane sensor AgrC

Virulence in Staphylococcus aureus is regulated via agr-dependent quorum sensing in which an autoinducing peptide (AIP) activates AgrC, a histidine protein kinase. AIPs are usually thiolactones containing seven to nine amino acid residues in which the thiol of the central cysteine is linked to the α-carboxyl of the C-terminal amino acid residue. The staphylococcal agr locus has diverged such that the AIPs of the four different S. aureus agr groups self-activate but cross-inhibit. Consequently, although the agr system is conserved among the staphylococci, it has undergone significant evolutionary divergence whereby to retain functionality, any changes in the AIP-encoding gene (agrD) that modifies AIP structure must be accompanied by corresponding changes in the AgrC receptor. Since AIP-1 and AIP-4 only differ by a single amino acid, we compared the transmembrane topology of AgrC1 and AgrC4 to identify amino acid residues involved in AIP recognition. As only two of the three predicted extracellular loops exhibited amino acid differences, site-specific mutagenesis was used to exchange the key AgrC1 and AgrC4 amino acid residues in each loop either singly or in combination. A novel lux-based agrP3 reporter gene fusion was constructed to evaluate the response of the mutated AgrC receptors. The data obtained revealed that while differential recognition of AIP-1 and AIP-4 depends primarily on three amino acid residues in loop 2, loop 1 is essential for receptor activation by the cognate AIP. Furthermore, a single mutation in the AgrC1 loop 2 resulted in conversion of (Ala5)AIP-1 from a potent antagonist to an activator, essentially resulting in the forced evolution of a new AIP group. Taken together, our data indicate that loop 2 constitutes the predicted hydrophobic pocket that binds the AIP thiolactone ring while the exocyclic amino acid tail interacts with loop 1 to facilitate receptor activation.     

Cross-Strain Quorum Sensing Inhibition by Staphylococcus aureus. Part 1: A Spatially Homogeneous Model

Staphylococcus aureus uses the agr quorum sensing (QS) system to regulate reciprocally colonisation and virulence factor production. S. aureus strains can be divided into four agr groups: those within a specific agr group activate the QS systems of strains belonging to the same group, while inhibiting agr expression in strains of other groups. Furthermore, agr homologues exist in many more species of gram-positive bacteria, raising the likelihood of cross-species interference. In principle, a non-pathogenic strain of S. aureus or other species of bacteria employing agr could be engineered to inhibit the QS systems of pathogenic strains using agr, thus down-regulating their production of virulence factors. We present three models of the agr operon belonging to strains competing for dominance, each comprising one of the three possible phosphorylation cascades governing the two component system (TCS) of the agr system. Bifurcation analyses clarify the aspects of QS most crucial in determining the efficacy of using a non-pathogenic strain for therapeutic purposes if the target TCS cascade is known and illustrate the qualitative and quantitative differences which occur as a result of mechanistic differences between the models. We highlight those results that, in concert with appropriate experimental data, would be most useful in ascertaining whether or not a classical TCS is in operation in a particular strain if this information is unknown.     

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.     

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.     

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.     

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.     

Occurrence of free D-amino acids and aspartate racemases in hyperthermophilic archaea.

The occurrence of free D-amino acids and aspartate racemases in several hyperthermophilic archaea was investigated. Aspartic acid in all the hyperthermophilic archaea was highly racemized. The ratio of D-aspartic acid to total aspartic acid was in the range of 43.0 to 49.1%. The crude extracts of the hyperthermophiles exhibited aspartate racemase activity at 70 degrees C, and aspartate racemase homologous genes in them were identified by PCR. D-Enantiomers of other amino acids (alanine, leucine, phenylalanine, and lysine) in Thermococcus strains were also detected. Some of them might be by-products of aspartate racemase. It is proven that D-amino acids are produced in some hyperthermophilic archaea, although their function is unknown.     

Distribution of two aminotransferases and D-amino acid oxidase within the nephron of young and adult rats.

In the adult rat kidney, alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC 2.6.1.1) and D-amino acid oxidase (EC 1.4.3.3) were measured in glomeruli, 4 parts of the proximal tubule, 2 parts of the distal tubule and in patches from the thin limb area and the papilla. These enzymes were measured in more limited parts of the nephron during postnatal development. Adult aspartate aminotransferase activities (percentage of the highest) ranged from 100 in the distal straight segment to 25 in the late part of the proximal straight segment to 10 in the thin limb and papillary area. Alanine aminotransferase (lower by a factor of 100 in absolute terms) was distributed as the mirror image of aspartate aminotransferase within proximal and distal tubules. D-Amino acid oxidase was 850-fold higher in proximal straight segments than in medullary structures. During development alanine aminotransferase increased 6-fold and D-amino acid oxidase, 4.5-fold in proximal straight tubules but aspartate aminotransferase increased in distal straight tubles 8-fold.     

Hemoglobin promotes Staphylococcus aureus nasal colonization

Staphylococcus aureus nasal colonization is an important risk factor for community and nosocomial infection. Despite the importance of S. aureus to human health, molecular mechanisms and host factors influencing nasal colonization are not well understood. To identify host factors contributing to nasal colonization, we collected human nasal secretions and analyzed their ability to promote S. aureus surface colonization. Some individuals produced secretions possessing the ability to significantly promote S. aureus surface colonization. Nasal secretions pretreated with protease no longer promoted S. aureus surface colonization, suggesting the involvement of protein factors. The major protein components of secretions were identified and subsequent analysis revealed that hemoglobin possessed the ability to promote S. aureus surface colonization. Immunoprecipitation of hemoglobin from nasal secretions resulted in reduced S. aureus surface colonization. Furthermore, exogenously added hemoglobin significantly decreased the inoculum necessary for nasal colonization in a rodent model. Finally, we found that hemoglobin prevented expression of the agr quorum sensing system and that aberrant constitutive expression of the agr effector molecule, RNAIII, resulted in reduced nasal colonization of S. aureus. Collectively our results suggest that the presence of hemoglobin in nasal secretions contributes to S. aureus nasal colonization.     

葡萄球菌毒力调控中的群体感应系统

葡萄球菌细胞密度依赖性的多基因表达调控(群体感应)系统,是通过自身诱导与信号转导途径使其感知环境信息,调节多种毒力因子的表达。这些毒力因子的表达受agr、sae以及arl等多种基因表达系统的紧密调控,同时也受Sar家族蛋白的调节。此外,葡萄球菌毒力及抗性密切相关的生物膜形成与发育,也受群体感应系统的影响。对群体感应系统的自身诱导作用的干扰,原则上可成为寻找新型抗菌药物较适合的途径。     

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.     

Fibrinogen depletion attenuates Staphyloccocus aureus infection by preventing density-dependent virulence gene up-regulation

Staphylococcus aureus undergoes a density-dependent conversion in phenotype from tissue-adhering to tissue-damaging and phagocyte-evading that is mediated in part by the quorum-sensing operon, agr, and its effector, RNAIII. Contributions of host factors to this mechanism for regulating virulence have not been studied. We hypothesized that fibrinogen, as a component of the inflammatory response, could create spatially constrained microenvironments around bacteria that increase density independently of bacterial numbers and thus potentiate quorum-sensing-dependent virulence gene expression. Here we show that transient fibrinogen depletion significantly reduces the bacterial burden and the consequential morbidity and mortality during experimental infection with wild-type S. aureus, but not with bacteria that lack expression of the quorum-sensing operon, agr. In addition, it inhibits in vivo activation of the promoter for the agr effector, RNAIII, and downstream targets of RNAIII, including alpha hemolysin and capsule production. Moreover, both in vitro and in vivo, the mechanism for promoting this phenotypic switch in virulence involves clumping of the bacteria, demonstrating that S. aureus responds to fibrinogen-mediated bacterial clumping by enhancing density-dependent virulence gene expression. These data demonstrate that down-modulation of specific inflammatory components of the host that augment bacterial quorum sensing can be a strategy for enhancing host defense against infection.     

Enantioselective synthesis of various D-amino acids by a multi-enzyme system

We have developed an effective method for the synthesis of various D-amino acids from the corresponding α-keto acids and ammonia by coupling four enzyme reactions catalyzed by D-amino acid aminotransferase, glutamate racemase, glutamate dehydrogenase, and formate dehydrogenase. In this system, D-glutamate is continuously regenerated from α-ketoglutarate, ammonia and NADH by the coupled reaction of glutamate dehydrogenase and glutamate racemase, and used as an amino donor for the enantioselective D-amino acid synthesis by the D-amino acid aminotransferase reaction. The unidirectional formate dehydrogenase reaction is also coupled to regenerate NADH consumed. Under the optimum conditions, D-enantiomers of valine, alanine, α-keto analogues with a molar yield higher than 80%.     

RNAIII inhibiting peptide (RIP) inhibits agr-regulated toxin production

Staphylococcal enterotoxins (SEs) are emetic toxins that cause food poisoning. SEs also function as powerful pyrogenic toxin superantigens that stimulate non-specific T-cell proliferation. Together with the hemolysins, SEs have been largely implicated as virulence factors in multiple infection models. Recent biochemical and genetic analyses have demonstrated that production of some of these toxins is partially regulated by quorum sensing mechanisms where proteins and peptides activate the accessory gene regulator (agr). Because toxin production is central to bacterial pathogenesis, therapeutic strategies alternative to antibiotics, and based on rational interference of the quorum sensing systems involved, are currently being developed. This approach would lead to repression of toxin production and, thus, to disease prevention. Here we provide evidence to conclude that synthetic analogs of the RNAIII inhibiting peptide (RIP) and antibodies to its target molecule TRAP function in vitro as efficient suppressors of agr-regulated exotoxin production by Staphylococcus aureus.     

Candidate targets of balancing selection in the genome of Staphylococcus aureus

Signatures of balancing selection can highlight polymorphisms and functions that are important to the long-term fitness of a species. We performed a first genome-wide scan for balancing selection in a bacterial species, Staphylococcus aureus, which is a common cause of serious antimicrobial-resistant infections of humans. Using a sliding window approach, the genomes of 16 strains of S. aureus, including 5 new genome sequences presented here, and 1 outgroup strain of S. epidermidis were scanned for signatures of balancing selection. A total of 195 short windows were investigated based on their extreme values of both Tajima's D (>2.03) and π/K ratios (>0.12) relative to the rest of the genome. To test the unusualness of these windows, an Approximate Bayesian Computation framework was used to select a null demographic model that better accounted for the observed data than did the standard neutral model. A total of 186 windows were demonstrated to be unusual under the null model and, thus, represented candidate loci under balancing selection. These 186 candidate windows were located within 99 candidate genes that were spread across 62 different loci. Nearly all the signal (97.2%) was located within coding sequences; balancing selection on gene regulation apparently occurs through the targeting of global regulators such as agr and gra/aps. The agr locus had some of the strongest signatures of balancing selection, which provides new insight into the causes of diversity at this locus. The list of candidate genes included multiple virulence-associated genes and was significantly enriched for functions in amino acid and inorganic ion transport and metabolism and in defense mechanisms against innate immunity and antimicrobials, highlighting these particular functions as important to the fitness of this pathogen.