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.     

Symmetric signalling within asymmetric dimers of the Staphylococcus aureus receptor histidine kinase AgrC

Virulence in Staphylococcus aureus is largely under control of the accessory gene regulator ( agr ) quorum-sensing system. The AgrC receptor histidine kinase detects its autoinducing peptide (AIP) ligand and generates an intracellular signal resulting in secretion of virulence factors. Although agr is a well-studied quorum-sensing system, little is known about the mechanism of AgrC activation. By co-immunoprecipitation analysis and intermolecular complementation of receptor mutants, we showed that AgrC forms ligand-independent dimers that undergo trans- autophosphorylation upon interaction with AIP. Remarkably, addition of specific AIPs to AgrC mutant dimers with only one functional sensor domain caused symmetric activation of either kinase domain despite the sensor asymmetry. Furthermore, mutant dimers involving one constitutive protomer demonstrated ligand-independent activity, irrespective of which protomer was kinase deficient. These results demonstrate that signalling through either individual AgrC protomer causes symmetric activation of both kinase domains. We suggest that such signalling across the dimer interface may be an important mechanism for dimeric quorum-sensing receptors to rapidly elicit a response upon signal detection.     

金黄色葡萄球菌agr系统受体蛋白AgrC的跨膜结构分析析

金黄色葡萄球菌agr 系统中,受体组氨酸蛋白激酶AgrC 能够被同源或非同源自诱导信号分子(autoinducing peptides,AIPs) 激活或抑制。通过TMHMM软件对四种agr 型AgrC蛋白的氨基酸序列进行分析及跨膜结构预测,发现agr玉型和agr郁型AgrC蛋白可能在胞外的第二个Loop 环与其AIP相互作用,agr域型可能与AIP域在胞外的第一个或第二个Loop 环相互作用。     

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.     

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.     

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.     

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.     

Reversible and specific extracellular antagonism of receptor-histidine kinase signaling.

Staphylococcal pathogenesis is regulated by a two-component quorum-sensing system, agr, activated by a self-coded autoinducing peptide (AIP). The agr system is widely divergent and is unique in that variant AIPs cross-inhibit agr activation in heterologous combinations. Cross-inhibition, but not self-activation, is widely tolerant of structural diversity in the AIPs so that these two processes must involve different mechanisms of interaction with the respective receptors. Herein, we have utilized this naturally occurring antagonism to demonstrate that both activation and inhibition are reversible and that activators and inhibitors interact at a common site on the receptor. These results suggest that molecules designed to compete with natural agonists for binding at receptor-histidine kinase sensor domains could represent a general approach to the inhibition of receptor-histidine kinase signaling.     

Ligand-receptor recognition for activation of quorum sensing in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The accessory gene regulator (agr) locus controls many of the virulence toxins involved in Staphylococcus aureus pathogenesis, and can be divided into four specificity groups. AgrC is the only group-specific receptor to mediate both intra-group activation and inter-group inhibition. We studied the ligand-receptor recognition of the agr system in depth by using a luciferase reporter system to identify the key residues responsible for AgrC activation in two closely related agr groups, AgrC-I, and AgrC-IV. Fusion PCR and site-directed mutagenesis were used to screen for functional residues of AgrC. Our data suggest that for AgrC-IV activation, residue 101 is critical for activating the receptor. In contrast, the key residues for the activation of AgrC-I are located at residues 49∼59, 107, and 116. However, three residue changes, T101A, V107S, I116S, are sufficient to convert the AIP recognizing specificity from AgrC-IV to AgrC-I.     

Structural basis for ligand recognition and discrimination of a quorum-quenching antibody

In the postantibiotic era, available treatment options for severe bacterial infections caused by methicillin-resistant Staphylococcus aureus have become limited. Therefore, new and innovative approaches are needed to combat such life-threatening infections. Virulence factor expression in S. aureus is regulated in a cell density-dependent manner using “quorum sensing,” which involves generation and secretion of autoinducing peptides (AIPs) into the surrounding environment to activate a bacterial sensor kinase at a particular threshold concentration. Mouse monoclonal antibody AP4-24H11 was shown previously to blunt quorum sensing-mediated changes in gene expression in vitro and protect mice from a lethal dose of S. aureus by sequestering the AIP signal. We have elucidated the crystal structure of the AP4-24H11 Fab in complex with AIP-4 at 2.5 Å resolution to determine its mechanism of ligand recognition. A key Glu^H95 provides much of the binding specificity through formation of hydrogen bonds with each of the four amide nitrogens in the AIP-4 macrocyclic ring. Importantly, these structural data give clues as to the interactions between the cognate staphylococcal AIP receptors AgrC and the AIPs, as AP4-24H11·AIP-4 binding recapitulates features that have been proposed for AgrC-AIP recognition. Additionally, these structural insights may enable the engineering of AIP cross-reactive antibodies or quorum quenching vaccines for use in active or passive immunotherapy for prevention or treatment of S. aureus infections.     

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.     

New insight into transmembrane topology of Staphylococcus aureus histidine kinase AgrC

Staphylococcus aureus accessory gene regulator (agr) locus controls the expression of virulence factors through a classical two-component signal transduction system that consists of a receptor histidine protein kinase AgrC and a cytoplasmic response regulator AgrA. An autoinducing peptide (AIP) encoded by agr locus activates AgrC, which transduces extracellular signals into the cytoplasm. Despite extensive investigations to identify AgrC–AIP interaction sites, precise signal recognition mechanisms remain unknown. This study aims to clarify the membrane topology of AgrC by applying the green fluorescent protein (GFP) fusion technique and the substituted cysteine accessibility method (SCAM). However, our findings were inconsistent with profile obtained previously by alkaline phosphatase. We report the topology of AgrC shows seven transmembrane segments, a periplasmic N-terminus, and a cytoplasmic C-terminus.     

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.     

Side-chain-to-tail thiolactone peptide inhibitors of the staphylococcal quorum-sensing system

The expression of many staphylococcal virulence factors are regulated by the agr locus via a two-component signal transduction system (TCSTS), which is activated in response to a secreted autoinducer peptide (AIP). By exploiting the unique chemical architecture of the naturally occurring AIP-1, several potent inhibitors of staphylococcal TCSTS were designed and synthesized using either a linear or branched solid-phase approach. These inhibitors are competitive binders and contain the crucial 16-membered side-chain-to-tail thiolactone peptide pharmacophore.     

Regulation of Staphylococcus aureus pathogenesis via target of RNAIII-activating Protein (TRAP)

Staphylococcus aureus can cause disease through the production of toxins. Toxin production is autoinduced by the protein RNAIII-activating protein (RAP) and by the autoinducing peptide (AIP), and is inhibited by RNAIII-inhibiting peptide (RIP) and by inhibitory AIPs. RAP has been shown to be a useful vaccine target site, and RIP and inhibitory AIPs as therapeutic molecules to prevent and suppress S. aureus infections. Development of therapeutic strategies based on these molecules has been hindered by a lack of knowledge of the molecular mechanisms by which they activate or inhibit virulence. Here, we show that RAP specifically induces the phosphorylation of a novel 21-kDa protein, whereas RIP inhibits its phosphorylation. This protein was termed target of RAP (TRAP). The synthesis of the virulence regulatory molecule, RNAIII, is not activated by RAP in the trap mutant strain, suggesting that RAP activates RNAIII synthesis via TRAP. Phosphoamino acid analysis shows that TRAP is histidine-phosphorylated, suggesting that TRAP may be a sensor of RAP. AIPs up-regulate the synthesis of RNAIII also in trap mutant strains, suggesting that TRAP and AIPs activate RNAIII synthesis via distinct signal transduction pathways. Furthermore, TRAP phosphorylation is down-regulated in the presence of AIP, suggesting that a network of signal transduction pathways regulate S. aureus pathogenesis.     

An equilibrium model of the metastable binding sites of alpha 2-macroglobulin and complement proteins C3 and C4

Two cyclic structures, the 15-membered thiolactone A and the 5-membered lactam P, have been proposed for the metastable binding sites of the serum proteins C3, C4, and alpha 2-macroglobulin. Neither structure alone adequately explains two unusual reactions of these sites, namely, covalent attachment to nucleophiles with liberation of a thiol group and spontaneous hydrolysis (autolysis) of an internal peptide bond. The metastable binding sites of these proteins were modeled with the 15-membered thiolactone 1 (Khan, S. A., and Erickson, B. W. (1981) J. Am. Chem. Soc. 103, 7374-7376) and the isomeric 5-membered lactam 2, which contains an internal pyroglutamyl (Glp) residue. Under physiologic conditions (phosphate-buffered saline, pH 7.3, 37 degrees C), thiolactone 1 and lactam 2 exist in dynamic equilibrium. Since the molar ratio of 2/1 is 11:1 at equilibrium, lactam 2 is 15 kcal/mol more stable than thiolactone 1. The activation energy for isomerization of 1 into 2 is 18 kcal/mol, which is about 5 kcal/mol lower than that for hydrolysis of the acyclic thiolester N,S-diacetyl-L-cysteine methylamide. Part of the chemistry of the metastable binding sites can be explained by an analogous equilibrium between protein structures A and B. Lactam B may be a key intermediate in the biosynthesis of thiolactone A. Under denaturating conditions, thiolactone A could either bind covalently to a receptive surface or isomerize into lactam B, which could undergo spontaneous hydrolysis of the Glu-Glp peptide bond.     

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.