Agr Activation Research Articles

The role of male hormones in bacterial infections: enhancing Staphylococcus aureus virulence through testosterone-induced Agr activation.

Staphylococcus aureus is a notorious pathogen predominantly involved in skin and soft tissue infections, exhibiting a distinct innate sex bias. This study explores the influence of testosterone on the virulence of S. aureus and elucidates its underlying mechanisms. Utilizing a skin abscess model in intact and castrated male mice, we assessed the effects of testosterone on S. aureus pathogenicity. Compared to controls, castrated mice showed significantly reduced abscess sizes and decreased bacterial loads, highlighting the role of testosterone in modulating the severity of S. aureus infections. In vitro experiments revealed that testosterone enhances the hemolytic activity, cytotoxicity, and oxidative stress resistance of S. aureus. Real-time quantitative PCR analysis showed a significant upregulation of the genes encoding α-hemolysin (hla) and phenol-soluble modulin (psmα). Importantly, testosterone treatment significantly enhanced the expression of the accessory gene regulator (Agr) quorum-sensing system components (agrC, agrA, agrB, agrD), while the SaeRS system (saeR, saeS, and sbi) exhibited only slight changes. Gene knockout experiments revealed that deletion of agrC, rather than saeRS and agrBD, abolishes the testosterone-induced enhancement of hemolysis and gene expression, underscoring the key role of AgrC. Molecular docking simulations indicated a direct interaction between testosterone and AgrC protein, with a strong binding affinity at the active site residue SER201. This study provides new insights into the mechanistic basis of how testosterone enhances the pathogenicity of S. aureus, potentially contributing to increased male susceptibility to S. aureus infections and offering a targeted approach for therapeutic interventions.

Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H2O2, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr resulted in decreased ATP levels and growth, despite increased rates of respiration or fermentation at appropriate oxygen tensions, suggesting that Δagr cells undergo a shift towards a hyperactive metabolic state in response to diminished metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H2O2 doses. Increased survival of wild-type agr cells during H2O2 exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H2O2. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived ‘memory’ of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Cybb−/−) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.

Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress.

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H2O2, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr resulted in decreased ATP levels and growth, despite increased rates of respiration or fermentation at appropriate oxygen tensions, suggesting that Δagr cells undergo a shift towards a hyperactive metabolic state in response to diminished metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H2O2 doses. Increased survival of wild-type agr cells during H2O2 exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H2O2. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived 'memory' of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Cybb-/-) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.

Characterization of an Autoinducing Peptide Signal Reveals Highly Efficacious Synthetic Inhibitors and Activators of Quorum Sensing and Biofilm Formation in Listeria monocytogenes.

Bacteria can use chemical signals to assess their local population density in a process called quorum sensing (QS). Many of these bacteria are common pathogens, including Gram-positive bacteria that utilize agr QS systems regulated by macrocyclic autoinducing peptide (AIP) signals. Listeria monocytogenes, an important foodborne pathogen, uses an agr system to regulate a variety of virulence factors and biofilm formation, yet little is known about the specific roles of agr in Listeria infection and its persistence in various environments. Herein, we report synthetic peptide tools that will enable the study of QS in Listeria. We identified a 6-mer AIP signal in L. monocytogenes supernatants and selected it as a scaffold around which a collection of non-native AIP mimics was designed and synthesized. These peptides were evaluated in cell-based agr reporter assays to generate structure-activity relationships for AIP-based agonism and antagonism in L. monocytogenes. We discovered synthetic agonists with increased potency relative to native AIP and a synthetic antagonist capable of reducing agr activity to basal levels. Notably, the latter peptide was able to reduce biofilm formation by over 90%, a first for a synthetic QS modulator in wild-type L. monocytogenes. The lead agr agonist and antagonist in L. monocytogenes were also capable of antagonizing agr signaling in the related pathogen Staphylococcus aureus, further extending their utility and suggesting different mechanisms of agr activation in these two pathogens. This study represents an important first step in the application of chemical methods to modulate QS and concomitant virulence outcomes in L. monocytogenes.

Accessory Gene Regulator (agr) Allelic Variants in Cognate Staphylococcus aureus Strain Display Similar Phenotypes.

The accessory gene regulator (agr) quorum-sensing system is an important global regulatory system of Staphylococcus aureus and contributes to its pathogenicity. The S. aureus agr system is divided into four agr groups based on the amino acid polymorphisms of AgrB, AgrD, and AgrC. The agr activation is group-specific, resulting in variations in agr activity and pathogenicity among the four agr groups. Strains with divergent agr system always have different phenotypes. In the present report, we, respectively, exchanged the agr system of a certain S. aureus with other three agr alleles and assessed the corresponding phenotypes of these congenic strains. Replacement of the agr system led to significant variations in hemolytic activity, protein expression, and virulence gene expression comparing with that of the parental strain. Interestingly, we found that the biological characteristics of these agr congenic strains in the same strain background were highly similar to each other, and the allele-dependent differences of the agr systems were weakened. These findings indicate that the allele-dependent agr predilections of S. aureus are determined by some factors in addition to the polymorphisms of AgrB, AgrD, and AgrC. Future studies may reveal the novel mechanism to improve our understanding of the agr network.

Quorum Sensing and Toxin Production in Staphylococcus aureus Osteomyelitis: Pathogenesis and Paradox.

Staphylococcus aureus is a Gram-positive pathogen capable of infecting nearly every vertebrate organ. Among these tissues, invasive infection of bone (osteomyelitis) is particularly common and induces high morbidity. Treatment of osteomyelitis is notoriously difficult and often requires debridement of diseased bone in conjunction with prolonged antibiotic treatment to resolve infection. During osteomyelitis, S. aureus forms characteristic multicellular microcolonies in distinct niches within bone. Virulence and metabolic responses within these multicellular microcolonies are coordinated, in part, by quorum sensing via the accessory gene regulator (agr) locus, which allows staphylococcal populations to produce toxins and adapt in response to bacterial density. During osteomyelitis, the Agr system significantly contributes to dysregulation of skeletal homeostasis and disease severity but may also paradoxically inhibit persistence in the host. Moreover, the Agr system is subject to complex crosstalk with other S. aureus regulatory systems, including SaeRS and SrrAB, which can significantly impact the progression of osteomyelitis. The objective of this review is to highlight Agr regulation, its implications on toxin production, factors that affect Agr activation, and the potential paradoxical influences of Agr regulation on disease progression during osteomyelitis.

Gaussia Luciferase as a Reporter for Quorum Sensing in Staphylococcus aureus.

Gaussia luciferase (GLuc) is a secreted protein with significant potential for use as a reporter of gene expression in bacterial pathogenicity studies. To date there are relatively few examples of its use in bacteriology. In this study we show that GLuc can be functionally expressed in the human pathogen Staphylococcus aureus and furthermore show that it can be used as a biosensor for the agr quorum sensing (QS) system which employs autoinducing peptides to control virulence. GLuc was linked to the P3 promoter of the S. aureus agr operon. Biosensor strains were validated by evaluation of chemical agent-mediated activation and inhibition of agr. Use of GLuc enabled quantitative assessment of agr activity. This demonstrates the utility of Gaussia luciferase for in vitro monitoring of agr activation and inhibition.

Adaptive expression of biofilm regulators and adhesion factors of Staphylococcus aureus during acute wound infection under the treatment of negative pressure wound therapy in vivo.

Negative pressure wound therapy (NPWT) is gaining acceptance as a physical therapy for a wide variety of infected wounds. To gain insight into the response of bacteria to NPWT in vivo, the adaptive expression of biofilm regulators and adhesion factors of Staphylococcus aureus (S. aureus), the most frequently isolated pathogen in the clinic, during acute wound infection was investigated. A 3 cm full-thickness dermal wound was created on each side of a rabbit back and inoculated with green fluorescent protein-labeled S. aureus. NPWT was initiated at 6 h post inoculation, with the wound on the contralateral side as the untreated self-control. The wounds were subjected to a 28 day observation period. Histological analysis, laser scanning confocal microscopy and scanning electron microscopy revealed a transition of S. aureus to a free-living phenotype in tissues treated with NPWT, compared with microcolonies in untreated wounds. Viable bacteria counts showed a modest reduction in the bioburden of NPWT group on day 8 (P<0.001), with ~1x106 colony-forming units/g tissue. Transcript analysis of biofilm- and colonization-related genes were investigated using reverse transcription-quantitative PCR on postoperative days 1, 2, 4 and 8. The poly-beta-1,6-N-acetyl-D-glucosamine synthase locus and holin-like protein CidA/antiholin-like protein LrgA network were less active in the NPWT group compared with the untreated control group. Accordingly, the expression profile switched to an elevated expression of the adhesive factors UDP-phosphate N-acetylglucosaminyl 1-phosphate transferase (at days 0-4) and fibronectin-binding protein A and iron-regulated surface determinant protein A at >4 days during both stages of colonization. Meanwhile, low expression levels of the effector molecule (RNAIII) of the accessory gene regulator type I (agr) system was detected in NPWT group, suggesting that the bacterial density in NPWT-treated wounds was under the threshold for agr activation, thus not leading to an active and invasive infection. The wounds treated by NPWT healed completely on day 28, compared with an average of an 8.11% defect area in the control group (P<0.001). The results of the current study indicated that S. aureus responds to NPWT by regulating gene expression, manifesting a decrease in biofilm formation and an increase in bacterial colonization in vivo, which potentially benefits the wound repair and healing process.

Candida albicans Impacts Staphylococcus aureus Alpha-Toxin Production via Extracellular Alkalinization.

Candida albicans and Staphylococcus aureus are common causes of nosocomial infections with severe morbidity and mortality. Murine polymicrobial intra-abdominal infection (IAI) with C. albicans and S. aureus results in acute mortality dependent on the secreted cytolytic effector alpha-toxin. Here, we confirmed that alpha-toxin is elevated during polymicrobial growth compared to monomicrobial growth in vitro Therefore, this study sought to unravel the mechanism by which C. albicans drives enhanced staphylococcal alpha-toxin production. Using a combination of functional and genetic approaches, we determined that an intact agr quorum sensing regulon is necessary for enhanced alpha-toxin production during coculture and that a secreted candidal factor likely is not implicated in elevating agr activation. As the agr system is pH sensitive, we observed that C. albicans raises the pH during polymicrobial growth and that this correlates with increased agr activity and alpha-toxin production. Modulation of the pH could predictably attenuate or activate agr activity during coculture. By using a C. albicans mutant deficient in alkalinization (stp2Δ/Δ), we confirmed that modulation of the extracellular pH by C. albicans can drive agr expression and toxin production. Additionally, the use of various Candida species (C. glabrata, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. krusei) demonstrated that those capable of raising the extracellular pH correlated with elevated agr activity and alpha-toxin production during coculture. Overall, we demonstrate that alkalinization of the extracellular pH by the Candida species leads to sustained activation of the staphylococcal agr system.IMPORTANCECandida albicans and Staphylococcus aureus are commonly coisolated from central venous catheters and deep-seated infections, including intra-abdominal sepsis. Thus, they represent a significant cause of nosocomial morbidity and mortality. Yet how these organisms behave in the context of polymicrobial growth remains poorly understood. In this work, we set out to determine the mechanism by which activation of the staphylococcal agr quorum sensing system and production of its major virulence effector alpha-toxin is enhanced during coculture with C. albicans Surprisingly, we likely ruled out that a secreted candidal factor drives this process. Instead, we demonstrated that alkalinization of the extracellular milieu by C. albicans and other Candida species correlated with elevated agr activity. Thus, we propose a mechanism where modulation of the extracellular pH by fungal opportunists can indirectly alter virulence of a bacterial pathogen. Uncovering molecular events that drive interkingdom pathogenicity mechanisms may enhance surveillance and treatment for devastating polymicrobial infections.

Timing Is Everything: Impact of Naturally Occurring Staphylococcus aureus AgrC Cytoplasmic Domain Adaptive Mutations on Autoinduction.

Mutations in the polymorphic Staphylococcus aureusagr locus responsible for quorum sensing (QS)-dependent virulence gene regulation occur frequently during host adaptation. In two genomically closely related S. aureus clinical isolates exhibiting marked differences in Panton-Valentine leukocidin production, a mutation conferring an N267I substitution was identified in the cytoplasmic domain of the QS sensor kinase, AgrC. This natural mutation delayed the onset and accumulation of autoinducing peptide (AIP) and showed reduced responsiveness to exogenous AIPs. Other S. aureus strains harboring naturally occurring AgrC cytoplasmic domain mutations were identified, including T247I, I311T, A343T, L245S, and F264C. These mutations were associated with reduced cytotoxicity, delayed/reduced AIP production, and impaired sensitivity to exogenous AIP. Molecular dynamics simulations were used to model the AgrC cytoplasmic domain conformational changes arising. Although mutations were localized in different parts of the C-terminal domain, their impact on molecular structure was manifested by twisting of the leading helical hairpin α1-α2, accompanied by repositioning of the H-box and G-box, along with closure of the flexible loop connecting the two and occlusion of the ATP-binding site. Such conformational rearrangements of key functional subdomains in these mutants highlight the cooperative response of molecular structure involving dimerization and ATP binding and phosphorylation, as well as the binding site for the downstream response element AgrA. These appear to increase the threshold for agr activation via AIP-dependent autoinduction, thus reducing virulence and maintaining S. aureus in an agr-downregulated "colonization" mode.IMPORTANCE Virulence factor expression in Staphylococcus aureus is regulated via autoinducing peptide (AIP)-dependent activation of the sensor kinase AgrC, which forms an integral part of the agr quorum sensing system. In response to bound AIP, the cytoplasmic domain of AgrC (AgrC-cyt) undergoes conformational changes resulting in dimerization, autophosphorylation, and phosphotransfer to the response regulator AgrA. Naturally occurring mutations in AgrC-cyt are consistent with repositioning of key functional domains, impairing dimerization and restricting access to the ATP-binding pocket. Strains harboring specific AgrC-cyt mutations exhibit reduced AIP autoinduction efficiency and a timing-dependent attenuation of cytotoxicity which may confer a survival advantage during established infection by promoting colonization while restricting unnecessary overproduction of exotoxins.

Towards a pan‐group activator of the quorum sensing system in the common pathogen Staphylococcus epidermidis

Quorum sensing (QS) is a chemical communication process that allows bacteria to regulate gene expression based on changes in population density and environmental cues, thereby enabling bacteria to coordinate their behaviors on a community‐wide scale. Among pathogens, QS is often found to play an important role in virulence. One such pathogen is the Gram‐positive bacteria Staphylococcus epidermidis. S. epidermidis is a leading cause of hospital‐acquired infections, and these infections are difficult to eradicate via traditional antibiotic therapy due to increasing antibiotic resistance and the formation of robust biofilms. The pathogenesis of S. epidermidis is mainly under the control of the accessory gene regulator (agr) QS system. agr activation is achieved by the binding of the cyclic peptide (an autoinducing peptide or AIP) signal to its cognate receptor AgrC. Synthetic compounds that target the agr system could represent valuable tools to elucidate the roles of QS and the agr system during S. epidermidis infections.S. epidermidis can be classified into three specificity groups (I, II, or III) based on the unique AIP sequence used by each group (AIP‐I, ‐II, or ‐III). A previous study characterizing the AIP‐I signal has yielded synthetic group‐specific and pan‐group inhibitors targeting the S. epidermidis agr system. The development of pan‐group activators of the agr system is of significant interest, however, as agr activation can reduce biofilm formation by S. epidermidis. To date, no such activators have been identified. The present study was directed toward the identification of synthetic pan‐group agr activators in S. epidermidis by studying and delineating the structure‐activity relationships (SARs) of AIPs‐I, ‐II, and ‐III. Our preliminary data show that the residues at the exocyclic tail region of AIP‐II and AIP‐III are important to maintain the efficacy and potency of the native AIPs, as well as the residues that are important for receptor activation, causing a switch in certain cases from activator to inhibitor. We have also identified endocyclic residues that are important to receptor binding and activation, as well as residues that are more tolerant to change. Utilizing the findings from these systematic SARs studies for all three native AIPs, we designed a small set of AIP analogs containing features hypothesized to be important for receptor activation in each agr group, and tested these AIP analogs in all three groups using reporter strains. From this focused set of AIP analogs, we discovered the first set of pan‐group activators of S. epidermidis agr system. Current efforts are directed toward improving the potency and the efficacy of these compounds, and their application to reduce biofilm accumulation of S. epidermidis on abiotic surfaces.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Linear peptidomimetics as potent antagonists of Staphylococcus aureus agr quorum sensing

Staphylococcus aureus is an important pathogen causing infections in humans and animals. Increasing problems with antimicrobial resistance has prompted the development of alternative treatment strategies, including antivirulence approaches targeting virulence regulation such as the agr quorum sensing system. agr is naturally induced by cyclic auto-inducing peptides (AIPs) binding to the AgrC receptor and cyclic peptide inhibitors have been identified competing with AIP binding to AgrC. Here, we disclose that small, linear peptidomimetics can act as specific and potent inhibitors of the S. aureus agr system via intercepting AIP-AgrC signal interaction at low micromolar concentrations. The corresponding linear peptide did not have this ability. This is the first report of a linear peptide-like molecule that interferes with agr activation by competitive binding to AgrC. Prospectively, these peptidomimetics may be valuable starting scaffolds for the development of new inhibitors of staphylococcal quorum sensing and virulence gene expression.

Structure-Function Analyses of a Staphylococcus epidermidis Autoinducing Peptide Reveals Motifs Critical for AgrC-type Receptor Modulation.

Staphylococcus epidermidis is frequently implicated in human infections associated with indwelling medical devices due to its ubiquity in the skin flora and formation of robust biofilms. The accessory gene regulator (agr) quorum sensing (QS) system plays a prominent role in the establishment of biofilms and infection by this bacterium. Agr activation is mediated by the binding of a peptide signal (or autoinducing peptide, AIP) to its cognate AgrC receptor. Many questions remain about the role of QS in S. epidermidis infections, as well as in mixed-microbial populations on a host, and chemical modulators of its agr system could provide novel insights into this signaling network. The AIP ligand provides an initial scaffold for the development of such probes; however, the structure-activity relationships (SARs) for activation of S. epidermidis AgrC receptors by AIPs are largely unknown. Herein, we report the first SAR analyses of an S. epidermidis AIP by performing systematic alanine and d-amino acid scans of the S. epidermidis AIP-I. On the basis of these results, we designed and identified potent, pan-group inhibitors of the AgrC receptors in the three S. epidermidis agr groups, as well as a set of AIP-I analogs capable of selective AgrC inhibition in either specific S. epidermidis agr groups or in another common staphylococcal species, S. aureus. In addition, we uncovered a non-native peptide agonist of AgrC-I that can strongly inhibit S. epidermidis biofilm growth. Together, these synthetic analogs represent new and readily accessible probes for investigating the roles of QS in S. epidermidis colonization and infections.

Apolipoprotein B48, the Structural Component of Chylomicrons, Is Sufficient to Antagonize Staphylococcus aureus Quorum-Sensing.

Serum lipoproteins (LP) are increasingly being recognized as dual purpose molecules that contribute to both cholesterol homeostasis and host innate defense. In fact, very low LP levels are associated with increased risk of bacterial infection in critically ill patients. In this respect, we reported that apolipoprotein B100 (apoB100), the 4536 amino acid structural protein of very low density lipoprotein (VLDL) produced by the liver, limits Staphylococcus aureus pathogenesis. S. aureus uses quorum-sensing (QS) via the accessory gene regulator (agr) operon and an autoinducing peptide (AIP) to coordinate expression of over 200 virulence genes. ApoB100 prevents agr activation by binding and sequestering secreted AIP. Importantly, human serum LP are produced not only by the liver, but are also produced by enterocytes, in the form of chylomicrons, during uptake of dietary lipids. In contrast to apoB100 in VLDL, human enterocytes use apoB48, the N-terminal 2152 amino acids (48%) of apoB100, as the structural component of chylomicrons. Interestingly, enteral feeding of critically ill patients has been associated with decreased risk of infectious complications, suggesting chylomicrons could contribute to host innate defense in critically ill patients when serum LP production by the liver is limited during the acute phase response. Therefore, we hypothesized that apoB48 would be sufficient to antagonize S. aureus QS. As expected, isolated apoB48-LP bound immobilized AIP and antagonized agr-signaling. ApoB48- and apoB100-LP inhibited agr activation with IC50s of 3.5 and 2.3 nM, respectively, demonstrating a conserved AIP binding site. Importantly, apoB48-LP antagonized QS, limited morbidity and promoted bacterial clearance in a mouse model of S. aureus infection. This work demonstrates that both naturally occurring forms of apolipoprotein B can antagonize S. aureus QS, and may suggest a previously unrecognized role for chylomicrons and enterocytes in host innate defense against S. aureus QS-mediated pathogenesis.

Increasing AIP Macrocycle Size Reveals Key Features of agr Activation in Staphylococcus aureus.

The agr locus in the commensal human pathogen, Staphylococcus aureus, is a two-promoter regulon with allelic variability that produces a quorum-sensing circuit involved in regulating virulence within the bacterium. Secretion of unique autoinducing peptides (AIPs) and detection of their concentrations by AgrC, a transmembrane receptor histidine kinase, coordinates local bacterial population density with global changes in gene expression. The finding that staphylococcal virulence can be inhibited through antagonism of this quorum-sensing pathway has fueled tremendous interest in understanding the structure-activity relationships underlying the AIP-AgrC interaction. The defining structural feature of the AIP is a 16-membered, thiolactone-containing macrocycle. Surprisingly, the importance of ring size on agr activation or inhibition has not been explored. In this study, we address this deficiency through the synthesis and functional analysis of AIP analogues featuring enlarged and reduced macrocycles. Notably, this study is the first to interrogate AIP function by using both established cell-based reporter gene assays and newly developed in vitro AgrC-I binding and autophosphorylation activity assays. Based on our data, we present a model for robust agr activation involving a cooperative, three-points-of-contact interaction between the AIP macrocycle and AgrC.

Early agr activation correlates with vancomycin treatment failure in multi-clonotype MRSA endovascular infections.

Persistent MRSA infections are especially relevant to endovascular infections and correlate with suboptimal outcomes. However, the virulence signatures of Staphylococcus aureus that drive such persistence outcomes are not well defined. In the current study, we investigated correlations between accessory gene regulator (agr) activation and the outcome of vancomycin treatment in an experimental model of infective endocarditis (IE) due to MRSA strains with different agr and clonal complex (CC) types. Twelve isolates with the four most common MRSA CC and agr types (CC5-agr II, CC8-agr I, CC30-agr III and CC45-agr I) were evaluated for heterogeneous vancomycin-intermediate S. aureus (hVISA), agr function, agrA and RNAIII transcription, agr locus sequences, virulence and response to vancomycin in the IE model. Early agr RNAIII activation (beginning at 2 h of growth) in parallel with strong δ-haemolysin production correlated with persistent outcomes in the IE model following vancomycin therapy. Importantly, such treatment failures occurred across the range of CC/agr types studied. In addition, these MRSA strains: (i) were vancomycin susceptible in vitro; (ii) were not hVISA or vancomycin tolerant; and (iii) did not evolve hVISA phenotypes or perturbed δ-haemolysin activity in vivo following vancomycin therapy. Moreover, agr locus sequence analyses revealed no common point mutations that correlated with either temporal RNAIII transcription or vancomycin treatment outcomes, encompassing different CC and agr types. These data suggest that temporal agr RNAIII activation and agr functional profiles may be useful biomarkers to predict the in vivo persistence of endovascular MRSA infections despite vancomycin therapy.

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.

Molecular Mechanisms of agr Quorum Sensing in Virulent Staphylococci

How to become virulent. The staphylococcal accessory gene-regulator (agr) quorum-sensing system is responsible for integrating growth-dependent signals with a complex genetic network to coordinate expression of a large variety of virulence factors. Our current understanding of the molecular mechanisms of agr activation and inhibition are described, along with implications for the in vivo role of agr in pathogenesis.

SvrA, a multi-drug exporter, does not control agr

The Staphylococcus aureus svrA gene was identified in a signature-tagged mutagenesis screen for Tn917 insertions attenuated for mouse virulence, and subsequently found to be defective in agr expression. Its attenuation of virulence was attributed to its failure to express the agr regulon. In addition to the Tn917 insertion in svrA, the original svrA mutant strain (P6C63) has an adventitious frame-shift in agrC, which results in truncation of the AgrC peptide. Separation of the svrA mutation from the agrC frame-shift revealed that svrA has no detectable affect on agr activation, as assessed by exoprotein profiles and the production of haemolytic toxins. These results indicate that svrA does not play a role in Staphylococcus aureus infections via an agr-mediated pathway.

A slipped‐mispairing mutation in AgrA of laboratory strains and clinical isolates results in delayed activation of agr and failure to translate δ‐ and α‐haemolysins

agr is a global regulator of staphylococcal virulence and other accessory gene functions, especially including the haemolysins. Lack of haemolysin production therefore generally represents a defect in agr function. An example of this is Staphylococcus aureus strain RN4220, a widely used laboratory strain that carries a nitrosoguanidine (MNNG)-induced mutation enabling it to accept DNA from Escherichia coli and other bacteria. We show here that the non-haemolytic phenotype of RN4220 is caused by an extra A residue in a run of seven As at the 3' end of agrA (agrA-8A). This causes a frameshift that results in the addition of three amino acyl residues to the C-terminal end of the protein. The 8A mutation does not inactivate the agr locus, but rather delays agr activation by 2-3 h, which results in failure to translate alpha- and delta-haemolysins, and hence, in a non-haemolytic phenotype. This mutation turned out not to be an adventitious consequence of MNNG mutagenesis, but rather had arisen in RN450, the immediate parent of RN4220. RN450 had become haemolytically heterogeneous in storage, and its non-haemolytic variants had the 8A mutation. The same mutation was also identified in a clinical isolate in which a non-haemolytic variant had arisen during the course of infection. Haemolytic activity in the mutant laboratory strains could be restored by the addition of auto-inducing peptide (AIP) early in growth, indicating that delayed production of RNAIII is responsible for the failure to translate alpha- and delta-haemolysins. Discovery of the 8A mutation has revealed the basis of the dissociation between agr activity and the non-haemolytic phenotype of RN4220, and has solved the long-standing mystery of the variable non-haemolytic phenotype of its immediate parent, RN450. The occurrence of this mutation in a clinical isolate indicates that it is not simply a laboratory phenomenon, and may represent a naturally occurring mechanism for the modulation of agr activity.