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

Laboratory of Synthetic Protein Chemistry, Training Program in Chemical Biology, The Rockefeller University, New York, NY 10065, USA.
Molecular Microbiology (Impact Factor: 5.03). 09/2009; 74(1):44-57. DOI: 10.1111/j.1365-2958.2009.06849.x
Source: PubMed

ABSTRACT 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The Staphylococcus aureus agr quorum sensing system plays a major role in the transition from the persistent to the virulent phenotype. S. aureus agr type (I-IV) strains are characterized by mutations in the sensor domain of the histidine kinase AgrC and differences in the sequence of the secreted auto-inducing peptides (AIP). Here we demonstrate that interactions between the cytosolic domain of AgrC (AgrCCyto) and the response regulator domain of AgrA (AgrARR) dictate the spontaneity of the cellular response to AIP stimuli. The crystal structure of AgrCCyto provided a basis for a mechanistic model to understand AgrC-AgrA interactions. This model enabled an analysis of the biochemical and biophysical parameters of AgrC-AgrA interactions in the context of the conformational features of the AgrC-AgrA complex. This analysis revealed distinct sequence and conformational features that determine the affinity, specificity and kinetics of the phosphotransfer reaction. This step, that governs the response time for transcriptional re-engineering triggered by an AIP stimulus, is independent of the agr type and similar for agonist or antagonist stimuli. These experimental data could serve as a basis to validate simulations of the quorum sensing response and for strategies that employ the agr quorum sensing system to combat biofilm formation in S. aureus infection.
    Journal of Bacteriology 05/2014; 196(15). DOI:10.1128/JB.01530-14 · 2.69 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Bacterial signaling systems are prime drug targets for combating the global health threat of antibiotic resistant bacterial infections including those caused by Staphylococcus aureus. S. aureus is the primary cause of acute bacterial skin and soft tissue infections (SSTIs) and the quorum sensing operon agr is causally associated with these. Whether efficacious chemical inhibitors of agr signaling can be developed that promote host defense against SSTIs while sparing the normal microbiota of the skin is unknown. In a high throughput screen, we identified a small molecule inhibitor (SMI), savirin (S. aureus virulence inhibitor) that disrupted agr-mediated quorum sensing in this pathogen but not in the important skin commensal Staphylococcus epidermidis. Mechanistic studies employing electrophoretic mobility shift assays and a novel AgrA activation reporter strain revealed the transcriptional regulator AgrA as the target of inhibition within the pathogen, preventing virulence gene upregulation. Consistent with its minimal impact on exponential phase growth, including skin microbiota members, savirin did not provoke stress responses or membrane dysfunction induced by conventional antibiotics as determined by transcriptional profiling and membrane potential and integrity studies. Importantly, savirin was efficacious in two murine skin infection models, abating tissue injury and selectively promoting clearance of agr+ but not Δagr bacteria when administered at the time of infection or delayed until maximal abscess development. The mechanism of enhanced host defense involved in part enhanced intracellular killing of agr+ but not Δagr in macrophages and by low pH. Notably, resistance or tolerance to savirin inhibition of agr was not observed after multiple passages either in vivo or in vitro where under the same conditions resistance to growth inhibition was induced after passage with conventional antibiotics. Therefore, chemical inhibitors can selectively target AgrA in S. aureus to promote host defense while sparing agr signaling in S. epidermidis and limiting resistance development.
    PLoS Pathogens 06/2014; 10(6):e1004174. DOI:10.1371/journal.ppat.1004174 · 8.06 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Staphylococcus aureus virulence is regulated when secreted autoinducing peptides (AIPs) are recognized by a membrane-bound receptor histidine kinase (RHK), AgrC. Some AIPs are agonists of virulence gene expression, while others are antagonists. It is unclear how AIP binding regulates AgrC activity. Here, we reconstitute an AgrC family member, AgrC-I, using nanometer-scale lipid bilayer discs. We show that AgrC-I requires membranes rich in anionic lipids to function. The agonist, AIP-I, binds AgrC-I noncooperatively in a 2:2 stoichiometry, while an antagonist ligand, AIP-II, functions as an inverse agonist of the kinase activity. We also demonstrate the kinase and sensor domains in AgrC are connected by a helical linker whose conformational state exercises rheostat-like control over the kinase activity. Binding of agonist or inverse-agonist peptides results in twisting of the linker in different directions. These two observations provide a view of the molecular motions triggered by ligand binding in an intact membrane-bound RHK.
    Molecular cell 03/2014; 53(6):929-40. DOI:10.1016/j.molcel.2014.02.029 · 14.46 Impact Factor

Full-text (2 Sources)

Available from
Mar 16, 2015