Influence of the AgrC-AgrA complex on the response time of Staphylococcus aureus quorum sensing

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 to IV strains are characterized by mutations in the sensor domain of the histidine kinase AgrC and differences in the sequences of the secreted autoinducing 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 of 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, which governs the response time for transcriptional reengineering triggered by an AIP stimulus, is independent of the agr type and similar for agonist and antagonist stimuli. These experimental data could serve as a basis on which 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 infections.

FIG 1

Conformational features of AgrC. (A) The ATP binding domain in the crystal structure of AgrC (PDB code 4BXI). Residues 389 to 393 could not be modeled in the electron density map (shown by a discontinuous line). (B) Structural superimposition of the ATP binding domain of AgrC and a representative ligand-bound ATP binding domain (TM0853; PDB code 2C2A; in gray). The lid of the ATP binding site adopts a conformation in AgrC that is different from that in other characterized domain structures in the apo- and ATP-bound forms. (C) AgrC sequence conservation across staphylococci. For this representation of sequence conservation on a structural model, the transmembrane segment of AgrC (residues 1 to 206) was modeled by using Acetabularia acetabulum rhodopsin AR2 (PDB code 3AM6) as a template in the LOMETS program (51). Mutations in the seventh transmembrane helix (inset) result in loss of specificity to self-activating peptides (3). The dimerization domain (residues 206 to 278) was modeled on the basis of T. maritima HK853 (PDB code 3DGE) by using the Phyre server (56). This figure was generated with ConSurf (52).

FIG 2

Kinetic parameters of phosphorylation and phosphotransfer. (A) The time course reaction used to calculate the initial rates of AgrC_Cyto∼P formation at different ATP concentrations. (B) Reaction velocity-versus-[ATP] curve for AgrC autophosphorylation. The kinetic parameters of this reaction are reported in the text. (C) Data used to calculate the kinetic parameters of the phosphotransfer reaction between AgrC_Cyto and AgrA_FL. (D) Comparison of phosphotransfer to AgrA_RR and phosphotransfer to AgrA_FL. The error bars represent standard deviations of five independent measurements.

FIG 3

Phosphorylation influences AgrC_Cyto-AgrA interactions. (A) Model of AgrC_Cyto-AgrA_RR interaction. The interacting residues in AgrC_Cyto and AgrA are shown in more detail in Fig. SA3, SA4, and SA5 in the supplemental material. (B) SPR spectroscopy experiments were performed by immobilizing AgrA_FL on a CM5 chip. Different concentrations of AgrC_Cyto were used as analytes. (C) AgrC_Cyto-AgrA_FL interactions in the presence of 2 mM ATP. (D) To quantify AgrC-AgrA interactions, Tyr-251 at the interface of AgrC and AgrA (inset highlighted in panel A) of AgrC_Cyto was mutated to a tryptophan residue. The emission spectrum (300 to 400 nm) of AgrC_CytoY251W (5 μM) suggests that the tryptophan fluorescence is sensitive to the conformation of AgrC_Cyto. (E) To monitor the interaction of AgrC with AgrA, 4 μM AgrC_CytoY251W was titrated with increasing concentrations of AgrA_FL (0 to 80 μM) and fluorescence emission at 344 nm was monitored (estimated K_d, 20.62 ± 2.98 μM). (F) ATP influences AgrC_Cyto-AgrA interactions. Fluorescence titration in the presence of 2 mM ATP resulted in a K_d of 9.02 ± 2.0 μM (error bars represent standard deviations; n = 6).

FIG 4

Quaternary association of AgrA_FL. (A) Phosphorylated AgrA_FL is a dimer in solution. Shown is the size exclusion profile of AgrA_FL on a Superdex 200-10/300GL column (GE Healthcare) (calibration shown in the inset). Phosphorylated AgrA was obtained by incubating this protein in 10 mM acetyl phosphate. (B) DLS measurements performed at different concentrations (0.3 to 2.0 μM) of AgrA_FL in the presence or absence of acetyl phosphate are consistent with the size exclusion results. The error bars represent the standard deviations of three measurements with different protein preparations.

FIG 5

(A) Effects of agonistic stimuli on the transcriptional levels of genes in the agr regulon. S. aureus clinical isolate 1437 (agr type II) was grown at 37°C in TSB, and cells were harvested at 3, 5, and 7 h. The mRNA levels of genes in the Agr regulon (agrA, agrC, RNAIII, hla [α-hemolysin], and spa [surface protein A]) were quantified. The error bars represent standard deviations of triplicates, and fold changes in expression are on a log scale. The inset shows the immunoprecipitation of AgrA from S. aureus cell lysates at different time points. Freshly purified recombinant AgrA was used as the control. (B) Effect of antagonistic stimuli on the transcriptional levels of genes in the agr regulon. The expression of agrA, agrC, RNAIII, hla, and spa was monitored after cells were subjected to antagonistic stimuli. In this representative illustration, clinical isolate 1437 (agr type II) was grown until the early exponential phase (3 h). Supernatants from S. aureus 559 (agr type I), 1039 (agr type III), and 368 (agr type IV) were added to the cultures, and they were grown further before the isolation of cells at 5 and 7 h. For clarity, the agr types are represented as I, II, III, and IV. (C) Effects of antagonistic stimuli on hemolysis. S. aureus strain 1437 was streaked onto 5% blood agar plates containing AIPs of other agr types. The clearance zones provide a measure of hemolysis. The effect of AIPs on hemolysis was visually monitored by comparison with blood agar plates without AIPs. These findings on the effects of agonist and antagonist stimuli were consistent across all of the clinical strains examined in this study (see Fig. SA17 in the supplemental material).

FIG 6

Schematic representation of the stages of the S. aureus agr quorum-sensing mechanism. This compilation, including the definition of steps I to V, is based on previous reports (8, 37, 53, 54). The focus of this study (inner dotted circle) is the intracellular signal transduction step regulated by the AgrC_Cyto-AgrA complex. The binding affinity for the AgrC_Cyto-AgrA interactions (including the effect of phosphorylation), as well as the enzyme kinetic parameters, rationalizes the spontaneity in triggering an intracellular response to an AIP stimulus. The signal amplification (larger circle) is slower, with a time frame of ca. 5 h. It is at this stage where the effect of the stability of RNAIII (half-life, >45 min [55]) is likely to be important. Our data (Fig. 5) suggest a linear AgrA concentration increase at both the mRNA and protein levels. This finding supports a mathematical model placing AgrA expression in a leading position before that of other genes in the agr regulon.