Deducing receptor signaling parameters from in vivo analysis: LuxN/AI-1 quorum sensing in Vibrio harveyi

Abstract

Quorum sensing, a process of bacterial cell-cell communication, relies on production, detection, and response to autoinducer signaling molecules. LuxN, a nine-transmembrane domain protein from Vibrio harveyi, is the founding example of membrane-bound receptors for acyl-homoserine lactone (AHL) autoinducers. We used mutagenesis and suppressor analyses to identify the AHL-binding domain of LuxN and discovered LuxN mutants that confer both decreased and increased AHL sensitivity. Our analysis of dose-response curves of multiple LuxN mutants pins these inverse phenotypes on quantifiable opposing shifts in the free-energy bias of LuxN for occupying its kinase and phosphatase states. To understand receptor activation and to characterize the pathway signaling parameters, we exploited a strong LuxN antagonist, one of fifteen small-molecule antagonists we identified. We find that quorum-sensing-mediated communication can be manipulated positively and negatively to control bacterial behavior and, more broadly, that signaling parameters can be deduced from in vivo data.

Figure 1. The V. harveyi Quorum-Sensing Circuit and the LuxN Trans-Membrane Domain

(A) The autoinducer receptor systems are CAI-1/CqsS, AI-1/LuxN, and AI-2/LuxPQ. CAI-1 is (S)-3-hydroxytridecan-4-one (squares), AI-1 is 3-hydroxybutanoyl homoserine lactone (ovals), and AI-2 is (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran-borate (triangles), and they are synthesized by CqsA, LuxM, and LuxS, respectively. At low cell densities, in the absence of appreciable autoinducer, CqsS, LuxN, and LuxQ act as kinases funneling phosphate via LuxU to LuxO (arrows). Phospho-LuxO activates expression of the qrr genes; the Qrr sRNAs (comb shapes) are transcribed and they bind to and facilitate the degradation of the mRNA encoding LuxR. Without LuxR, there is no quorum sensing, and thus no light production. At high cell density, in the presence of autoinducers, the receptors act as phosphatases, draining phosphate from LuxO via LuxU. Transcription of the qrr genes is terminated, the LuxR mRNA is stabilized, and LuxR protein is produced. By activating and repressing a variety of genes, LuxR facilitates the transition of the cells into quorum-sensing mode. One operon activated by LuxR at high cell density encodes luciferase, so in the presence of autoinducers, V. harveyi produces light. (B) The cartoon depicts the putative topology of the N-terminal region of LuxN. Amino acids in red, when mutated, confer a dark phenotype. Amino acids in blue denote sites where mutations enhance sensitivity of LuxN to AI-1. The amino acid in green represents the LuxN* suppressor mutation that prevents C450-0730 antagonism.

Figure 2. LuxN AI-1 Dose-Response Curves

(A) Light production at various AI-1 concentrations is shown for wild-type LuxN and for representative LuxN mutants that have increased AI-1 EC-50 values. The data were fit with a variable-slope sigmoidal dose-response curve to determine the EC₅₀ values. (B) Light production at various AI-1 concentrations is shown for wild-type LuxN and for representative LuxN mutations that cause constitutive dark phenotypes at all AI-1 concentrations. EC₅₀ values were not determined for these mutants.

Figure 3. Molecules that Antagonize LuxN-AI-1 Binding or Signaling

(A) Structures and designations of five molecules that inhibit LuxN signaling in response to AI-1. The IC₅₀ value for each antagonist molecule is given below its structure. (B) Light production from wild-type LuxN and LuxN F163A was measured at the specified AI-1 concentrations in the presence of 0 µM, 1 µM, and 10 µM C450-0730. Data were fit as described above. (C) The light production values in panel B were collapsed as a function of f-Δε_WT as described in Experimental Procedures. f is the ligand-dependent free-energy difference between the kinase active (on) and kinase inactive (off) states of LuxN, and Δε_WT is the wild type value of f in the absence of ligand. The binding parameters used are as follows: $K_{\text{off}}^{\text{AI}-1}=1\text{nM},K_{\text{on}}^{\text{AI}-1}=1\text{mM},K_{\text{off}}^{\mathrm{C}450-0730}=1\text{mM},K_{\text{on}}^{\mathrm{C}450-0730}=500\text{nM}.$ The collapse was obtained by using Δε−Δε_WT = 3.2 for the LuxN F163A mutant.

Figure 4. AI-1 Dose-response Curves of the LuxN* Suppressor Mutants

(A) Light production of the wild-type LuxN, the LuxN* mutants, and LuxN F163A at various AI-1 concentrations. The data were fit with a variable-slope sigmoidal dose-response curve to determine the EC₅₀ value for each LuxN* mutant. (B) Light production of the dark LuxN F163A mutant harboring combinations of LuxN* mutations. Data were fit and AI-1 EC₅₀ value was determined as above. An EC₅₀ value could not be determined for the quadruple mutant because it is constitutively bright at all AI-1 concentrations.

Figure 5. LuxN Signal Transduction Can Be Described by a Two-State Model

(A) Wild-type LuxN toggles between two conformations indicated by the open and closed periplasmic domains. At low cell density, when the AI-1 concentration is negligible, LuxN is strongly biased toward its kinase state represented by the open periplasmic structure. At high cell density, in the presence of AI-1 (dark ovals), LuxN is biased toward the phosphatase state represented by the closed periplasmic structure. (B) This two-state model is represented by a free-energy diagram that describes the two ligand-free forms of the protein as on (open periplasmic domain) or off (closed periplasmic domain). The free energies of these two states are independent of ligand concentration and are represented by horizontal black lines. The free energy of the on state is lower than the free energy of the off state, producing the bias toward the kinase mode at low cell densities (i.e. low autoinducer concentration). The free energy of LuxN in its phosphatase state and bound to ligand (off_L) is represented by the descending solid curve. The point at which the free energy of the off_L state equals the free energy of the on state (solid circle) corresponds to the EC₅₀ value for AI-1. LuxN mutants identified in the genetic screen that possess increased AI-1 EC₅₀ values are represented as on⁻. Compared to wild-type LuxN, they have lower on state free energies and therefore exhibit larger AI-1 EC₅₀ values. By contrast, the three LuxN* mutants that exhibit a bias toward the phosphatase state are represented as on⁺. These mutants possess higher on state free energies than wild-type LuxN and therefore have decreased AI-1 EC₅₀ values. The EC₅₀ values of the on⁻ and on⁺ mutants are represented by the open circles.

Figure 6. Data Collapse for LuxN*, LuxN Bias, and Combined LuxN*-Bias Mutants

(A) Collapse of the dose-response data from LuxN* R245L and G271D mutants with the combined wild-type/LuxN F163A antagonist collapse from Figure 3C. These LuxN* curves were collapsed by adjusting only the bias Δε−Δε_WT to +0.5. (B) Collapse of dose-response curves from representative dark LuxN mutants with the combined wild-type/LuxN F163A antagonist collapse from Figure 3C. The LuxN W224A and LuxN T214I dose-response curves were collapsed by adjusting only the bias Δε−Δε_WT to −1.5 and −4.3, respectively. The LuxN F155A and LuxN F162A dose-response curves were collapsed by adjusting the bias Δε−Δε_WT parameter and increasing the $K_{\text{off}}^{\text{AI}-1}$ for LuxN F155A, Δε−Δε_WT = −1.0 and $K_{\text{off}}^{\text{AI}-1}=10\mathrm{nM}$, for LuxN F162A, Δε−Δε_WT = −1.0 and $K_{\text{off}}^{\text{AI}-1}=100\mathrm{nM}$.