Identification of QS-activating compounds in <i>V. cholerae</i>.

<p>(A) Chemical structures of the eleven QS-activating compounds. The structure of CAI-1 is shown for reference. (B) Differential responses to Class 1 and Class 2 compounds by the <i>V. cholerae</i> Δ<i>cqsA</i> Δ<i>luxS</i> double synthase mutant (BH1578) and the <i>luxO</i>^D47E mutant (BH1651). The normalized light (RLU, relative light units) produced was monitored in the absence (white) and presence of Class 1 (gray) or Class 2 (black) compounds. A representative experiment is shown using compound 1 (Class 1) and compound 11 (Class 2) from (A). (C) QS dose-response curves of <i>V. cholerae</i>. The normalized light (RLU, relative light units) produced by the <i>V. cholerae</i> Δ<i>cqsA</i> Δ<i>luxS</i> mutant carrying the <i>lux</i> operon (BH1578) is plotted as a function of the concentration of the eleven QS-activating compounds shown in (A). Black curves denote responses to Class 1 compounds. Blue curves denote responses to Class 2 compounds. The red curve denotes the response to the native autoinducer CAI-1, which is the positive control. Error bars are present, but are too small to be observed in the plot. The bars represent standard errors of the mean for three independent trials. (D) Effect of compound 11 on expression of <i>qrr</i>4. Expression of <i>qrr</i>4 was monitored in a <i>V. cholerae luxO</i>^D47E strain carrying a <i>qrr</i>4-<i>gfp</i> transcriptional reporter (SLS353). The response is shown in the presence and absence of 50 µM compound 11. Expression of <i>qrr</i>4-<i>gfp</i> from the Δ<i>luxO</i> mutant (SLS373) is shown for reference. AU denotes arbitrary units. Error bars represent standard errors of the mean for three independent trials.</p

The LuxO Inhibitor does not affect DNA binding.

<p>LuxO D47E DNA binding in the presence and absence of compounds 11 and 12 was investigated by gel mobility shift assays (A) and fluorescent anisotropy assays (B). In (A), LuxO D47E was present at 1 µM. Compounds 11 and 12 were present at 200 µM. In (B), LuxO D47E was present at the indicated concentrations and compounds 11 and 12 were present at 200 µM. Error bars are present, but are too small to be observed in the plot. The bars represent standard errors of the mean for three independent trials.</p

Enzyme kinetic analyses of LuxO ATPase inhibition.

<p>(A) Michaelis-Menton enzyme kinetic analysis of LuxO ATPase activity. The LuxO D47E ATP hydrolysis rate is plotted as a function of the concentration of ATP in the presence of the indicated amounts of compound 11. Error bars represent standard errors of the mean for at least three independent trials. (B) Lineweaver-Burk plot derived from the assay described in (A). (C) Lineweaver-Burk plot derived from a LuxO D47E ATPase assay in the presence of the indicated amounts of compound 12. (D) Correlation between % inhibition of LuxO D47E ATPase activity (2.5 mM ATP and 30 µM inhibitors) and EC₅₀ of QS-activation potency (derived from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002767#ppat-1002767-g003" target="_blank">Figure 3</a>) for the different LuxO inhibitors.</p

The Quorum-Sensing Circuit in <i>Vibrio cholerae</i>.

<p>The CqsA/CqsS signal transduction system is shown as the example for the <i>V. cholerae</i> QS circuit. (Left) At low cell density (LCD), the CAI-1 autoinducer concentration is below the detection threshold, and the membrane bound CqsS receptor functions as a kinase. The LuxO response regulator is phosphorylated and it activates the transcription of genes encoding the four Qrr sRNA genes. Aided by the RNA chaperone Hfq, the Qrr sRNAs activate and repress translation of the AphA and HapR proteins, respectively. (Right) At high cell density (HCD), binding of CAI-1 to CqsS inhibits its kinase activity. LuxO is not phosphorylated and transcription of the <i>qrr</i> genes is terminated. Translation of AphA is inhibited and HapR is derepressed. Hundreds of genes are controlled by AphA and HapR, including genes required for biofilm formation and virulence. HapR also functions as a transcriptional activator of the heterologous <i>V. harveyi lux</i> operon <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002767#ppat.1002767-Miller1" target="_blank">[22]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002767#ppat.1002767-Rutherford1" target="_blank">[24]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002767#ppat.1002767-Hammer1" target="_blank">[26]</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002767#ppat.1002767-Zhu2" target="_blank">[30]</a>. Dotted lines denote components that are not expressed while solid lines represent those that are produced.</p

Structure-Activity-Relationship of LuxO inhibitors.

<p>The core chemical structure of the LuxO inhibitors is shown at the top. All analogs possess the identical 6-thio-5-azauracil moiety with modifications in the terminal side chains (denoted R). Variations in the side chain are shown on the right. Normalized light (RLU, relative light units) produced by the <i>V. cholerae luxO</i>^D47E strain (BH1651) carrying the <i>lux</i> operon is plotted as a function of concentration of the eight different analogs. Error bars are present, but are too small to be observed in the plot. The bars represent standard errors of the mean for three independent trials.</p

The LuxO inhibitors activate QS in different Vibiro species.

<p>(A) Normalized light (RLU, relative light units) produced by the <i>V. harveyi luxO</i>^D47E strain in the absence and presence of 50 µM of compounds 11 and 12. (B) Colony morphology of the constitutively active <i>V. parahaemolyticus luxO^*</i> mutant (LM4476) and the isogenic <i>V. parahaemolyticus</i> Δ<i>luxO</i> mutant (LM9688) in the absence and presence of 500 µM compounds 11 and 12. Each strain was inoculated four times on the same plate.</p

Mechanism of Vibrio cholerae Autoinducer-1 Biosynthesis

Vibrio cholerae, the causative agent of the disease cholera, uses a cell to cell communication process called quorum sensing to control biofilm formation and virulence factor production. The major V. cholerae quorum-sensing signal CAI-1 has been identified as (<i>S</i>)-3-hydroxytridecan-4-one, and the CqsA protein is required for CAI-1 production. However, the biosynthetic route to CAI-1 remains unclear. Here we report that (<i>S</i>)-adenosylmethionine (SAM) is one of the two biosynthetic substrates for CqsA. CqsA couples SAM and decanoyl-coenzyme A to produce a previously unknown but potent quorum-sensing molecule, 3-aminotridec-2-en-4-one (Ea-CAI-1). The CqsA mechanism is unique; it combines two enzymatic transformations, a β,γ-elimination of SAM and an acyltransferase reaction into a single PLP-dependent catalytic process. Ea-CAI-1 is subsequently converted to CAI-1, presumably through the intermediate tridecane-3,4-dione (DK-CAI-1). We propose that the Ea-CAI-1 to DK-CAI-1 conversion occurs spontaneously, and we identify the enzyme responsible for the subsequent step: conversion of DK-CAI-1 into CAI-1. SAM is the substrate for the synthesis of at least three different classes of quorum-sensing signal molecules, indicating that bacteria have evolved a strategy to leverage an abundant substrate for multiple signaling purposes

Role of the CAI‑1 Fatty Acid Tail in the <i>Vibrio cholerae</i> Quorum Sensing Response

Quorum sensing is a mechanism of chemical communication among bacteria that enables collective behaviors. In <i>V. cholerae</i>, the etiological agent of the disease cholera, quorum sensing controls group behaviors including virulence factor production and biofilm formation. The major <i>V. cholerae</i> quorum-sensing system consists of the extracellular signal molecule called CAI-1 and its cognate membrane bound receptor called CqsS. Here, the ligand binding activity of CqsS is probed with structural analogues of the natural signal. Enabled by our discovery of a structurally simplified analogue of CAI-1, we prepared and analyzed a focused library. The molecules were designed to probe the effects of conformational and structural changes along the length of the fatty acid tail of CAI-1. Our results, combined with pharmacophore modeling, suggest a molecular basis for signal molecule recognition and receptor fidelity with respect to the fatty acid tail portion of CAI-1. These efforts provide novel probes to enhance discovery of antivirulence agents for the treatment of <i>V. cholerae</i>

Development of Potent Inhibitors of Pyocyanin Production in <i>Pseudomonas aeruginosa</i>

The development of new approaches for the treatment of antimicrobial-resistant infections is an urgent public health priority. The <i>Pseudomonas aeruginosa</i> pathogen, in particular, is a leading source of infection in hospital settings, with few available treatment options. In the context of an effort to develop antivirulence strategies to combat bacterial infection, we identified a series of highly effective small molecules that inhibit the production of pyocyanin, a redox-active virulence factor produced by <i>P. aeruginosa</i>. Interestingly, these new antagonists appear to suppress <i>P. aeruginosa</i> virulence factor production through a pathway that is independent of LasR and RhlR

Structure, Regulation, and Inhibition of the Quorum-Sensing Signal Integrator LuxO

<div><p>In a process called quorum sensing, bacteria communicate with chemical signal molecules called autoinducers to control collective behaviors. In pathogenic vibrios, including <i>Vibrio cholerae</i>, the accumulation of autoinducers triggers repression of genes responsible for virulence factor production and biofilm formation. The vibrio autoinducer molecules bind to transmembrane receptors of the two-component histidine sensor kinase family. Autoinducer binding inactivates the receptors’ kinase activities, leading to dephosphorylation and inhibition of the downstream response regulator LuxO. Here, we report the X-ray structure of LuxO in its unphosphorylated, autoinhibited state. Our structure reveals that LuxO, a bacterial enhancer-binding protein of the AAA+ ATPase superfamily, is inhibited by an unprecedented mechanism in which a linker that connects the catalytic and regulatory receiver domains occupies the ATPase active site. The conformational change that accompanies receiver domain phosphorylation likely disrupts this interaction, providing a mechanistic rationale for LuxO activation. We also determined the crystal structure of the LuxO catalytic domain bound to a broad-spectrum inhibitor. The inhibitor binds in the ATPase active site and recapitulates elements of the natural regulatory mechanism. Remarkably, a single inhibitor molecule may be capable of inhibiting an entire LuxO oligomer.</p></div