Figure 1.
The Quorum-Sensing Circuit in Vibrio cholerae.
The CqsA/CqsS signal transduction system is shown as the example for the V. cholerae 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 qrr 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 V. harveyi lux operon [22], [24], [26]–[30]. Dotted lines denote components that are not expressed while solid lines represent those that are produced.
Figure 2.
Identification of QS-activating compounds in V. cholerae.
(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 V. cholerae ΔcqsA ΔluxS double synthase mutant (BH1578) and the luxOD47E 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 V. cholerae. The normalized light (RLU, relative light units) produced by the V. cholerae ΔcqsA ΔluxS mutant carrying the lux 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 qrr4. Expression of qrr4 was monitored in a V. cholerae luxOD47E strain carrying a qrr4-gfp transcriptional reporter (SLS353). The response is shown in the presence and absence of 50 µM compound 11. Expression of qrr4-gfp from the ΔluxO mutant (SLS373) is shown for reference. AU denotes arbitrary units. Error bars represent standard errors of the mean for three independent trials.
Figure 3.
Structure-Activity-Relationship of LuxO inhibitors.
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 V. cholerae luxOD47E strain (BH1651) carrying the lux 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.
Figure 4.
The LuxO Inhibitor does not affect DNA binding.
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.
Figure 5.
Enzyme kinetic analyses of LuxO ATPase inhibition.
(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 Figure 3) for the different LuxO inhibitors.
Figure 6.
Isolation of LuxO mutants resistant to inhibition.
(A) Normalized light (RLU, relative light units) produced by the V. cholerae ΔluxO strain carrying luxOD47E and luxOD47E harboring additional mutations in the absence (white) or presence of 100 µM of compound 11 (black) or compound 12 (gray). Error bars represent standard errors of the mean for three independent trials. Western blot analyses demonstrate that the wild type and all mutants produce comparable amounts of LuxO protein. (B) The locations of the resistance-conferring mutations are inferred from the ATP-bound Aquifex aeolicus NtrC1 structure (3M0E). Two monomers of NtrC1 are shown (cyan and green). The residues predicted to form the Walker B motif are shown in blue. The four resistance-conferring mutations (I211, L215, L242, and V294) are shown in orange. The catalytic arginine residue and ATP are shown in magenta (with side chain) and yellow, respectively.
Figure 7.
The LuxO inhibitors activate QS in different Vibiro species.
(A) Normalized light (RLU, relative light units) produced by the V. harveyi luxOD47E strain in the absence and presence of 50 µM of compounds 11 and 12. (B) Colony morphology of the constitutively active V. parahaemolyticus luxO* mutant (LM4476) and the isogenic V. parahaemolyticus ΔluxO mutant (LM9688) in the absence and presence of 500 µM compounds 11 and 12. Each strain was inoculated four times on the same plate.
Figure 8.
Control of virulence factor production by LuxO inhibitors.
(A) Western blot analysis of TcpA (Top), HapR (middle), and LuxS (bottom, loading control) in a V. cholerae luxOD47E mutant in the presence of 0, 12.5, 25, 50, 100, and 200 µM compound 12. (B) Western blot analysis of the cytoplasmic and secreted VopD in the V. parahaemolyticus constitutively active luxO* strain (LM4476) in the presence of 0, 200, and 500 µM compound 12. An isogenic V. parahaemolyticus ΔluxO mutant (LM9968) is included as the control. (C) Cytotoxicity of V. parahaemolyticus LM4476 (luxO*) on cultured HeLa cells in the absence and presence of 500 µM compound 12. Cytotoxicity was measured by lactate dehydrogenase (LDH) release from HeLa cells. 100% cytotoxicity denotes LDH activity released upon treatment with 0.45% (v/v) Triton-X100. The V. parahaemolyticus ΔluxO mutant LM9968 and the no-bacteria control are included for comparison. Error bars represent standard errors of the mean for three independent trials.