Quadruple Quorum-Sensing Inputs Control <i>Vibrio cholerae</i> Virulence and Maintain System Robustness

<div><p>Bacteria use quorum sensing (QS) for cell-cell communication to carry out group behaviors. This intercellular signaling process relies on cell density-dependent production and detection of chemical signals called autoinducers (AIs). <i>Vibrio cholerae</i>, the causative agent of cholera, detects two AIs, CAI-1 and AI-2, with two histidine kinases, CqsS and LuxQ, respectively, to control biofilm formation and virulence factor production. At low cell density, these two signal receptors function in parallel to activate the key regulator LuxO, which is essential for virulence of this pathogen. At high cell density, binding of AIs to their respective receptors leads to deactivation of LuxO and repression of virulence factor production. However, mutants lacking CqsS and LuxQ maintain a normal LuxO activation level and remain virulent, suggesting that LuxO is activated by additional, unidentified signaling pathways. Here we show that two other histidine kinases, CqsR (formerly known as VC1831) and VpsS, act upstream in the central QS circuit of <i>V</i>. <i>cholerae</i> to activate LuxO. <i>V</i>. <i>cholerae</i> strains expressing any one of these four receptors are QS proficient and capable of colonizing animal hosts. In contrast, mutants lacking all four receptors are phenotypically identical to LuxO-defective mutants. Importantly, these four functionally redundant receptors act together to prevent premature induction of a QS response caused by signal perturbations. We suggest that the <i>V</i>. <i>cholerae</i> QS circuit is composed of quadruple sensory inputs and has evolved to be refractory to sporadic AI level perturbations.</p></div

VpsS and CqsR contribute to the QS response in <i>V</i>. <i>cholerae</i>.

<p>(A-B) The QS response in different <i>V</i>. <i>cholerae</i> mutants missing multiple QS receptors was measured with a HapR-dependent bioluminescence operon. Normalized light production was measured in different receptor mutants in triplicates. RLU denotes relative light units. (A) Black lines and symbols represent the wild-type. Red lines and symbols represent the Δ<i>cqsS</i> Δ<i>luxQ</i> strain. Blue lines and symbols represent the Δ<i>vpsS</i> Δ<i>cqsR</i> strain. (B) Black lines and symbols represent the wild-type. Purple lines and symbols represent the Δ<i>cqsS</i> Δ<i>luxQ</i> Δ<i>vpsS</i> strain (CqsR only). Red lines and symbols represent the Δ<i>cqsS</i> Δ<i>luxQ</i> Δ<i>cqsR</i> strain (VpsS only). Orange lines and symbols represent the Δ<i>luxQ</i> Δ<i>vpsS</i> Δ<i>cqsR</i> strain (CqsS only). Blue lines and symbols represent the Δ<i>cqsS</i> Δ<i>vpsS</i> Δ<i>cqsR</i> strain (LuxQ only). Green lines and symbols represent the strain lacking all four receptors (Δ<i>cqsS</i> Δ<i>luxQ</i> Δ<i>vpsS</i> Δ<i>cqsR</i>). (C) Effect of receptor mutations in <i>V</i>. <i>cholerae</i> infections. Competitive indices (CI) were determined between wild-type Δ<i>lacZ</i> and the indicated <i>V</i>. <i>cholerae</i> mutants in infant mice 24 hr post-infection. Each symbol represents the CI in an individual mouse and the horizontal lines indicate the median for each competition. The open symbols represent data below the limit of detection for the mutant strain. Δ3 represents triple receptor mutants with the remaining receptor shown in parentheses. Δ4 represents a <i>V</i>. <i>cholerae</i> mutant missing all four receptors.</p

Multiple sensory inputs maintain <i>V</i>. <i>cholerae</i> QS system robustness.

<p>(A-H) The QS response in different <i>V</i>. <i>cholerae</i> CqsS-expressing strains was measured with a HapR-dependent bioluminescence operon. Normalized light production was measured in different strains in duplicates. RLU denotes relative light units. The genotype of the strain used is listed above each graph. Black lines and symbols represent samples without additional CAI-1. Red lines and symbols represent samples with additional 20 μM CAI-1.</p

Activated LuxO and Qrr1-4 sRNAs are required for <i>V</i>. <i>cholerae</i> host colonization.

<p>Competitive indices (CI) were determined between wild-type Δ<i>lacZ</i> and the indicated <i>V</i>. <i>cholerae</i> mutants in infant mice 24 hr post-infection. Each symbol represents the CI in an individual mouse and horizontal lines indicate the median for each competition. Open symbols represent data below the limit of detection for the mutant strain. In that case, it was assumed that there was 1 mutant CFU present at the next lowest dilution of the wild-type sample to calculate the CIs.</p

LuxU is the key HPT protein in the <i>V</i>. <i>cholerae</i> QS signal transduction system.

<p>(A-B) The QS response in different <i>V</i>. <i>cholerae</i> mutants missing LuxU was measured with a HapR-dependent bioluminescence operon. Normalized light production was measured in different Δ<i>luxU</i> mutants in triplicates. RLU denotes relative light units. (A) Black lines and symbols represent the original Δ<i>luxU</i> mutant (WN3045). Red lines and symbols represent the new Δ<i>luxU</i> mutant (WN3557). (B) Blue lines and symbols represent the new Δ<i>luxU</i> strain with an empty vector. Red lines and symbols represent the new Δ<i>luxU</i> strain expressing WT <i>luxO</i>. Black lines and symbols represent the new Δ<i>luxU</i> strain expressing <i>luxO</i>^G333S. (C) Effect of different Δ<i>luxU</i> mutations in <i>V</i>. <i>cholerae</i> infections. Competitive indices (CI) were determined between WT Δ<i>lacZ</i> and the indicated <i>V</i>. <i>cholerae</i> mutants in infant mice 24 hr post-infection. Each symbol represents the CI in an individual mouse and horizontal lines indicate the median for each competition. Open symbols represent data below the limit of detection for the mutant strain.</p

VpsS and CqsR activities are modulated by molecules secreted by <i>V</i>. <i>cholerae</i>.

<p>Qrr4 production in <i>V</i>. <i>cholerae</i> expressing only VpsS (A and C) or CqsR (B and D) was measured with a <i>qrr</i>4-<i>lux</i> bioluminescence reporter in the presence and absence of spent culture media harvested from wild-type (A-B) or from the Δ<i>cqsA</i> Δ<i>luxS</i> mutants (C-D). Normalized light production was measured at least in triplicates. RLU denotes relative light units. Black lines and symbols indicate samples grown in fresh medium. Blue and red lines and symbols indicate samples grown in the presence of 80% (v/v) spent culture medium harvested from the wild-type and the Δ<i>cqsA</i> Δ<i>luxS</i> mutants, respectively, supplemented with 20% of 5× LB.</p

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

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