Cholera Quorum: HTS for inducers of light production in the absence of autoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_Dose_CherryPick_Activity
Assay Overview: A modified strain of Vibrio cholerae used in this assay uses light production to signal quorum sensing. Vibrio cholerae is not naturally bioluminescent but the closely related species, Vibrio harveyi, produces light when the population is at a high density (i.e. a quorum is sensed). In the V. cholerae BH1578 strain, the V. harveyi luxCDABE (luciferase) operon was introduced as a more ..
Keywords: V. cholerae, cqsA, luxS mutant, quorum sensing, luminescence
Assay Overview: A modified strain of Vibrio cholerae used in this assay uses light production to signal quorum sensing. Vibrio cholerae is not naturally bioluminescent but the closely related species, Vibrio harveyi, produces light when the population is at a high density (i.e. a quorum is sensed). In the V. cholerae BH1578 strain, the V. harveyi luxCDABE (luciferase) operon was introduced as a cosmid and the operon is activated by endogenous mechanisms to elicit light upon reaching a quorum. In addition, the BH1578 strain is a cqsA, luxS double mutant that lacks both autoinducer synthases (CAI-1 and AI-2). BH1578 does not generate light in the absence of exogenous autoinducers but bioluminescence can be stimulated up to 10,000-fold by adding 1 uM (saturating) CAI-1. Bacteria are plated into 384 well plates and compound is added by the pin transfer method. CAI-1 will be used as the positive control. In addition to luminescence, the confluency of each well is measured at an absorbance of 600 nM.
Expected Outcome: Molecules that induce light production in BH1578 in the absence of exogenous autoinducers will cause an increased signal and register as hits in this assay. As a reminder of the rationale for the screen, the accumulation of autoinducers normally represses virulence factor expression and biofilm formation in V. cholerae.
Vibrio cholerae quorum-sensing modulator bioassay
1.BH1578 (V. cholerae del cqsA del luxQ carrying the pBB1 cosmid, which contains the V. harveyi luxCDABE luciferase operon)
LB Medium: Dissolve in 10 g/L Tryptone, 5 g/L Yeast Extract, and 10 g/L NaCl in distilled water, autoclave, store at room temperature
Tetracycline (10 mg/mL): Dissolve 10 mg tetracycline in 1 mL 100% ethanol, store at -20 degrees C, protect from light
LB/tet: add 1 mL tetracycline (10 mg/mL) to 1L of LB medium. Final concentration of tetracycline is 10 microg/mL. Prepare it fresh on a daily basis.
CAI-1 stock: Dissolve CAI-1 in DMSO to 50 mM (10.7 mg/mL), store at -20oC
1.Grow up the BH1578 reporter strain in 100 mL LB/Tet at 30 oC for >16 hours with shaking (200 rpm). The final OD600 of each culture should be > 3.0
2.Dilute the culture to an OD600 of 0.9 with LB/Tet, mix well. (Note: avoid biofilm aggregates in the culture, a low speed centrifugation (200 rpm for 1 min) should remove most aggregates)
3.Add 20 uL of LB-Tet per 384 well assay plate with the Thermo Combi fluid dispenser. Add 150 nL of compound per well.
4.Dispense 10 microL of diluted culture into each well of a 384 well plate (Black with clear bottom Greiner 781096 plates). Compounds are screened at 20 microM final concentration. 1 uM CAI-1 was used as the positive control.
5.Incubate the plates at 30 degrees C for 6 hours.
5.Measure bioluminescence (USLum(384)) and OD600 in a Perkin-Elmer Envison Multilabel Reader
PRESENCE OF CONTROLS: Neutral control wells (NC; n=36) and positive control wells (PC; n=36) were included on every plate.
EXPECTED OUTCOME: Active compounds result in increasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal Max_Concentration.
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold 40.0
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= 30% but <= 70%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)