Cholera Quorum: HTS for inducers of light production in the absence ofautoinducers using BH1578 (luxS deficient, cqsA deficient) Measured in Microorganism System Using Plate Reader - 2132-01_Agonist_SinglePoint_HTS_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 ..
BioActive Compounds: 553
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 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 AcqsA AluxQ carrying 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 -20 degrees C
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) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in increasing readout signal.
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.
This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 30.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 50.
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
tSamples passing PAR_T only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
NOTE: Raw signal values for inactive compounds were outside the linear detection range of the plate reader; therefore, no REPRODUCIBILITY_COSINE_TRANSFORM was calculated for inactive compounds.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)