Absorbance Microorganism-Based Retest to Identify Inhibitors of Vibrio harveyi
Keywords: Quorum Sensing, Auto-inducer 2 (AI-2), Vibrio harveyi, LuxS, LuxPQ, LuxO, LuxU,LuxR, luciferase, Luminescense ..more
BioActive Compounds: 18
Keywords: Quorum Sensing, Auto-inducer 2 (AI-2), Vibrio harveyi, LuxS, LuxPQ, LuxO, LuxU,LuxR, luciferase, Luminescense
Assay Overview: A modified strain of Vibrio harveyi with constitutive on quorum sensing system will be exposed to small molecules identified from the HTS screen used to identify LuxS inhibitors and LuxPQ antagonists. Growth of the organism post exposure will be followed using optical density and disruption of down stream quorum sensing elements will be observed based on decreased luminescent signal.
Expected Outcome: Identification of AI-2 quorum sensing system inhibitors with modes of action which either antagonize the LuxPQ receptor or inhibit LuxS synthase. Such inhibitors should not perturb growth (observed by optical density) nor the constitutive on quorum sensing (observed by luminescence).
Add 45 nL 10 mM sample / well to a sterile 384 well black clear bottom assay plate (Greiner microclear). Add 60 uL / well screening culture using a combi dispenser (Thermo) and read OD600 on Envision plate reader (Perkin-Elmer), incubate plate 9 h 30C in a humid incubator, read OD600 and luminescence on Envision plate reader.
Quorum Sensing Reagents
BB721 Vibrio harveyi (Constitutive On Quorum Sensing )
5 mL LM medium plus one colony BB721, 30C 250 rpm
Overnight Culture diluted to OD600 0.0005 in AB medium
LM Medium (Rich medium for overnights and agar plates)
20 g NaCl (JT Baker, 3624-19)
10 g bactotryptone (BD, 211705)
5 g yeast extract (EMD, 1.03753)
brought to 1 liter, 0.22 u sterile filter
For plates, add 7.5 g agar (BD, 28130) / 500 ml LM autoclave 15 min, plate and flame
AB Medium (Autoinducer Bioassay Medium)
100 ml 10X AB salt
2 g casamino acids (BD, 223050)
10 mL 1 M Phosphate Buffer pH 7
10 mL 0.1 M Arginine
100 uL 1 M Borate
brought to 1 liter, 0.22 u sterile filter
10X AB salt (3M NaCl, 0.5 M MgSO4)
175 g NaCl (JT Baker, 3624-19)+ 123 g MgSO4 * 7 H20 (Sigma, 230391) brought to 1 liter, 0.22 u sterile filter
1 M Phosphate Buffer pH 7
61.5 mL 1 M K2HPO4 (Sigma, P3786 ) + 38.5 mL 1 M KH2PO4 (Sigma, P0662)
1 M Borate
15.5 g Boric Acid (Sigma, B1934) brought to 500 mL 0.22 u sterile filter
100 mM Arginine
8.71 g Arginine (free base) (Calbiochem, 1820) brought to 500 mL 0.22 u sterile filter
Single-Point Cherrypick Data Analysis
Negative control wells (NC) and positive controls wells (PC) were included on every plate.
Initial and final absorbance readings were taken for each well. The difference between these two measurements (dABS) was smaller for compounds which resulted in reduced growth of cells in the well. Therefore active compounds result in decreased readout signal.
Analysis used to determine PubChem Activity Score and Outcome
dABS was normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median calculated signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Therefore a negative activity value is expected when signal decreases relative to NC.
No plate pattern correction algorithm was used.
The final PUBCHEM_ACTIVITY_SCORE was calculated by multiplying the mean of all valid replicate values (which can be described in units of negative percent activity) by -1, resulting in a score between 0 and 100 (in units of 'percent inhibition').
The PUBCHEM_ACTIVITY_OUTCOME class was assigned as described below, based on an activity threshold of -50%:
Activity_Outcome = 1 (inactive)
<50% of replicates fall outside threshold.
Activity_Outcome = 2 (active)
100% of replicates fall outside threshold.
Activity_Outcome = 3 (inconclusive)
>= 50% and <= 75% of replicates fall outside threshold.
Compounds ideal for further study were inactive in this assay, and active in 2035-02_INHIBITORS_SINGLE-POINT_MLPCN-CHERRYPICK.
Categorized Comment - additional comments and annotations
Data Table (Concise)