Whole cell secondary assay to determine compounds inducing growth delay in presence of metal chelator in Pseudomonas Aeruginosa (absorbance 405 nm) Measured in Whole Organism System Using Plate Reader - 2091-05_Inhibitor_Dose_DryPowder_Activity_Set5
Assay Overview: Pseudomonas Aeruginosa growth delay assay (absorbance) in presence of metal chelator ..more
Keywords: pyoverdine, PvdQ, iron, metal chelator
Assay Overview: Pseudomonas Aeruginosa growth delay assay (absorbance) in presence of metal chelator
Bacteria typically require total iron concentrations in the micromolar range to support growth. Many pathogens such as P. aeruginosa produce siderophores (e.g. pyoverdine) with molecular weights below 1500 Da that bind to iron with remarkably high affinities. The PvdQ acylase is responsible for removal of the fatty acid on pyoverdine. Interestingly, mutant strains of P. aeruginosa with a deletion in the PvdQ gene do not make pyoverdine and cannot grow on iron limiting media. This secondary assay aims to selectively identify small molecules delaying the growth of Pseudomonas Aeruginosa in presence of iron chelator ethylenediamine-di (o-hydroxyphenulacetic acid) EDDHA . The small molecules will reduce the absorbance signal observed at a wavelength of 600nm mediated by the growth or absorbance signal observed at a wavelength of 405nm (pyoverdine production) of Pseudomonas Aeruginosa in presence of EDDHA.
Expected outcome: Identification of probe(s) delaying growth of Pseudomonas Aeruginosa in presence of iron chelator EDDHA. Compounds reducing the absobance by 35% at 405nm and 600nm in duplicate will be considered hits.
Protocol Whole cell Pseudomonas Aeruginosa growth delay assay in presence of metal chelator
Day 1 (Thaw bacteria):
1.Thaw frozen vial Pseudomonas aeruginosa ((Schroter), Migula)(ATCC), 15692) and grow overnight bacteria in Succinate M9 media at 37degreeC with shaking.
Day 2 (Pinning compound in assay plate and dispense bacteria):
2.Dispense 30 ul of Succinate M9 media with 2 uM (final concentration) of ethylenediamine-di (o-hydroxyphenulacetic acid) EDDHA (Complete Green Company) in 384-well black clear bottom assay plates (Aurora Biotechnologies, 32441) using MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific). The dose response compound plate (8 concentrations, 3 fold dilution, starting concentration 10 mM) are pinned twice using 384 well pin tool (100 nl) on pin table (Walkup Cybi Well) and transferred to assay plate. Pins are washed with methanol and DMSO between each pinning. Then, 10 ul of Pseudomonas aeruginosa with 2uM EDDHA (starting OD (600nm) of 0.000002 to a final OD (600 nm) of 0.0000005) is dispensed with MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific) in the same assay plates.
3.Incubate bacteria in humidified environment at 37degreeC for 24-30h (Cytomat, High res).
Day 3 (Read on Envision)
4.Read absorbance (405 nm) on Envision (Perkin Elmer).
Succinate M9 media recipe:
6g Potassium phosphate dibasic (34.5 mM)(KH2PO4)(Sigma, P3786)
3g Potassium phosphate monobasic (22 mM)(KH2PO4)(Fisher Scientific, M-10522)
1g Ammonium sulfate (7.5 mM)((NH4)2SO4)(Sigma, A4418)
4g Succinic acid (34 mM)(Sigma, S3674)
Adjust pH at 7.0 with Potassium hydroxide (KOH)(VWR,BDH0262)
After autoclaving media, add 0.25g Magnesium sulfate (MgSO4)(Sigma, M2643)
PRESENCE OF CONTROLS: Neutral control wells (NC; n=150) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
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 (2)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw 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).
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 -35.0
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 >= -70% but <= -30%, 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.
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
Assay Format: Organism-based
Assay Cell Type: Pseudomonas aeruginosa (Schroeter) Migula
* Activity Concentration. ** Test Concentration.
Data Table (Concise)