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BioAssay: AID 624033

Whole cell secondary assay to identify compounds reducing the production of iron chelator pyoverdine using Chromeazurol S dye in Pseudomonas Aeruginosa Measured in Whole Organism System Using Imaging - 2091-11_Inhibitor_Dose_DryPowder_Activity

Pyoverdine is a siderophore with a low molecular-weight iron (III) specific ligand known as metal chelator present in Pseudomonas Aeruginosa. PvdQ is an enzyme involved in the synthesis of Pyoverdine and the deletion of this enzyme results in the generation of an immature form of pyoverdine,which cannot scavenger external iron. Chrome azurol S (CAS) is a method that can be used to detect the more ..
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 Tested Compounds
 Tested Compounds
All(11)
 
 
Active(8)
 
 
Inactive(3)
 
 
 Tested Substances
 Tested Substances
All(11)
 
 
Active(8)
 
 
Inactive(3)
 
 
AID: 624033
Data Source: Broad Institute (2091-11_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2012-04-09
Hold-until Date: 2013-04-05
Modify Date: 2013-04-05

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 8
Depositor Specified Assays
AIDNameTypeComment
488968Broad Institute Inhibitors of Pyoverdine Production Projectsummarysummary assay
Description:
Keywords: Pyoverdine, PvdQ, Pseudomonas Aeruginosa


Assay Overview: Chrome Azurol Assay in Pseudomonas aeruginosa
Pyoverdine is a siderophore with a low molecular-weight iron (III) specific ligand known as metal chelator present in Pseudomonas Aeruginosa. PvdQ is an enzyme involved in the synthesis of Pyoverdine and the deletion of this enzyme results in the generation of an immature form of pyoverdine,which cannot scavenger external iron. Chrome azurol S (CAS) is a method that can be used to detect the mobilization of iron in the media. In this assay, we will measure the iron level in the supernatant of Pseudomonas Aeruginosa grown with in presence of compounds inhibitors of PvdQ using Chrome Azurol S as a dye. In nomal low iron level conditions, pyoverdine wil reducing the iron content in the media by transporting iron inside the cells. However, in presence of PvdQ inhibitors, immature pyoverdine will not be secreted and iron content in the media will not be affected. In presence of iron, CAS will change color and the difference can be measured through absorbance at 665 nm.

Expected Outcome: Identifying compounds increasing absorbance at 665 nm. Compounds increasing by 50% compared to the PvdQ mutant strain in duplicate with an IC50 below 50uM will be considered actives hits.
Protocol
Chrome Azurol Assay Protocol

Protocol taken from (Characterization of pseudomonas achromobactin in Pseudomonas syringae pv. phaseolicola 1448a, BMC Microbiology 2011, 11:218, Owen and Ackerly).
Day 1 (Overnight culture):
P.aeruginosa PAO and PvdQ mutant (provided by Dr. Andrew Gulick) cells are grown overnight in 5 mL CAA Media (see recipe below) to an OD of ~0.6-0.8.
Day 2
The P. aeruginosa cells are pelleted through centrifugation and washed with CAA media twice. At the same time, the test compounds are dissolved in media in dose (Starting concentration 125uM, 2 fold dilution, 10 dose points). 185uL of the cells diluted to an OD of around 0.1 at 595nm in presence of the test compound is then dispensed into 96 well plates in triplicate. The bacteria are grown at 30degreeC with slow shake for 6 hours to give an OD at 595nm around 0.5-0.6. After the 6h incubation, the plate is spun down (3,000 rpm, 25degreeC) for 15 minutes to pellet the cells. 150 uL of the media supernatant is collected and transferred into a flat bottom 96-well plate. 30 uL of the CAZS solution (see recipe below) is added and incubated for 30 minutes.
Absorbance is read at 665 nm using Spectrophotometer. Cell number is normalized by measuring absorbance at 595nm. Value set up as percentage of positive control (PvdQ mutant, setup at 100%).
Reagents
Chrome Azurol S (CAZS) solution
2 mM Chromeazurol S in ddH20 (Santa Cruz Biotech,AC252601)
1mM FeCl3 in 10mM HCL {hexahydrate}(Sigma, F2877)
5mM Hexadecyltrimethyl-ammonium bromide (HDTMA) in ddH20 (Sigma, H6269)
Add 10ml FeCl3 solution to 50ml of the 2mM CAS solution and slowly pour this mix into 40mL 5mM HDTMA while stirring. Sterilize, store at 4degreeC in foil.
CasAmino Acid Media (CAA Media)
5 g/L (low iron) Bacto Casamino Acids (Difco, BD, Ref # 223050)
1.54 g/L K2HPO4 (3H2O) (Fisher Scientific, M-10522)
Sterilize
0.25 g/L MgSO4 (7H2O) (Sigma, M2643) (autoclave a stock separately and add to media after it cools)
Comment
The dose response curves were generated through Excel and the active concentration (AC50) for each compound was estimated at a concentration where the dose response curve crosses a predetermined activity (in this case 50% of the activity of the positive control PvdQ mutant). The cutoff for the AC50 values was set to 10X (extrapolation) above the maximum concentration tested. Each dose response curve in the in vitro primary assay was normalized to the neutral control (DMSO) and the positive control (PvdQ mutant strain), which their values were set at 0% (DMSO) and 100% (PvdQ mutant) respectively. Each compound/dose points was compared to the neutral and positive control and the raw value attributed to the compound was converted as a percent effect value (%).

The activity score was set as a maximal score of 100 for a compound having an AbsAC50 of 5uM or less on this assay and no score for a compound with an activity beyond 50uM. The ranked score of all compounds is the percentage between these 2 concentrations.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_uM *The concentration at which activity reaches 50% of the maximumFloatμM

* Activity Concentration.
Additional Information
Grant Number: 1 R03 MH092076-01

Data Table (Concise)
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