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BioAssay: AID 624060

Orcinol Secondary Screening for Inhibitors of Bacterial Capsule Biogenesis (2)

Assay Rationale and Summary: Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs). Over 100 million UTIs occur annually throughout the world, including more than 7 million cases in U.S. adolescents and adults. UTIs in younger children are associated with greater risk of morbidity and mortality than in older children and adults. more ..
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 Tested Compounds
 Tested Compounds
All(1)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Active(1)
 
 
 Related BioAssays
 Related BioAssays
AID: 624060
Data Source: Southern Research Specialized Biocontainment Screening Center (ECPhage_Orincol2)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-04-11
Hold-until Date: 2013-04-10
Modify Date: 2013-04-10

Data Table ( Complete ):           Active    All
BioActive Compound: 1
Depositor Specified Assays
Show more
AIDNameTypeComment
488966Primary and Confirmatory Screening for Inhibitors of Bacterial Capsule Biogenesisconfirmatoryprimary and confirmatory screen
602408Confirmatory Screen for Inhibitors of Bacterial Capsule Biogenesis (2)confirmatoryConfirmatory screen
493020Cytotoxicity screening for potential Inhibitors of Bacterial Capsule BiogenesisconfirmatoryCytotoxicity counter screen
504769Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis (2)confirmatoryCytotoxicity counter screen
504831Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis (3)confirmatoryCytotoxicity counter screen
588399Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis (4)confirmatoryCytotoxicity counter screen
602414Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis (5)confirmatoryCytotoxicity counter screen
504358Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 controlconfirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control
504543Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control (2)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control
504675Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control (3)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control
504768Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control (4)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 with C7 control
588321Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 (5).confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189
588386Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 (6)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189
588395Screening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189 (7)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis E.coli strain UT189
504349Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis InhibitionconfirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (Specificity Screen)
504538Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (2)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (Specificity Screen)
504676Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (3)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (Specificity Screen)
504767Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (4)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (Specificity Screen)
588322Screening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (5)confirmatoryScreening for Inhibitors of Bacterial Capsule Biogenesis - T7 Lysis Inhibition (Specificity Screen)
504733Orcinol Secondary Screening for Inhibitors of Bacterial Capsule BiogenesisotherOrcinol Secondary Screening for Inhibitors of Bacterial Capsule Biogenesis
488970Screening for Inhibitors of Bacterial Caspule Biogenesis - Summarysummary
652021Cytotoxicity screening for potential Inhibitors of Bacterial Capsule Biogenesis (4)confirmatory
Description:
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: Dr. Patrick Seed, Duke University, Durham, NC
Award: 1 R03 MH090791-01

Assay Rationale and Summary: Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs). Over 100 million UTIs occur annually throughout the world, including more than 7 million cases in U.S. adolescents and adults. UTIs in younger children are associated with greater risk of morbidity and mortality than in older children and adults. Antimicrobial resistance among UPEC is on the rise, driving efforts to elucidate vulnerable targets in the molecular pathogenesis of infection. New insights into the roles of K capsules in UPEC virulence during UTI make capsules an attractive target.
During UTI, UPEC lives in intracellular and extracellular locales. UPEC adheres to the apical bladder epithelium and then invades this layer of cells. Within the bladder epithelium, UPEC typically replicates in a biofilm-like state called intracellular bacterial communities (IBC). After maturation of IBCs, UPEC disperses away from the IBC and exits the infected cells. Extracellular UPEC must then re-adhere, initiating the invasion and intracellular replication phases again. Past studies have revealed bacteria encased in the IBC within a complex matrix of fibrous protein assemblies and polysaccharides. Prior studies have also shown that disruption of the IBC pathway aborts experimental UTI, highlighting the importance of this intracellular lifecycle. A detailed study of urine samples from women with acute UTI demonstrated IBC in shed bladder epithelial cell, showing that the pathway is conserved in humans. Investigations have shown that K capsule contributes to multiple aspects of pathogenesis, including IBC formation.

Of the different K types, the Group 2 and Group 3 capsules are most prevalent among UPEC isolates, with K1 and K5 being leading types. Although the capsules have different compositions, they are synthesized, assembled, and exported by functionally homologous factors, leading us to hypothesize that we can develop small molecular inhibitors of K-type encapsulation that target the most medically important K types. This secondary assay will identify the phenotypic specificity of hits from which we may determine the optimal targets for capsule biogenesis inhibition and develop analogues with pharmacologically optimized properties.
Protocol
Secondary Assay Protocol: The biochemical determination of cell-surface associated capsule was performed using UTI89 or the DeltakpsM mutant bacterial strains. The strains are grown grown with and without the test inhibitor (100 uM), were recovered by centrifugation, and the cell pellets were washed in PBS and resuspended in Tris buffer, pH5. Low pH released surface polysaccharide without lysing the bacteria. Released polysaccharide was harvested by separation from whole bacteria by centrifugation followed by deproteination with phenol/chloroform and precipitation with ethanol. The precipitated material was subjected to acid hydrolysis (pH 2 at 80 degrees C for 1 hr) and incubated with orcinol, which reacts with periodic intermediates to produce a violet color quantified at OD 572 nm. Inhibitors reducing or abrogating surface encapsulation will yield low orcinol levels similar to the capsule export control strain DeltakpsM. The positive control drug C7 was used in this screen.
Comment
Outcome: Percentage of capsule formation was calculated relative to the mean of the bacterial (no drug) control. Capsule formation at 100 uM was tested. Compounds that showed <50% capsule formation were considered Active.

Score: In this secondary assau using purified compounds, active compounds were scored on a scale of 81-100 based on thier ability to inhibit capsule formation. Compounds that were not confirmed as active were given the score 0.
Categorized Comment
BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: secondary: selectivity

BAO: bioassay specification: assay biosafety level: BSL2

BAO: assay format: cell-based format

BAO: bioassay specification: assay measurement type: endpoint assay

BAO: format detail: reagent: inducer: none

BAO: meta target: molecular target: protein target:enzyme

BAO: meta target: biological process target: biofilm formation

BAO: meta target detail: binding reporter specification: interaction: protein-drug

BAO: assay design: viability reporter: cell number

BAO: detection technology: spectrophotometry: absorbance

Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Percent Capsule Formation @ 100 uMFloat%
Additional Information
Grant Number: 1 R03 MH090791-01

Data Table (Concise)
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