Document Detail


Development and application of a cellular, gain-of-signal, bioluminescent reporter screen for inhibitors of type II secretion in Pseudomonas aeruginosa and Burkholderia pseudomallei.
MedLine Citation:
PMID:  21602485     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The type II secretion (T2S) system in gram-negative bacteria comprises the Sec and Tat pathways for translocating proteins into the periplasm and an outer membrane secretin for transporting proteins into the extracellular space. To discover Sec/Tat/T2S pathway inhibitors as potential new therapeutics, the authors used a Pseudomonas aeruginosa bioluminescent reporter strain responsive to SecA depletion and inhibition to screen compound libraries and characterize the hits. The reporter strain placed a luxCDABE operon under regulation of a SecA depletion-responsive upregulated promoter in a secA deletion background complemented with an ectopic lac-regulated secA copy. Bioluminescence was indirectly proportional to the isopropyl-β-D-thiogalactopyranoside concentration and stimulated by azide, a known SecA ATPase inhibitor. A total of 96 compounds (0.1% of 73,000) were detected as primary hits due to stimulation of luminescence with a z score ≥5. Direct secretion assays of the nine most potent hits, representing five chemical scaffolds, revealed that they do not inhibit SecA-mediated secretion of β-lactamase into the periplasm but do inhibit T2S-mediated extracellular secretion of elastase with IC(50) values from 5 to 25 µM. In addition, seven of the nine compounds also inhibited the T2S-mediated extracellular secretion of phospholipase C by P. aeruginosa and protease activity by Burkholderia pseudomallei.
Authors:
Donald T Moir; Ming Di; Erica Wong; Richard A Moore; Herbert P Schweizer; Donald E Woods; Terry L Bowlin
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2011-05-20
Journal Detail:
Title:  Journal of biomolecular screening     Volume:  16     ISSN:  1552-454X     ISO Abbreviation:  J Biomol Screen     Publication Date:  2011 Aug 
Date Detail:
Created Date:  2011-07-26     Completed Date:  2011-12-12     Revised Date:  2014-09-22    
Medline Journal Info:
Nlm Unique ID:  9612112     Medline TA:  J Biomol Screen     Country:  United States    
Other Details:
Languages:  eng     Pagination:  694-705     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Adenosine Triphosphatases / genetics
Bacterial Proteins / genetics,  metabolism
Burkholderia pseudomallei / drug effects*,  genetics
Enzyme Inhibitors / pharmacology
Gene Expression Regulation, Bacterial / drug effects
Genes, Reporter / genetics*
High-Throughput Screening Assays*
Luminescent Proteins / analysis*,  genetics
Membrane Transport Proteins / genetics
Pseudomonas aeruginosa / drug effects*,  genetics
Secretory Pathway / drug effects,  genetics
Small Molecule Libraries / pharmacology
Sodium Azide / pharmacology
Grant Support
ID/Acronym/Agency:
AI-056644/AI/NIAID NIH HHS; R44 AI056644/AI/NIAID NIH HHS; R44 AI056644-06/AI/NIAID NIH HHS
Chemical
Reg. No./Substance:
0/Bacterial Proteins; 0/Enzyme Inhibitors; 0/Luminescent Proteins; 0/Membrane Transport Proteins; 0/Small Molecule Libraries; 119129-39-4/SecA protein, Bacteria; 968JJ8C9DV/Sodium Azide; EC 3.6.1.-/Adenosine Triphosphatases
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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