Development and application of a cellular, gain-of-signal, bioluminescent reporter screen for inhibitors of type II secretion in pseudomonas aeruginosa and burkholderia pseudomallei

Donald T. Moir, Ming Di, Erica Wong, Richard A. Moore, Herbert P. Schweizer, Donald E. Woods, Terry L. Bowlin

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

Abstract

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 IC50 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.

Original languageEnglish (US)
Pages (from-to)694-705
Number of pages12
JournalJournal of Biomolecular Screening
Volume16
Issue number7
DOIs
StatePublished - Aug 2011
Externally publishedYes

Keywords

  • high-throughput screening
  • inhibitors
  • Pseudomonas aeruginosa
  • type II secretion

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biotechnology
  • Biochemistry
  • Molecular Medicine
  • Pharmacology
  • Drug Discovery

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