title: Development and application of a cellular, gain-of-signal, bioluminescent reporter screen for inhibitors of type II secretion in Pseudomonas aeruginosa and Burkholderia pseudomallei.
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 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: Microbiotix, Inc., Worcester, MA 01605, USA. dmoir@microbiotix.com
authors: Donald T
authors: DT
authors: Moir
authors: Ming
authors: M
authors: Di
authors: Erica
authors: E
authors: Wong
authors: Richard A
authors: RA
authors: Moore
authors: Herbert P
authors: HP
authors: Schweizer
authors: Donald E
authors: DE
authors: Woods
authors: Terry L
authors: TL
authors: Bowlin
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