The type II secretion (T2S) system in Gram-negative bacteria is comprised of 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, we 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 up-regulated promoter in a secA deletion background complemented with an ectopic lac-regulated secA copy. Bioluminescence was indirectly proportional to the IPTG 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 9 most potent hits, representing 5 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 – 25 μM. In addition, 7 of the 9 compounds also inhibited the T2S-mediated extracellular secretion of phospholipases C by P. aeruginosa and of protease activity by Burkholderia pseudomallei.

A homogeneous, sensitive, cellular bioluminescent high throughput screen was developed for inhibitors of gyrase and other DNA damaging agents in Pseudomonas aeruginosa. The screen is based on a Photorhabdus luminescens luciferase operon transcriptional fusion to a promoter that responds to DNA damage caused by reduced gyrase levels and fluoroquinolone inhibition. This reporter strain is sensitive to levels of ciprofloxacin as low as ¼-MIC with Z’ scores above 0.5, indicating the assay is suitable for high-throughput screening. This screen combines the benefits of a whole cell assay with a sensitivity and target specificity superior to those of traditional cell-based screens for inhibitors of viability or growth. In duplicate pilot screens of 2,000 known bioactive compounds, 13 compounds generated reproducible signals ≥50% of that of the control (ciprofloxacin at ¼-MIC) using bioluminescence readings after 7h of incubation. Ten are fluoroquinolones known to cause accumulation of cleaved DNA-enzyme complexes in bacterial cells; the other three are known to create DNA adducts. Therefore, all 13 hits inhibit DNA synthesis, but by a variety of different DNA damaging mechanisms. This convenient, inexpensive screen will be useful for rapidly identifying DNA gyrase inhibitors and other DNA damaging agents, which may lead to potent new anti-bacterials.