Cell-Based Assay to Determine Type 3 Secretion System Translocon Assembly in Pseudomonas aeruginosa Using Split...

Area of Science:

• Microbiology and Infectious Diseases
• Molecular Biology
• Drug Discovery

Background:

• Multi-drug-resistant *Pseudomonas aeruginosa* is a significant threat in healthcare settings.
• The Type 3 secretion system (T3SS) is a key virulence factor in *P. aeruginosa* pathogenesis.
• T3SS translocon assembly is essential for virulence and a potential therapeutic target.

Purpose of the Study:

• To develop a novel whole-cell-based assay for detecting T3SS translocon assembly.
• To establish a high-throughput screening method for identifying inhibitors of T3SS assembly.

Main Methods:

• Utilized a split NanoLuc luciferase reporter system for translocon assembly readout.
• Developed a whole-cell assay to monitor the protrusion of PopD N-terminus into host cells.
• Assessed assay performance using signal/noise ratio and Z'-factor for robustness.

Main Results:

• The novel assay successfully detected functional T3SS translocon assembly.
• Achieved a favorable signal/noise ratio of 13.6.
• Demonstrated assay robustness with a Z'-factor of 0.67, suitable for high-throughput screening.

Conclusions:

• A novel, robust whole-cell assay for T3SS translocon assembly has been developed.
• This assay is suitable for high-throughput screening of small-molecule inhibitors.
• The findings facilitate the development of new therapeutic strategies against *P. aeruginosa* infections.