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Cell-Based Assay to Determine Type 3 Secretion System Translocon Assembly in Pseudomonas aeruginosa Using Split
Hanling Guo1, Emily J Geddes2, Timothy J Opperman2
1Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003, United States.
Abstract:
Multi-drug-resistant Pseudomonas aeruginosa poses a serious threat to hospitalized patients. This organism expresses an arsenal of virulence factors that enables it to readily establish infections and disseminate in the host. The Type 3 secretion system (T3SS) and its associated effectors play a crucial role in the pathogenesis of P. aeruginosa, making them attractive targets for the development of novel therapeutic agents. The T3SS translocon, composed of PopD and PopB, is an essential component of the T3SS secretion apparatus. In the properly assembled translocon, the N-terminus of PopD protrudes into the cytoplasm of the target mammalian cell, which can be exploited as a molecular indicator of functional translocon assembly. In this article, we describe a novel whole-cell-based assay that employs the split NanoLuc luciferase detection system to provide a readout for translocon assembly. The assay demonstrates a favorable signal/noise ratio (13.6) and robustness (Z' = 0.67), making it highly suitable for high-throughput screening of small-molecule inhibitors targeting T3SS translocon assembly.
Insights
A new assay detects the assembly of the Type 3 secretion system (T3SS) translocon in Pseudomonas aeruginosa. This tool aids in developing new drugs to combat dangerous, drug-resistant bacterial infections.
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.
