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

Multi-drug-resistant 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 pathog...

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Bibliographic Details
Published in:ACS infectious diseases 2023-12, Vol.9 (12), p.2652-2664
Main Authors: Guo, Hanling, Geddes, Emily J, Opperman, Timothy J, Heuck, Alejandro P
Format: Article
Language:English
Subjects:
Animals
Bacterial Proteins - genetics
Humans
Mammals
Pseudomonas aeruginosa - physiology
Pseudomonas Infections
Type III Secretion Systems
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Summary:Multi-drug-resistant 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 , 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 ( ' = 0.67), making it highly suitable for high-throughput screening of small-molecule inhibitors targeting T3SS translocon assembly.
ISSN:2373-8227
2373-8227
DOI:10.1021/acsinfecdis.3c00482