RT Journal Article SR Electronic T1 Cell-based assay to determine Type 3 Secretion System translocon assembly in Pseudomonas aeruginosa using split luciferase JF bioRxiv FD Cold Spring Harbor Laboratory SP 2023.06.22.546099 DO 10.1101/2023.06.22.546099 A1 Guo, Hanling A1 Geddes, Emily A1 Opperman, Timothy J. A1 Heuck, Alejandro P. YR 2023 UL http://biorxiv.org/content/early/2023/06/22/2023.06.22.546099.abstract AB Multidrug 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 to disseminate in the host. The Type III 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, comprised 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 manuscript, 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 (17.9) and robustness (z’=0.73), making it highly suitable for high-throughput screening of small molecule inhibitors targeting T3SS translocon assembly.Competing Interest StatementTJP and EJG are employees of Microbiotix, Inc.