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Lookup NU author(s): Yaping Yang, Dr Felicity AlcockORCiD, Professor Tracy Palmer FRS FRSE FMedSciORCiD
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
© 2024 The Authors.Successful colonization by the opportunistic pathogen Staphylococcus aureus depends on its ability to interact with other microorganisms. Staphylococcus aureus strains harbour a T7b subtype of type VII secretion system (T7SSb), a protein secretion system found in a wide variety of Bacillota, which functions in bacterial antagonism and virulence. Assessment of T7SSb activity in S. aureus has been hampered by low secretion activity under laboratory conditions and the lack of a sensitive assay to measure secretion. Here, we have utilized NanoLuc binary technology to develop a simple assay to monitor protein secretion via detection of bioluminescence. Fusion of the 11 amino acid NanoLuc fragment to the conserved substrate EsxA permits its extracellular detection upon supplementation with the large NanoLuc fragment and luciferase substrate. Following miniaturization of the assay to 384-well format, we use high-throughput analysis to demonstrate that T7SSb-dependent protein secretion differs across strains and growth temperature. We further show that the same assay can be used to monitor secretion of the surface-associated toxin substrate TspA. Using this approach, we identify three conserved accessory proteins required to mediate TspA secretion. Co-purification experiments confirm that all three proteins form a complex with TspA.
Author(s): Yang Y, Scott AA, Kneuper H, Alcock F, Palmer T
Publication type: Article
Publication status: Published
Journal: Open Biology
Year: 2024
Volume: 14
Issue: 8
Print publication date: 01/08/2024
Online publication date: 14/08/2024
Acceptance date: 28/06/2024
Date deposited: 27/08/2024
ISSN (electronic): 2046-2441
Publisher: Royal Society Publishing
URL: https://doi.org/10.1098/rsob.240060
DOI: 10.1098/rsob.240060
Data Access Statement: The datasets supporting this article have been uploaded to Figshare [52]. Alcock F, Palmer T, Yang Y, Scott A . 2024 NanoBit assay data [dataset]. Figshare. (doi:10.6084/m9.figshare.25368598) Supplementary material is available online [53]. Yang Y, Scott AA, Kneuper H, Alcock F, Palmer T . 2024 Data from: High throughput functional analysis provides novel insight into type VII secretion in Staphylococcus aureus. Figshare. (doi:10.6084/m9.figshare.c.7397761)
PubMed id: 39139050
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