研究目的
To overcome the lack of specificity of T3SS-dependent protein secretion and translocation into eukaryotic cells by controlling T3SS activity by external light.
研究成果
The LITESEC system presented in this work uses light-controlled sequestration of an essential dynamic T3SS component to precisely regulate the activity of the T3SS. This approach provides a new method for highly time- and space-resolved protein secretion and delivery into eukaryotic cells.
研究不足
The LITESEC-act system was less efficient for heterologous cargo expressed from plasmid and activated more slowly than LITESEC-supp2, indicating that parts of SctQ remain tethered to the membrane after illumination.
1：Experimental Design and Method Selection:
The study exploited the dynamic nature of the T3SS to control its activity using optogenetic interaction switches to regulate the availability of the cytosolic T3SS component SctQ by light.
2：Sample Selection and Data Sources:
The Gram-negative enterobacterium Yersinia enterocolitica strain IML421asd was used, which is nonpathogenic but possesses a functional T3SS.
3：List of Experimental Equipment and Materials:
Fluorescence microscopy was used to visualize the components of iLID- and LOV-based sequestration systems in live Y. enterocolitica.
4：Experimental Procedures and Operational Workflow:
The localization of mCherry-bait fusions was determined by fluorescence microscopy in live Y. enterocolitica expressing the corresponding unlabeled anchor proteins.
5：Data Analysis Methods:
The change of the normalized fluorescence signal across the bacterial cells was quantified by line scans.
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