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A colorimetric assay for studying effector secretion through the bacterial type III secretion system
Masami Miyake1, Sadatsugu Sakane, Chiho Kobayashi
1Department of Molecular Bacteriology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan. mami@biken.osaka-u.ac.jp
FEMS Microbiology Letters
|November 13, 2007
Summary
Researchers developed a simple cell viability assay to monitor bacterial effector protein secretion via the type III secretion system. This method offers a cost-effective, high-throughput alternative for studying bacterial virulence mechanisms.
Area of Science:
- Microbiology
- Cell Biology
- Molecular Biology
Background:
- Bacterial type III secretion systems (T3SS) are crucial for virulence, enabling direct injection of effector proteins into host cells.
- Monitoring effector translocation is essential for understanding bacterial pathogenesis and host-pathogen interactions.
- Existing methods for studying T3SS function can be complex or lack high-throughput capabilities.
Purpose of the Study:
- To develop a novel, simple, and cost-effective colorimetric method for monitoring bacterial effector protein secretion through the T3SS.
- To evaluate the utility of this method for studying effector translocation and secretion signals.
- To provide a high-throughput alternative for T3SS research.
Main Methods:
- Constructing bacterial effectors fused with Bordetella adenylate cyclase as a reporter.
- Quantifying host cell viability as an indicator of effector translocation, rather than measuring intracellular cAMP.
- Utilizing commercially available reagents and standard laboratory equipment for cell death quantification.
- Testing the method with enteropathogenic Escherichia coli and its effectors (Tir, EspF, Map).
Main Results:
- Host cell viability decreased upon infection with secretion-competent bacteria expressing effector-adenylate cyclase fusion proteins.
- Cell death was exacerbated by ATP depletion (2-deoxyglucose, sodium azide), indicating partial involvement of ATP depletion.
- The method successfully monitored translocation of multiple effectors (Tir, EspF, Map) from enteropathogenic E. coli.
- The assay was effective in studying the secretion signal sequence for the effector Tir.
Conclusions:
- A novel, simple, and inexpensive colorimetric cell viability assay effectively monitors bacterial effector protein secretion via T3SS.
- This method is suitable for high-throughput screening and mechanistic studies of T3SS.
- The assay provides a valuable alternative to existing procedures for studying T3SS effector translocation and secretion signals.
