Journal article
Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, Vol.152, e60488
10/01/2019
DOI: 10.3791/60488
PMID: 31710035
Abstract
Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.
Details
- Title: Subtitle
- Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens
- Creators
- Robert Faris - University of IowaMary M. Weber - University of Iowa
- Resource Type
- Journal article
- Publication Details
- JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, Vol.152, e60488
- Publisher
- Journal Of Visualized Experiments
- DOI
- 10.3791/60488
- PMID
- 31710035
- ISSN
- 1940-087X
- eISSN
- 1940-087X
- Number of pages
- 8
- Grant note
- University of Iowa Department of Microbiology and Immunology
- Language
- English
- Date published
- 10/01/2019
- Academic Unit
- Molecular Physiology and Biophysics; Microbiology and Immunology
- Record Identifier
- 9984297332702771
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