A functional genomic yeast screen to identify pathogenic bacterial proteins

Area of Science:

• Microbiology
• Molecular Biology
• Genomics

Background:

• Bacterial pathogens inject effector proteins into host cells to manipulate cellular processes, crucial for disease.
• Identifying these translocated proteins is vital for understanding pathogenesis but remains challenging due to sequence uniqueness and lack of clear phenotypes.
• Existing methods for identifying translocated proteins have limitations, necessitating novel approaches.

Purpose of the Study:

• To develop and validate a functional genomic screen using yeast growth inhibition as an indicator for bacterial protein translocation.
• To assess the sensitivity and specificity of yeast growth inhibition for detecting translocated proteins from bacterial pathogens.
• To identify novel translocated proteins using this yeast-based assay.

Main Methods:

• Development of a semi-automated quantitative assay to monitor the growth of hundreds of yeast strains in parallel.
• Expression of Shigella and Francisella tularensis proteins in Saccharomyces cerevisiae to observe growth inhibition phenotypes.
• Analysis of yeast growth inhibition as a correlate for protein translocation.

Main Results:

• Expression of approximately half of tested Shigella translocated proteins significantly inhibited yeast growth.
• Expression of non-translocated Shigella proteins and most Francisella tularensis proteins did not significantly inhibit yeast growth.
• Identified IpaJ as a novel Shigella type III secretion system substrate, previously missed by other methods.

Conclusions:

• Yeast growth inhibition is a sensitive and specific indicator for identifying translocated bacterial effector proteins.
• The developed assay complements existing methods and is valuable for studying difficult-to-culture or genetically intractable pathogens.
• The assay's reliance on genomic DNA makes it broadly applicable for pathogen research.