A random mutagenesis screen enriched for missense mutations in bacterial effector proteins

Area of Science:

• Microbiology
• Systems Biology
• Molecular Biology

Background:

• Pathogen effectors are crucial for host manipulation and immune evasion.
• Studying these effectors provides insights into pathogen biology and host pathways.
• Saccharomyces cerevisiae (yeast) is a powerful model organism for effector research.

Purpose of the Study:

• To develop and validate a yeast-based screening method for identifying informative, in-frame, missense mutations in effector proteins.
• To combine in silico protein modeling with mutagenesis to uncover effector structure-function relationships.
• To characterize novel functional domains and activities of Legionella pneumophila effectors.

Main Methods:

• Utilized a yeast growth inhibition assay to screen random effector mutants.
• Employed AlphaFold2 for in silico protein structure prediction.
• Performed missense-directed mutagenesis and in vitro biochemical assays.

Main Results:

• Identified known active sites in the metalloprotease RavK.
• Pinpointed a putative active site in SdbB.
• Discovered previously unknown functional motifs in the C-terminal domain of SdbA, suggesting glycosyltransferase activity.

Conclusions:

• The developed yeast-based method efficiently enriches for functional effector mutations.
• Integrating computational modeling and experimental mutagenesis reveals critical effector features.
• The study elucidates novel functions for Legionella pneumophila effectors, advancing our understanding of host-pathogen interactions.