Genome-Wide Libraries for Protozoan Pathogen Drug Target Screening Using Yeast Surface Display

Area of Science:

• Parasitology
• Molecular Biology
• Drug Discovery

Background:

• Limited genetic tools hinder genome-wide studies of protozoan pathogens, restricting the identification of drug and vaccine targets.
• Understanding protozoan biology and developing new treatments are crucial for global health.

Purpose of the Study:

• To develop an efficient method for constructing genome-wide libraries for yeast surface display (YSD).
• To establish a YSD fitness screen (YSD-FS) for identifying drug targets in protozoan pathogens.
• To demonstrate the utility of YSD libraries and YSD-FS in discovering drug targets and understanding drug mechanisms.

Main Methods:

• Construction of genome-wide YSD libraries for *Trypanosoma brucei*, *Trypanosoma cruzi*, and *Giardia lamblia*.
• Utilizing Nanopore sequencing for assessing genome coverage of the constructed libraries.
• Employing YSD-FS assay to identify drug targets by screening for yeast fitness changes in response to drugs.
• Western blot and imaging analysis to confirm protein expression and localization.

Main Results:

• Generated diverse YSD libraries (10^5-10^6 clones) with extensive genome coverage for three key protozoan parasites.
• Confirmed successful surface expression of *G. lamblia* proteins in yeast.
• Identified known interactors of metronidazole using the YSD-FS assay.
• Discovered enrichment of nucleotide-binding domain sequences linked to metronidazole resistance, suggesting novel drug targets.

Conclusions:

• The developed YSD library construction method and YSD-FS assay are effective for identifying drug targets in protozoan pathogens.
• These libraries serve as valuable resources for discovering drug/vaccine targets, receptors, and interactions.
• The approach is adaptable to other organisms and expression systems, potentially accelerating drug discovery for neglected diseases.