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

The lack of genetic tools to manipulate protozoan pathogens
has
limited the use of genome-wide approaches to identify drug or vaccine
targets and understand these organisms’ biology. We have developed
an efficient method to construct genome-wide libraries for yeast surface
display (YSD) and developed a YSD fitness screen (YSD-FS) to identify
drug targets. We show the efficacy of our method by generating genome-wide
libraries for Trypanosoma brucei, Trypanosoma cruzi, and Giardia lamblia parasites. Each library has a diversity of ∼105 to 106 clones, representing ∼6- to 30-fold of
the parasite’s genome. Nanopore sequencing confirmed the libraries’
genome coverage with multiple clones for each parasite gene. Western
blot and imaging analysis confirmed surface expression of the G. lamblia library proteins in yeast. Using the YSD-FS
assay, we identified bonafide interactors of metronidazole, a drug
used to treat protozoan and bacterial infections. We also found enrichment
in nucleotide-binding domain sequences associated with yeast increased
fitness to metronidazole, indicating that this drug might target multiple
enzymes containing nucleotide-binding domains. The libraries are valuable
biological resources for discovering drug or vaccine targets, ligand
receptors, protein–protein interactions, and pathogen–host
interactions. The library assembly approach can be applied to other
organisms or expression systems, and the YSD-FS assay might help identify
new drug targets in protozoan pathogens.