Genome-wide Screen for Aggregatibacter actinomycetemcomitans-Cytolethal Distending Toxin Hypersensitivity in Yeast

Method: Based on our previously established yeast model, the catalytic subunit AaCdtB was conditionally expressed under the control of a galactose-inducible promoter in haploid yeast cells. We performed a genome-wide screen for host mutations that confer hypersensitivity to AaCdtB using the yeast deletion library, a collection of strains with single-gene-deletion of virtually all non-essential open reading frames (ORFs) in the genome. Yeast strains that showed hypersensitivity to AaCdtB were selected and rescreened by plate sensitivity assay in comparison to the same strains transformed with empty vector control.

Results: Approximately 5,000 yeast strains in the deletion library were transformed and screened for hypersensitivity to AaCdtB expression. Over 300 mutant strains were selected for rescreening; among these, 133 strains showed hypersensitivity when compared to their respective controls.  Gene Ontology analyses of these genes suggested that they are enriched for genes involved in cellular response to stress, protein modification process, DNA metabolic process, and chromosome organization.  In particular, they are highly enriched for DNA damage/repair related genes.  Furthermore, the list of genes required for the response to AaCdtB partially overlaps with those of other DNA damaging agents.

Conclusion: Our genome-wide analysis showed enrichments of genes involved in cellular stress response, particularly of DNA damage/repair genes, among genes required for cell survival in response to AaCdtB.  Further analyses and investigations in the yeast model could lead to a better understanding on the mechanisms of AaCdtB cytotoxicity and novel ways to counteract its activity.