IADR Abstract Archives
Screening for Potential Effectors from Tannerella forsythia Genome in Yeast
Objectives: Tannerella forsythia (Tf) is an important periodontopathic bacteria, but little is known about its virulence factors. This is partly because Tf is difficult to cultivate and manipulate genetically. To overcome these problems, we performed a functional genomic screen of Tf genome for potential effector genes using the budding yeast Saccharomyces cerevisiae as a surrogate host.
Methods: We constructed a conditional expression library with 2.5-3 kb fragments of partially digested Tf genomic DNA in a galactose-inducible yeast expression vector (pYES2). The library was transformed into yeast cells and then replica plated onto media containing galactose (inducing condition) and glucose (repressing condition). Colonies that failed to grow on galactose-containing media when the expression was induced were selected for secondary screening by spotting serial dilutions of yeast cells on inducing and control media. The plasmids were recovered and sent for DNA sequencing. The sequences of the inserts were mapped on the Tf genomic sequence in GenBank. The open reading frames (ORFs) found in the inserts were cloned into pYES2 vector and tested for the ability to inhibit yeast growth.
Results: In the primary screenings of 3 sets of Tf genomic library, accounting for approximately 10-fold Tf genome coverage, we identified over 30 clones with the phenotype of interest. In the secondary screening, we isolated 4 clones with plasmid-dependent, galactose-inducible toxicity to yeast cells. Among these, 2 ORFs were cloned and shown to inhibit yeast growth in inducing condition.
Conclusions: The yeast-based screen yielded 2 ORFs from Tf genome that could encode for putative effector proteins. Further tests will be required to test if these proteins could act as a toxin on mammalian cells.
Methods: We constructed a conditional expression library with 2.5-3 kb fragments of partially digested Tf genomic DNA in a galactose-inducible yeast expression vector (pYES2). The library was transformed into yeast cells and then replica plated onto media containing galactose (inducing condition) and glucose (repressing condition). Colonies that failed to grow on galactose-containing media when the expression was induced were selected for secondary screening by spotting serial dilutions of yeast cells on inducing and control media. The plasmids were recovered and sent for DNA sequencing. The sequences of the inserts were mapped on the Tf genomic sequence in GenBank. The open reading frames (ORFs) found in the inserts were cloned into pYES2 vector and tested for the ability to inhibit yeast growth.
Results: In the primary screenings of 3 sets of Tf genomic library, accounting for approximately 10-fold Tf genome coverage, we identified over 30 clones with the phenotype of interest. In the secondary screening, we isolated 4 clones with plasmid-dependent, galactose-inducible toxicity to yeast cells. Among these, 2 ORFs were cloned and shown to inhibit yeast growth in inducing condition.
Conclusions: The yeast-based screen yielded 2 ORFs from Tf genome that could encode for putative effector proteins. Further tests will be required to test if these proteins could act as a toxin on mammalian cells.