Use of Yeast Mutant Collection to Study MUC7-12-mer Peptide Targets

Objectives: To investigate the antimicrobial activity of the human salivary mucin MUC7 derived 12-mer peptide using the Saccharomyces cerevisiae deletion pool and determine the targets of this cationic antimicrobial peptide.

Methods: S. cerevisiae genome-wide pool of 4,653 homozygous diploid deletion strains (each “bar-coded” by a unique oligonucleotide uptag and downtag) was used in two methods, 1) direct selection and 2) fitness profiling. For direct selection, the resistant strains were selected through a prolonged exposure to the peptide. Selected clones were identified by DNA sequencing of the unique barcodes. For fitness profiling, the pool was grown in the presence (5 µM) and absence of the peptide. Cells were removed at different time points for DNA isolation and PCR amplification of the unique uptags and downtags are to be used in tag array hybridizations. We have designed the tag microarrays based on the previously published studies and had them manufactured by NimbleGen.

Results: By direct selection method, one MUC7-12-mer-resistant clone that has been selected was identified as a knock-out of the mitochondrial C1-tetrahydrofolate synthase gene (involved in interconversion between different oxidation states of tetrahydrofolate). The resistance was confirmed using a separately purchased identical deletion mutant. For fitness profiling, thus far we have isolated DNA from cells collected at different time points, and are currently working on obtaining an asymmetric Cy-dye labeled PCR products for microarray analysis.

Conclusion: Yeast genome-wide mutant collection provides us with a useful tool for identification of deletions leading to fitness advantage or disadvantage over the parental strain in the presence of the MUC7-12-mer peptide. The fitness profiling is in progress. Supported by NIH/NIDCR grant DE009820 (LAB) and its Supplement (JRF).