Use of yeast mutant collection to study MUC7-12-mer peptide targets
Fuss, Jason R.
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Objectives . To investigate the antimicrobial activity/mode of action of the human salivary mucin MUC7-derived 12-mer peptide, using the Saccharomyces cerevisiae genome-wide deletion mutants. 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 genomic DNA isolation and the DNA was used as a template for asymmetric PCR amplification of the unique uptags and downtags using fluorescent dye-labeled primers. The products were hybridized to tag arrays (containing complementary sequences to the uptags and downtags). Tag intensity was used to determine the abundance/fitness of each strain within the pool exposed to the peptide. 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, preliminary data showed that deletions of several genes associated with the ESCRT (endosomal sorting complex required for transport) and RIM101 signaling pathway are highly sensitive to the peptide. The RIM101 signaling pathway regulates response to pH and other environmental conditions and requires some of the components of ESCRT. Conclusion . Yeast genome-wide deletion 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. Although further microarray analysis is required, the fitness profiling has provided us with preliminary data showing that strains with deletions of genes associated with the RIM101 pathway confer sensitivity to the peptide. This implies that this pathway plays a major role in protection of S. cerevisiae against damage inflicted by the peptide. Supported by NIH/NIDCR grant RO1 DE009820 (LAB) and its Supplement (JRF).