Exploring the Mode of Action of Antimicrobial Peptide MUC7 12-Mer by Fitness Profiling of Saccharomyces cerevisiae Genomewide Mutant Collection

Abstract

The MUC7 12-mer (RKSYKCLHKRCR) is a cationic antimicrobial peptide derived from the human salivary mucin MUC7. To study its effect/mechanism of action on fungi, we performed a fitness screen of a tagged, diploid, homozygous gene deletion mutant pool of the yeast Saccharomyces cerevisiae grown in the presence of the MUC7 peptide. Forty-five strains exhibiting reduced fitness and 13 strains exhibiting increased fitness (sensitivity or resistance, respectively) were identified by hybridization intensities to tag arrays. The strongest fitness defects were observed with deletions in genes encoding elements of the RIM101 signaling pathway (regulating response to alkaline and neutral pH and other environmental conditions) and of the endosomal sorting complex required for transport (ESCRT; functioning mainly in protein sorting for degradation, but also required for activation of the RIM101 pathway). Other deletions identified as conferring fitness defect or gain are in genes associated with a variety of functions, including transcription regulation, protein trafficking, transport, metabolism, and others. The results of the pool fitness screen were validated by a set of mutant strains tested individually in the presence of the MUC7 12-mer. All tested RIM101-related deletion strains showing fitness defects confirmed their sensitivities. Taken together, the results led us to conclude that deletions of genes associated with the RIM101 pathway confer sensitivity to the peptide by preventing activation of this pathway and that this stress response plays a major role in the protection of S. cerevisiae against damage inflicted by the MUC7 12-mer peptide.

Chemical Genomic Screening of a Saccharomyces cerevisiae Genomewide Mutant Collection Reveals Genes Required for Defense against Four Antimicrobial Peptides Derived from Proteins Found in Human Saliva

“…Prominent among the 45 strains responsive to all treatments (Table 2) are mutants whose deleted genes are involved in the induction of Rim101p, a transcription factor regulating response to, among others, alkaline and neutral pH (18)(19)(20), as well as those encoding several components of the ESCRT sorting machinery (32). These two groups of deletion mutants together constitute 30% of all strains responsive to all four peptides, and both were identified as the most sensitive in our earlier screen of S. cerevisiae nonessential-gene mutants exposed to the MUC7 peptide (9). We argued previously that hypersensitivity of ESCRT deletion mutants was also associated with the inability to induce the RIM101 response, a claim supported by the well-documented fact that, in addition to its major function in sorting proteins for degradation, some elements of ESCRT machinery participate in the process leading to proteolytic activation of the Rim101p (33).…”

“…Among the strains exhibiting altered fitness when treated with the MUC7 peptide, those associated with induction of the RIM101 signaling pathway exhibited particularly strong fitness defects. The RIM101 pathway regulates the response to alkaline and neutral pH and other environmental conditions (18)(19)(20), and our results suggest that this pathway is also respon-sive to, and leads to protection from, the type of stress imposed on yeasts by MUC7 peptide (9).…”

“…Five replicates of each probe were distributed randomly on the array as described previously (17). DNA isolation, asymmetric PCR, and tag array hybridizations were carried out as described earlier (9), except that the template for PCR consisted of DNA isolated from samples of mutant pools with nonessential and essential-gene deletions mixed at a 2.1:1 ratio. This specific ratio was applied to account for the difference in the number of strains and in the number of tags in each pool.…”

“…In a previous study (9), we performed this screen with the MUC7 12-mer, the most potent of a series of CAMPs derived from the N-terminal region of the human mucin MUC7. This peptide is active against a broad range of microorganisms, including pathogenic fungi, and has long been studied in our laboratory (10)(11)(12)(13).…”

“…It kills fungal cells by disrupting the plasma membrane, resulting in its depolarization (12) and subsequent release of small molecules such as ATP (our unpublished results), accompanied by rapid accumulation of large quantities of the peptide inside the cell (14). The MUC7 12-mer peptide fitness screen was performed on nearly five thousand homozygous, diploid strains of Saccharomyces cerevisiae containing deletions of nonessential genes (9). The effect of the tested condition on the growth of each strain was measured in parallel via oligonucleotide microarray technology, made possible by built-in, strain-specific DNA tags (15)(16)(17).…”