Methods: A set 3633 heterozygous C. albicans deletion strains marked by gene-specific DNA-tags (barcodes) are grown in medium in the presence/absence of the peptides. Following four consecutive 24h growth, cells from each culture are collected. DNA is isolated, molecular barcodes amplified by PCR and amplicons quantified by parallel sequencing of deletion tags. The number of times a specific DNA tag is sequenced is proportional to the content of the respective mutant strain in the pool at a given time-point. The rate of decline or increase in the relative contents of a strain in the pool over several time-points is thus a measure of its sensitivity or resistance to the peptides.
Results: C. albicans mutant pool was profiled with four antimicrobial peptides of salivary origin: MUC7-12-mer, Histatin5-12-mer (P113), cathelicidin-KR20 and lactoferricin1-11. We have determined the optimal concentration of each peptide for the profiling, and collected cells at each experimental time point. Currently, we are in the process of DNA isolation, molecular barcode amplification and sequencing used to assess fitness (sensitivity or resistance) of each strain in the pool.
Conclusions: C. albicans growth is more sensitive to the peptides than S. cerevisiae, thus peptide concentrations had to be lowered to obtain appropriate amount of cells. Upon completion of this study, we expect to identify deletion strains conferring sensitivity and resistance to the peptides reflecting both similarities and differences between C. albicans and S. cerevisiae.
Keywords: Antimicrobial agents/inhibitors, Fungi, Saliva and fitness profiling
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